Symposium: RNA Structure and Function · 2018-01-24 · Symposium: RNA Structure and Function...

366a Tuesday, February 20, 2018

Symposium: RNA Structure and Function

1805-SympCoupling between Transcription & Splicing Tunes Gene ExpressionKarla Neugebauer.Yale University, New Haven, CT, USA.RNA is synthesized by RNA polymerases that travel along template DNA.The growing RNA polymers are referred to as nascent. As transcription pro-ceeds, nascent RNA grows in length and sequence complexity, increasing op-portunity for folding and interacting with RNA binding proteins. Ineukaryotes, the capping enzymes modify the mRNA 5’ end, the spliceosomeassembles and removes introns, and the polyadenylation machinery cleaves 3’ends to initiate polyadenylation of mRNA and terminate transcription. All ofthis takes place in a nascent RNP, which lies close to the DNA axis. I willillustrate how recent single molecule RNA-Seq strategies have revealed thein vivo kinetics of pre-mRNA splicing relative to the progress of RNA poly-merase II (Pol II). In budding and fission yeast, the spliceosome can act onintrons as soon as they emerge from Pol II, showing that splicing and tran-scription rates are matched. This predicts that introns present in multi-intron transcripts may be removed in the order of their transcription. Instead,sequencing of full-length nascent RNA molecules in S. pombe revealed thatintron removal is equally likely to occur ‘‘not in order’’, showing regulation.Surprisingly, partially spliced transcripts were rare; most nascent RNA de-tected was either fully spliced or unspliced, indicating that splicing of anygiven intron may depend on the splicing status of the other introns in the tran-script. Fully unspliced transcripts failed to cleave at the polyA site, underwenttranscriptional read-through at gene 3’ends, and were degraded by the exo-some. These observations suggest crosstalk among spliceosomes and 3’endprocessing machineries assembling on the same nascent transcript. I willdiscuss an example of regulated gene expression in which reduced splicingleads to lower reduced mRNA levels. These findings highlight coordinationbetween transcription and RNA processing steps along the pathway of geneexpression.

1806-SympCryo-Em Snapshots of the SpliceosomeKiyoshi Nagai.MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.In the past two years we have determined near atomic resolution structuresof yeast spliceosomes in several key intermediate states. These structureshave begun to reveal the detailed mechanical working of this intricate mo-lecular machine. The yeast pre-catalytic B complex structure (1) showsthat U2 snRNP is held over the tri-snRNP by U2/U6 helix II and thebranch helix is bound to SF3b. The single stranded region of U4 snRNAis already bound to the active site of the Brr2 helicase ready to unwindthe U4/U6 snRNA duplex suggesting how U6 snRNA freed from U4snRNA may fold to form the active site. In the C complex structure imme-diately after the first trans-esterification reaction (2) the cleaved 5’exon isheld between the Large and N-terminal domains of Prp8 and the 5’-phos-phate of the first intron nucleotide (G1) positioned at the catalytic center islinked to the branch point adenosine through the 2’-5’ phosphodiester link-age. The branch helix is docked into the active site by step 1 factors. ThePrp16 helicase located near the intron exit site is poised to release the step1 factors and the branch helix. The C* complex structure after Prp16 ac-tion (3) shows that the dissociation of step 1 factors from the active siteinduce conformational change of the active site to create a space for theincoming 3’exon. The RNA-based catalytic core of the spliceosomeformed during the B to Bact transition remains unchanged throughoutthe catalytic phase of the splicing and catalyzes both branching andexon-ligation and . The DEAH-box RNA helicases and step specific factorsregulate docking and undocking of the substrates (branch site and 3’ splicesite) to the single RNA-based active site to catalyze the two trans-esterification reactions.

1807-SympCrystal Structures of a Group II Intron Lariat and Implications for theSpliceosomeMaria Costa1, H�elene Walbott1, Dario Monachello1, Eric Westhof2,Francois Michel1.1Institute for Integrative Biology of the Cell - CNRS, CEA, University Paris-Sud, University Paris-Saclay, Gif-sur-Yvette, France, 2Institute of Molecularand Cellular Biology of the CNRS, Strasbourg, France.Group II introns are catalytic RNAs that can excise by themselves from precur-sor RNA molecules. These large ribozymes of bacterial origin are believed to

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have played a crucial role in eukaryotic evolution as the ancestors of nuclearpremessenger introns and their splicing machinery (the spliceosome). A hall-mark of group II introns, which they share with spliceosomal introns, is theirexcision as branched molecules called ‘lariats’. This particular conformationresults from a specific 2’-5’ phosphodiester bond between a conserved intronadenosine and the first intron nucleotide, most often a guanosine. Most bacterialgroup II introns encode a reverse transcriptase enzyme that associates with theintron to promote its genomic mobility through ‘retrotransposition’. Thismobility pathway is initiated by ‘reverse splicing’ of the excised intron lariatinto a DNA target.Our recent crystal structures of a group II intron lariat reveal for the firsttime that the 2’-5’ branch structure organizes the intron active site for effi-cient and accurate ligation of the flanking exons during the last stage ofsplicing. Moreover, after the release of the ligated exons, the terminal 3’-hy-droxyl group of the excised lariat remains docked in the reaction center, in aconfiguration poised for catalysis of the reverse-splicing reaction, the firststep in retrotransposition. Interestingly, these structures also reveal thatactive-site assembly and catalysis are under tight conformational control:(1) a rearrangement of the base-pairing pattern within the adenosine-branchpoint helix promotes docking of the 2’-5’ branch into the activesite, (2) binding of the 5’-exon drives an induced fit that contributes tocoordination of one of the two catalytic metal ions. Finally, this work ex-tends the parallels between group II introns and the spliceosome by suggest-ing new homologies between active-site nucleotides conserved in bothsystems.

1808-SympA Solid View on RNA: Solid-State NMR of RNA and RNP ComplexesAlexander Marchanka, Mumdooh Ahmed, Teresa Carlomagno.Leibniz University of Hanover, Hanover, Germany.To date, substantial progresses have been made in the structure determinationof membrane proteins and amyloid fibrils by solid-state NMR, while signifi-cantly fewer studies have addressed the structure of RNA or protein-RNA com-plexes (RNP). Nevertheless, the application of ssNMR to study large RNPcomplexes holds excellent promises, due to the independence of the ssNMRline widths from the molecular size. Here, I will present the ssNMR-basedstructure of the complex consisting of the the 26mer box C/D RNA and the pro-tein L7Ae, together with the experimental strategy that we used to obtain it.This includes experiments for the assignment of RNA resonances and collec-tion of structural restraints, as well as experiments to determine the protein-RNA contacts. In addition I will discuss the application of proton-detectionin ssNMR of RNA.Finally, I will show first ssNMR spectra of a RNA in the context of the 400 kDabox C/D RNP.

Symposium: Interrogating MembraneOrganization and Dynamics

1809-SympInsight Into Plasma Membrane Organization Acquired with SecondaryIon Mass Spectrometry (SIMS)Mary L. Kraft.Chem & Biomol Engr, University of Illinois, Urbana, IL, USA.The plasma membrane is compartmentalized into different domains thathave specialized compositions and functions. Identifying the compositionsof these domains, the mechanisms that establish them, and how they relateto cellular function is currently a major challenge. Significant advances inimaging and biotechnology have enabled imaging the distributions of pro-teins in cell membranes with unprecedented spatial resolution and speci-ficity. Although the cellular abundances of cholesterol, sphingolipids, andother lipid species influence many important biological processes, less isknown about the distributions of cholesterol and distinct lipid species withinthe plasma membrane. We are addressing this issue with a high-resolutionsecondary ion mass spectrometry (SIMS) approach for imaging isotope-labeled cholesterol and sphingolipids in parallel with immunolabeled pro-teins in the plasma membranes of mammalian cells with %100-nm-lateralresolution. With this approach, we have found that sphingolipids are concen-trated in micrometer-scale domains that are dependent on the cytoskeleton,but cholesterol is evenly distributed within the plasma membrane. Wehave also used this approach to image the distribution of cholesterol, sphin-golipids, and influenza viral envelope proteins in the plasma membranes ofcells infected with the influenza virus. These experiments are a direct test ofthe long-standing hypotheses that influenza virus assembles and buds from

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domains enriched with cholesterol and sphingolipids. The results of these ex-periments will be presented.

1810-SympComputational Modeling of Realistic Cell MembranesSiewert J. Marrink.Univ Groningen, Groningen, Netherlands.Recent advances in coarse-grain modeling allow us to zoom out from in-dividual atoms and molecules to supramolecular complexes and subcellularcompartments that contain tens of millions of particles and capture thecomplexity of the crowded environment of real cell membranes. Here Iwill describe the state-of-the-art of modelling cell membrane processeswith the coarse-grain Martini model developed in our lab. I will illustratethe power of the model by providing a few in-depth examples oflarge-scale simulations, including the exchange of electron carriers in thephotosystem II complex embedded in the thylakoid membrane, and thelateral organization of lipids and proteins in a realistic plasma membranemodel.

1811-SympMixing Water, Tranducing Energy, Shaping MembranesWan-Chih Su1, Doug Gettel2, Shiva Emami2, Sowmya Purushothaman2,3,Morgan Chabanon4, Padmini Rangamani4, Atul N. Parikh2,3.1Chemistry, University of California, Davis, CA, USA, 2BiomedicalEngineering and Materials Science & Engineering, University ofCalifornia, Davis, CA, USA, 3Centre for Biomimetic Sensor Science,Materials Science Engineering, Nanyang Technological University,Singapore, Singapore, 4Mechanical Engineering, University of California,San Diego, CA, USA.A solute, excluded from or confined within a spatial ‘‘compartment’’embedded in an aqueous continuum, creates a gradient in the chemical activ-ity of water. This in turn prompts a directed flow of water pushing it into thesolute-laden compartment and out of the solute-starved one. Serving as a non-specific entropic force, this osmotic stress acts on the vesicular boundariesproducing long-lived out-of-equilibrium morphologies and cooperativebehaviors.Drawing from recent experiments in our lab employing giant vesicles contain-ing (or excluding) molecular (e.g., sugars) and colligatively non-ideal macro-molecular (e.g., PEG and Dextran) osmolytes, this talk considers how theosmotic activity of water dynamically remodels the membrane, inducing mem-brane shapes (including protrusions, invaginations, and buds), driving topolog-ical division (producing colonies of daughter vesicles), and organizingmembrane domains in a pulsatile manner - all while dissipating the osmotic en-ergy in theoretically predictable manners.

1812-SympUncovering Theorganelle Interactome: Dynamic Imaging of MultipleOrganellesJennifer Lippincott-Schwartz.Janelia Research Campus, HHMI Janelia Research Campus, Ashburn, VA,USA.Numerous aspects of cell biology and biophysics are becoming clearer dueto emerging visualization technologies, which can capture processes at thelevel of whole organisms down to single molecules. Here, I will discuss de-velopments in probes and microscopes that are dramatically expanding pro-ductive imaging. To surmount fluorophore bleed-through, a combinedexcitation-based spectral unmixing and lattice light sheet microscopy wasused to visualize up to six organelles (i.e., ER, Golgi, mitochondria, lyso-somes, peroxisomes and lipid droplets) simultaneously within cells. This al-lowed us to track these organelles through time and analyze their inter-organelle contacts, providing a systems-level map of the organelle interac-tome and how it is perturbed under different physiological conditions. Toincrease temporal resolution during imaging, we employed total internalreflection fluorescence combined with structured illumination microscopyto visualize organelle dynamics at very high temporal-spatial resolution.Examining the ER, we observed that many peripheral ER sheets seen usingdiffraction-limited imaging are actually highly perforated structurescomprised of tightly latticed groups of dynamic tubules. Within the latticedER tubule meshwork, subdiffraction-limited holes were observed (�150-250 nm diameter) having transient lifespans (�250 msec). Viewed at higherresolution using lattice light sheet microscopy combined with point accumu-lation for nanoscale topology (PAINT), the peripheral ER sheets representeda complex meshwork of tightly cross-linked ER tubules, with potentiallyimportant roles in ER function.

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Platform: Intrinsically Disordered Proteins (IDP)and Aggregates I

1813-PlatInferring Properties of Disordered Chains From FRET Transfer Effi-cienciesWenwei Zheng1, G€ul Zerze2, Alessandro Borgia3, Jeetain Mittal2,Benjamin Schuler4, Robert B. Best5.1College of Integrative Sciences and Arts, Arizona State University, Mesa,AZ, USA, 2Department of Chemical and Biomolecular Engineering, LehighUniversity, Bethlethem, PA, USA, 3Department of Biochemistry, Universityof Zurich, Zurich, Switzerland, 4Department of Biochemistry andDepartment of Physics, University of Zurich, Zurich, Switzerland,5Laboratory of Chemical Physics, National Institute of Diabetes andDigestive and Kidney Diseases, National Institutes of Health, Bethesda, MD,USA.Forster resonance energy transfer (FRET) is a powerful tool for elucidatingboth structural and dynamic properties of unfolded or disordered biomole-cules, especially in single-molecule experiments. However, the key observ-ables, namely the mean transfer efficiency and fluorescence lifetimes of thedonor and acceptor chromophores, are averaged over a broad distribution ofdonor-acceptor distances. The inferred average properties of the ensembletherefore depend on the form of the model distribution chosen to describethe distance, as has been widely recognized. In addition, while the distributionfor one type of polymer model may be appropriate for a chain under a givenset of physico-chemical conditions, it may not be suitable for the same chainin a different environment. An apparently consistent application of the samepolymer model over all conditions may distort the apparent changes in chaindimensions with variation of temperature or solution composition. Here, wepresent an alternative and straightforward approach to determining ensembleproperties from FRET data, in which the polymer scaling exponent is allowedto vary with solution conditions. In its simplest form, it requires either themean FRET efficiency or fluorescence lifetime information. In order to testthe accuracy of the method, we have utilized both synthetic FRET datafrom implicit and explicit solvent simulations for 30 different protein se-quences, and experimental single-molecule FRET data for an intrinsicallydisordered and a denatured protein. In all cases, we find that the inferred radiiof gyration are within 10% of the true values, thus providing higher accuracythan simpler polymer models. In addition, the scaling exponents obtained byour procedure are in good agreement with those determined directly from themolecular ensemble. Our approach can in principle be generalized to treatingother ensemble-averaged functions of intramolecular distances from experi-mental data.

1814-PlatThe Collapsed Conformational Landscape of the Hnrnpa1 LowComplexity Region Revealed by SAXS, NMR and SimulationErik W. Martin, Ivan Peran, Tanja Mittag.Structural Biology, St Jude Children’s Research Hospital, Memphis, TN,USA.Liquid-liquid phase separation (LLPS), a process in which a protein solutiondemixes into protein-dense and light phases, is likely critical in the forma-tion of stress granules and other membrane-less organelles. Recent workfurther suggests that LLPS may promote protein fibrillization in neurodegen-erative disease related processes, stressing the importance of a detailed un-derstanding of the molecular interactions mediating LLPS and theconformational properties that predispose proteins to undergo LLPS. Pro-teins regions of low sequence complexity (LCR) appear to be particularlywell poised to phase separate at physiologically relevant solution conditions.Aiming to deconvolute the sequence specific driving forces for LLPS, theLCR of hnRNPA1 is used as a model system. It is reasonable to assumethat many contacts that exist between molecules in protein dense stateswill be recapitulated intramolecularly, making the conformational landscapeof monodisperse hnRNPA1 LCR particularly interesting. Small angle x-rayscattering (SAXS) and nuclear magnetic resonance (NMR) measurementsreveal that this LCR is significantly collapsed compared with the vast major-ity of the disordered proteome, yet appears to be dynamic and lacking persis-tent structure. All-atom Monte Carlo simulations and NMR measurementssuggest that interplay between glycine, aromatic and charged amino acidslead to this unique conformational behavior. Aromatic residue side chaincontacts and clustering are observed by NMR and simulations and appearsto drive the collapse. These forces are balanced by like charge repulsion,which is potentially crucial to maintaining solubility and dynamics. We

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expect that the molecular interactions revealed in disperse hnRNPA1 arecritical to LLPS.

1815-PlatProbing the Conformational Dynamics of the Disordered 4E-BP2 Proteinin Different Phosphorylation States using Single-Molecule FluorescenceSpencer Smyth1,2, Zhenfu Zhang1,2, Alaji Bah3,4, Julie D. Forman-Kay3,4,Claudiu C. Gradinaru1,2.1Department of Physics, University of Toronto, Toronto, ON, Canada,2Chemical and Physical Sciences, University of Toronto, Mississauga, ON,Canada, 3Biochemistry, University of Toronto, Toronto, ON, Canada,4Molecular Structure and Function Program, The Hospital or Sick Children,Toronto, ON, Canada.Intrinsically disordered proteins (IDPs) are a class of proteins that lack a well-defined 3D structure while carrying out a diverse range of biological functions.They play a crucial role in regulatory pathways and in mediating interactionswith multiple partners. Cap-dependent initiation of translation is regulated bythe interaction of 120-residue eukaryotic initiation factor 4E (eIF4E) withdisordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependentmanner.Fluorescence correlation spectroscopy (FCS) and fluorescence anisotropydecay (FAD) were used to study the conformations, dynamics and binding of4E-BP2. Anisotropy data informed the local chain flexibility at various pointswithin the 4E-BP2. The segmental flexibility is hindered in the folded 18-62region upon phosphorylation, whereas the rest of the chain becomes more flex-ible. The local segments become more flexible upon denaturation, which sug-gests that the native protein, whereas not having a stable 3D fold, containssignificant secondary structure. Segmental rotational correlation times andwobbling cone angles extracted for different labelling sites along the chain pro-vide a rigidity map of 4E-BP2 and are essential for elucidating the bindingmode to eIF4E.Longer range intra-chain reorganization dynamics were assessed by FCSvia photoblinking/quenching of the fluorophore by aromatic residues. Het-erogeneous quenching kinetics were observed: multi-site phosphorylationof the protein slows the proximal chain motions and modulates thekinetics of the distal regions. Our results paint a complex behavior of4E-BP2 upon phosphorylation and binding and suggest that electrostaticsplay a crucial role in modulating its dimensions and compactness andthus its activity.

1816-PlatSequence-Encoded Heterogeneity of Interactions Decouples DifferentMeasures of Protein Sizes and Reconciles the Discrepant Inferencesfrom Saxs versus Fret ExperimentsKiersten M. Ruff1, Gustavo Fuertes2, Niccolo Banterle2, Dmitri I. Svergun3,Edward A. Lemke2, Rohit V. Pappu1.1Biomedical Engineering, Washington University in St. Louis, Saint Louis,MO, USA, 2European Molecular Biology Laboratory, Heidelberg, Germany,3European Molecular Biology Laboratory, Hamburg, Germany.Quantifying the conformational properties of folded proteins as a function ofdenaturant concentration is important for understanding the mechanism ofprotein folding. Additionally, quantifying the conformational properties ofintrinsically disordered proteins (IDPs) can yield insights regarding the func-tions of IDPs. Radius of gyration, RG, which quantifies the average size of aprotein, and end-to-end distance, REE, are two commonly used conformationaldescriptors as these values can be determined using small-angle x-ray scat-tering (SAXS) and single molecule Forster resonance energy transfer(smFRET), respectively. However, for several proteins, the RG profile deter-mined directly from SAXS as a function of denaturant concentration differsfrom the RG profile inferred using REE values determined from smFRETand a given homopolymer model. Here, we show that this discrepancy isnot due to any inherent flaws of either measurement, but instead arises as aresult of sequence-specific interactions that exist within proteins under lowdenaturant conditions. Sequence-specific interactions induce heterogeneitywithin the conformational ensemble of a protein, which in turn decouplesglobally averaged (e.g., RG) and length-scale-specific (e.g., REE) conforma-tional descriptors. The extent of decoupling of conformational descriptors,and thus the appropriateness of using a homopolymer model to convert be-tween RG and REE, is sequence specific and is often unknown a priori.Here, we investigate the relationship between sequence-complexity and devi-ations from homopolymer models. Additionally, we show that by combiningresults from experimental measurements that yield distinct conformationaldescriptors with molecular simulations we can provide complete conforma-tional descriptions of heterogeneous ensembles.

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1817-PlatSaxs Confirms that FRET Dyes Promote Collapse of an Otherwise FullyDisordered ProteinJoshua A. Riback1, Micayla A. Bowman2, Adam M. Zmyslowski3,Kevin W. Plaxco4, Patricia L. Clark2, Tobin R. Sosnick3.1Graduate Program in the Biophysical Sciences, University of Chicago,Chicago, IL, USA, 2Department of Chemistry and Biochemistry, Universityof Notre Dame, Notre Dame, IN, USA, 3Department of Biochemistry andMolecular Biology, University of Chicago, Chicago, IL, USA, 4Departmentof Chemistry and Biochemistry, University of California, Santa Barbara, CA,USA.Significance: The dimensions of intrinsically disordered proteins (IDPs) re-mains controversial. Using SAXS, we previously found that even relatively hy-drophobic IDPs remain expanded in the complete absence of denaturant,contrary to conclusions drawn from FRET studies that foldable sequencescollapse in water. Here we use our methodology to demonstrate that labelingwith Alexa488 causes the IDP to undergo significant dye-induced contraction.These results demonstrate that the addition of dyes influences the properties ofIDPs and is likely the primary origin of the discrepancy between prior SAXSand FRET results.ABSTRACT: The origins of the discrepancy between SAXS and FRET mea-surements of IDP dimensions remain controversial. To address this, we recentlydeveloped a robust analysis for extracting Rg and n (the Flory exponent;Rg�Nn) from single SAXS experiments using a molecular form factor derivedfrom simulations. Performing this analysis on a 334 AA IDP, we find that theaddition of a dye pair promotes collapse, decreasing Rg from 51 to 45 A and nfrom 0.54 to 0.50. This observation is consistent with prior studies on PEG,which found the addition of dyes induces similar contraction1 despite thefact that, as we show here, the polymer behaves as a self-avoiding randomwalk. Recent studies have suggested that FRET and SAXS results can be recon-ciled if SAXS reports on Rg while remaining relatively insensitive to Rend-to-end,essentially decoupling the two. To the contrary, our analysis finds that the entireSAXS profile is sensitive to conformational ensembles enriched in shortenedend-to-end distances, such as those produced by dye-dye interactions. Morebroadly, our simulations define a route to characterize the deviations from ho-mopolymeric behavior inherent in disordered proteins.1Watkins et al, PNAS 2015.

1818-PlatCharacterization of the Aggregation-Prone Ensemble of Tau in the Pres-ence of PolyphosphatesSanjulaWickramasinghe1, Hope Merens2, Justine Lempart3, Ursula Jakob3,Elizabeth Rhoades2.1Biochemistry and Molecular Biophysics Graduate Group, University ofPennsylvania, Philadelphia, PA, USA, 2Department of Chemistry, Universityof Pennsylvania, Philadelphia, PA, USA, 3Department of Molecular, Cellularand Developmental Biology, University of Michigan, Ann Arbor, MI, USA.Tau is a microtubule associated protein implicated in Alzheimer’s diseasethrough its aggregation and deposition as neurofibrillary tangles. Tau is bothan intrinsically disordered protein and it aggregates very slowly in vitro makingit challenging to study the mechanism of aggregation. Typically highly anionicmolecular aggregation inducers, such as heparin, are used to induce aggrega-tion in vitro, although these molecules are unlikely to have much physiologicalrelevance. Recently, polyphosphates, linear chain of phosphate residues foundin all prokaryotic and eukaryotic cells, have been proposed to act as a universalscaffold for the initiation of aggregation of amyloidogenic proteins. Polyphos-phates have been shown to both increase the rate of amyloid fiber formation andto stabilize the fibers. Here, we use single molecule FRET to characterizeconformational changes in tau in the presence of polyphosphates. The low con-centrations (pM) of protein utilized in single molecule measurements allow forthe population of aggregation-prone conformations, while inhibiting aggrega-tion. In the presence of polyphosphates, tau undergoes domain specific confor-mational changes in which there is an overall expansion of the protein with aconcomitant compaction of the microtubule binding region. Conservation ofconformational shifts in the presence of different aggregation inducers will pro-vide a model of the consensus conformational features that potentiate tau foraggregation in disease.

1819-PlatDisordered Protein Linkers: Predicting Effective Concentrations usingPolymer PhysicsCharlotte S. Sørensen, Magnus Kjaergaard.Aarhus University, Aarhus C, Denmark.The most common function of intrinsically disordered regions of proteins isperhaps to act as a flexible linker. Linkers can tether functional modules

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such as for example a protein and its inhibitor as in ball-and-chain regula-tion. Such tethering will profoundly change reaction rates by convertingconcentration-dependent inter-molecular reactions to intra-molecular reac-tions. Instead of concentration, the intra-molecular interaction will becontrolled by the properties of the linker. Despite of the ubiquity of suchlinkers they are rarely studied quantitavely. The ‘‘closeness of tethering’’can be described via the effective concentration: The concentration of freeligand necessary to provide the same encounter rate as the intra-molecularreaction. If a flexible linker is sufficiently long, the effective concentrationbecomes a property solely of the linker, and not of what is being linked. Thisopens the possibility studying effective concentrations in an assay of ourchoice, and extrapolating to experimentally intractable systems. We presenta FRET competition assay to measure the effective concentration enforcedby a protein linker. Automated protein production using an open-sourceliquid handling robot allows us to analyze effective concentrations at highthrough-put. We find that effective concentration measured in this systemfollow polymer scaling laws similar to those described for protein size.We explore how sequence composition of the linker affects the scaling co-efficients by systematically varying to content and patterning of e.g.charges, hydrophobic residues and prolines. These scaling coefficients arecurrently being used to develop an in silico effective concentrationpredictor.

1820-PlatThe Liquid-Like Structure of ElastinSarah Rauscher, R�egis Pomes.University of Toronto, Toronto, ON, Canada.The protein elastin imparts extensibility, elastic recoil, and resilience to tis-sues including arterial walls, skin, lung alveoli, and the uterus. Elastin andelastin-like peptides are hydrophobic, disordered, and undergo liquid-liquid phase separation upon self-assembly. Despite extensive study, thestructure of elastin remains controversial. We use molecular dynamics sim-ulations on a massive scale to elucidate the structural ensemble of aggre-gated elastin-like peptides. Consistent with the entropic nature of elasticrecoil, the aggregated state is stabilized by the hydrophobic effect. However,self-assembly does not entail formation of a hydrophobic core. The polypep-tide backbone forms transient, sparse hydrogen-bonded turns and remainssignificantly hydrated even as self-assembly triples the extent of nonpolarside-chain contacts. Individual chains in the assembly approach amaximally-disordered, melt-like state which may be called the liquid stateof proteins. These findings resolve long-standing controversies regardingelastin structure and function and afford insight into the phase separationof disordered proteins.

Platform: EPR, NMR, Electron Microscopy,Diffraction, and Scattering

1821-PlatIntracellular EPR Spectroscopy and Genetically Encoded Spin LabelsMalte Drescher.University of Konstanz, Konstanz, Germany.In-cell EPR in combination with site-directed spin-labeling is a very powerfultool to monitor the structure and dynamics of bio-macromolecules in their nat-ural environment.We have demonstrated that it can be used for precise intracellular distance mea-surements as proven by a model compound, which consists of a spacer of well-known stiffness. Additionally, as model peptide an oligoproline peptide wassite-directedly spin labeled and analyzed by in-cell EPR. The results suggestthat the peptide is inserted into cell membranes, coinciding with a conforma-tional change.We investigated the in-cell conformations of the Parkinson-Protein alpha-Syn-uclein and its disease variants.The most elegant approach for site-directed spin labeling is the geneticencoding of a noncanonical, spin-labeled amino acid. This enables theintracellular biosynthesis of spin-labeled proteins and obviates the needfor any chemical labeling step usually required for protein EPR studies.We have developed a genetically encoded spin label that can be intro-duced at multiple, user-defined sites of a protein. It can report intramolec-ular distance distributions in proteins by EPR measurements. Thisprovides elegant new perspectives for in-cell EPR studies of endogenousproteins.

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1822-PlatBinding of VAMP2 to Membranes of Mammalian Cells Probed by In-CellNMRChuchu Wang, Shengnan Zhang, Cong Liu.Interdisciplinary Research Center on Biology and Chemistry, Shanghaiinstitute of organic chemsitry, Shanghai, China.The synaptic vesicle associated membrane protein 2 (VAMP2), one of themain components of the SNARE complex, regulates the fusion of synapticvesicles with the presynaptic membrane. The SNARE motif of VAMP2,which is essential in mediating SNARE complex formation, recognizesand binds to only certain types of membrane mimic (e.g. DPC micellesbut not nanodisc) in vitro. Whether and how VAMP2 is associated withmembrane in vivo are key questions to understand the regulatory mecha-nism of VAMP2-mediated SNARE complex assembly. In this study, wesystemically characterized the structural changes of VAMP2 in differentmammalian cells at residue-resolution by using in-cell NMR. Combiningwith immunofluorescence microscopy, membrane fractionation and in so-lution NMR, we found that the SNARE motif and juxtamembrane regionof VAMP2 partially bind to membranes, while the rest is highly flexiblein cellular environment. Our work demonstrates the dynamical bindingmode of VAMP2 to membrane in cells, and highlights the potentialimportance of this binding mode in regulating assembly of SNAREcomplex.

1823-PlatA New Wavelet Approach to Remove Noise from Experimental Signals:Reducing Signal Acquisition Times and Improving Resolution in Biophys-ical MethodsMadhur Srivastava1, Jack H. Freed2.1Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY,USA, 2Chemistry and Chemical Biology, Cornell University, Ithaca, NY,USA.The structure, dynamics and function of a biomolecule play a key role indetermining disease mechanisms, which is essential for early diagnosis,drug development and effective treatment. However, the physical methodsavailable for such study (e.g., Electron Spin Resonance (ESR), NuclearMagnetic Resonance (NMR), and Electron Microscopy (EM)) can onlyprobe the biological sample in constrained environments. Despite majoradvances in their technology, they still lack sufficient sensitivity and res-olution to conduct biological studies in the native environment. Suchstudies are currently challenging because the experimental signals areheavily dominated by noise. To address the challenge of noise removalin experimental signals, we developed a new wavelet denoising method(1) and a novel approach to remove/reduce noise (2). Our method isable to objectively identify and eliminate noise coefficients in the waveletdomain, overcoming the limitations of the standard wavelet denoisingmethods and other filtering methods. It has been applied to different typesof experimental signals, and it is found to increase the SNR by about twoorders of magnitude, while preserving the integrity of the signal. In addi-tion, its computation time is more than six times faster than the earliermethods. We will show how applying this method has revealed differentconformations in proteins and enabled studies at low sampleconcentrations.(1) Srivastava, Anderson, Freed (2016) IEEE Access 4, 3862(2) Srivastava, Georgieva, Freed (2017) J. Phys. Chem. A.121, 2452

1824-PlatLipid Bilayer Structure in Native Cell Membrane Nanoparticles of Multi-drug Exporter ACRBWeihua Qiu1,2, Ziao Fu3, Guoyan Xu1, Robert A. Grassucci4,5,Wayne A. Hendrickson4,6, Yan Zhang1, Joachim Frank4,7, Youzhong Guo1,2.1Department of Medicinal Chemistry, Virginia CommonwealthUniversity, Richmond, VA, USA, 2Institute for Structural Biology, DrugDiscovery and Development, Virginia Commonwealth University,Richmond, VA, USA, 3Integrated Program, Columbia University, New York,NY, USA, 4Department of Biochemistry and Molecular Biophysics,Columbia University, New York, NY, USA, 5Howard Hughes MedicalInstitute, Columbia University, New York, NY, USA, 6New York StructuralBiology Center, New York, NY, USA, 7Department of Biological Sciences,Columbia University, New York, NY, USA.Structural information for cell membrane and protein-lipid interaction is inhigh-demand for understanding the function of many membrane proteins;however, this information is still rare because currently there is no

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effective approach to obtain this information. We recently developed anovel native cell membrane nanoparticles system. With this system, wedetermined the structure of native cell membrane lipid bilayer that associ-ated with a membrane protein, multidrug exporter AcrB, using single-particle cryo-EM. Our cell membrane lipid bilayer model consists of 12lipid molecules in the inner leaflet and 12 lipid molecules within the outerleaflet of the lipid bilayer. The lipid bilayer interacts with protein specif-ically through both phosphate head and hydrocarbon tails. The hydrocarbontails within the inner leaflet of the lipid bilayer are in a well-organized hex-agonal pattern. Our results provided valuable structural information for theinteraction between AcrB and cell membrane. We demonstrated the greatpotential of using native cell membrane nanoparticles system in membraneprotein structural biology.

1825-PlatCorrelated Cryo-Fluorescence and Cryo-Electron Microscopy can Iden-tify Sites of Membrane FusionLauren Ann Metskas1,2, John A.G. Briggs1,2.1Structural Studies Unit, MRC Laboratory of Molecular Biology, Cambridge,United Kingdom, 2Structural and Computational Biology, EuropeanMolecualr Biology Laboratory, Heidelberg, Germany.Advances in cryo-electron microscopy have recently been coupled withcorrelated light and electron microscopy (CLEM), where fluorescence isused to locate regions of interest and characterize their molecular composi-tion prior to data collection. While the goal of cryo-CLEM is typically thelocalization of a cell, organelle or protein complex of interest within a vitri-fied sample, recent advances of cryo-fluorescence microscopy into single-molecule regimes [Wolff et al., Biol. Cell. 2016] and the thin depth of sam-ples suggest that fluorescence could also be used to probe function or dy-namics similar to TIRF-based single-particle assays. In this way, cryo-CLEM has the ability to assist not only in efficient data collection, butalso a description of functional state that might not be apparent by visuali-zation alone. We have established that resonant energy transfer is possible atcryo temperatures, highlighting the potential of cryo-CLEM for applicationsrequiring fluorophore autoquenching or FRET. Using autoquenching, wehave adapted an influenza fusion assay for cryo-CLEM. During fusion be-tween an influenza virus-like particle and a unilamellar vesicle, the two lipidbilayers enter into apposition before beginning lipid exchange, progressingto hemifusion and finally opening a fusion pore. By incorporating cryo-CLEM, fusion events are localized and identified based on the fluorescentsignatures for these three stages, before being targeted for cryo-electron to-mography. The fluorescence can then place fusion events from the cryo-CLEM experiment within either a bulk kinetic fusion assay time coursecompleted with the same sample batch, or into published single-moleculetime courses.

1826-PlatQuantitative Analysis of Immature Secretory Granules in Beta Cells ofMouse Pancreatic Islets by Serial Block-Face Scanning Electron Micro-scopyRichard D. Leapman1, Maria A. Aronova1, Amith Rao1,Emma L. McBride1, Guofeng Zhang1, Huanyu Xu2, Abner L. Notkins2,Tao Cai2.1NIBIB, National Institutes of Health, Bethesda, MD, USA, 2NIDCR,National Institutes of Health, Bethesda, MD, USA.Serial block-face scanning electron microscopy (SBF-SEM) can providehigh-resolution structural information from large volumes of biological tis-sue at the nanometer scale. By using SBF-SEM with a �1 kV electronprobe that is scanned across the surface of a plastic embedded block oftissue, it is possible to image tissue volumes as large as 100,000 cubic mi-crometers with 5 nanometer voxel size in the plane of the block face and25 nanometer normal to the block face. A sensitive detector collects back-scattered electrons from heavy atoms staining ultrastructure near the sur-face of the block, and a microtome built in to the specimen stage cuts athin layer from block face to expose a surface for imaging. Repetition ofthese steps produces an image stack that can be aligned, analyzed, andsegmented.We have used SBF-SEM to study immature secretory granules in beta cellsof mouse islets. Secretory granules contain insulin that is released inresponse to elevated blood glucose levels. Our objective was to use granulemorphology to define their state of maturity. Immature granules contain pro-insulin, and have lightly stained cores surrounded by thin halos, in contrastto mature granules that have more densely stained insulin cores surroundedby wide halos. From the SBF-SEM image stacks, we identified mature and

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immature secretory granules and measured the ratio of core volume to totalgranule volume. We also measured the relative abundances of immaturegranules and mature granules, which enabled us to estimate the maturationtime for the beta granules by considering the measured release times ofisotopically labeled insulin.

1827-PlatResonant Soft X-Ray Scattering of Proteins in SolutionDan Ye1, Thinh Le1, Cheng Wang2, Peter H. Zwart3, Chenhui Zhu2,Esther W. Gomez4, Enrique D. Gomez5.1Chemical Engineering, Penn State University, University Park, PA, USA,2Advanced Light Source, Lawrence Berkeley National Laboratory,Berkeley, CA, USA, 3Berkeley Center for Structural Biology, MolecularBiophysics and Integrated Bioimaging, Lawrence Berkeley Laboratory,Berkeley, CA, USA, 4Chemical Engineering, Biomedical Engineering, PennState University, University Park, PA, USA, 5Chemical Engineering,Materials Research Institute, Penn State University, University Park,PA, USA.Protein structure is crucial for biological function and may suggest importantparameters for the design of therapeutics and enzymes. We propose resonantsoft X-ray scattering (RSoXS) as an approach to study proteins and otherbiological assemblies in solution. Calculation of the scattering contrast sug-gests that soft X-ray scattering is more sensitive than hard X-ray scattering,because of contrast generated at the absorption edges of constituent elementssuch as carbon, nitrogen, and oxygen. As a proof-of-principle, we haveexamined the structure of bovine serum albumin (BSA) in solution byRSoXS. We find that by varying incident X-ray energies, we are able toachieve higher scattering contrast near the absorption edge. Furthermore,different signatures of the shape of the protein are revealed by tuning theX-ray energy within the carbon absorption edge. From our RSoXS scatteringresult we are able to reconstruct the structure of BSA in three-dimensions.The RSoXS results also agree with hard X-ray experiments, including crys-tallographic data. Our study demonstrates the potential of RSoXS for study-ing protein structure in solution and highlights advantages of this techniqueover traditional X-ray scattering.

1828-PlatCryo-Em Structure of Type 1 PilusWeili Zheng1, Caitlin N. Spaulding2, Henry L. Schreiber IV,2,Karen W. Dodson2, Matt S. Conover2, Fengbin Wang1, Pontus Svenmarker3,Areli Luna-Rico4, Olivera Francetic4, Magnus Andersson3, Scott J. Hultgren2,Edward H. Egelman1.1Department of Biochemistry and Molecular Genetics, University ofVirginia, Charlottesville, VA, USA, 2Center for Women’s Infectious DiseaseResearch and Department of Molecular Microbiology, WashingtonUniversity School of Medicine, St. Louis, MO, USA, 3Department ofPhysics, Umea University, Umea, Sweden, 4Biochemistry ofMacromolecular Interactions Unit, Department of Structural Biology andChemistry, Institut Pasteur, Paris, France.Urinary tract infections (UTIs) are caused by a wide range of pathogens, butthe most common causative agent of UTIs is uropathogenic Escherichia coli(UPEC). Virtually all uropathogenic strains of E. coli encode filamentoussurface adhesive organelles called type 1 pili, which are a subset ofChaperone-usher pathway (CUP) pili. CUP pili are also ubiquitously ex-pressed on the surface of many other Gram-negative bacterial pathogens.They are important virulence factors facilitating host-pathogen interactionsthat are crucial for the establishment and persistence of an infection, andinvolved in regulating other key processes such as biofilm formation. Wehave solved the 4.2 A resolution cryo-EM structure of the type 1 pilus,which was present as a background contaminant in a prep of type 4 pili.We have taken advantage of the strength of cryo-EM to separate differentmolecules and conformations present in solution to show that filament im-ages which might otherwise have been discarded as a contaminant can actu-ally be used to build an atomic model. The model reveals the residues thatallow a long chain of FimA subunits, linked by the insertion of a b-strand ofone subunit into the b-sheet of an adjacent subunit, to coil into a rigid rod.We show that site-specific mutation of these residues reduces the forceneeded to unwind the rod. Strikingly, one mutation (A22R) which showedthe greatest reduction in unwinding force, eliminated bladder infections ina mouse model. This is presumably due to the fact that the altered mechanicsof the A22R pilus rod cannot withstand the shear forces due to urinary flowin the bladder and bacteria harboring this mutation are cleared from thebladder. This provides new insights into the important role of pili mechanicsin bacterial pathogenesis.

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Platform: Cell Mechanics and Motility II

1829-PlatInvestigating the Effect of Matrix Porosity on the Mechanics of NeutrophilMigration in Three-Dimensional Extracellular MatricesJoshua Francois1, Juan Carlos del Alamo2, Richard Firtel3,Juan C. Lasheras2.1Bioengineering, University of California, San Diego, La Jolla, CA, USA,2Mechanical & Aerospace Engineering, University of California, San Diego,La Jolla, CA, USA, 3Division of Cell and Developmental Biology, Universityof California, San Diego, La Jolla, CA, USA.While much research has been dedicated to identifying the cascade of specificbiochemical processes involved in the recruitment of neutrophils, much less isknown about the mechanical events driving their migration; in particular, howthey generate the necessary traction forces to migrate across three-dimensional (3-D) extravascular spaces and the effect of matrix porosity dur-ing this process is unclear. In this study, we investigate the importance ofextracellular matrix properties on the mechanics of 3-D neutrophil motilityin collagen gels using Elastographic 3D Force Microscopy (E3DFM). Weembedded neutrophil-like differentiated human promyelocytic leukemia(dHL-60) cells in collagen matrices of different concentrations containingfluorescent micro-beads. Neutrophil motility was induced via the introductionof the neutrophil chemokine formyl-Methionyl-Leucyl-Phenylalanine (fMLP)in a custom build device. Both Confocal and Fluorescent microscopy tech-niques were used to image the movement of the embedded micro-beads aswell as fluorescently labeled cells. Particle Image Velocimetry (PIV) andFinite Deformation Theory were used to compute displacement fields in thecollagen matrices. Stress fields in the matrices were computed using ourE3DFM method. We will present data showing that morphological changesand migratory patterns differed depending on the porosity of the collagenmatrices. We will also provide data showing a clear relationship betweenthe aforementioned migratory characteristics and computed displacementand stress fields around migrating neutrophils in collagen matrices. The resultsfrom our study show that neutrophils migrating in 3-D environments employdistinct mechanical mechanisms that depend on the structure of their mechan-ical environments.

1830-PlatRevealing Bacterial Surface Physiology using Dual Atomic Force and Op-tical Time-Lapse MicroscopyHaig A. Eskandarian.Sciences de la Vie - Global Health Institute, Ecole Polytechnique F�ed�erale deLausanne, Lausanne, Switzerland.The manifestation of many bacterial phenotypes has traditionally beendescribed using techniques engineered to illuminate molecular mechanisms.However, the molecular mechanisms thought to govern several bacterial pro-cesses can be deleted without phenotypic consequences. Physical principlesplay an important role in making molecular mechanisms relevant. Describingthose physical principles has remained largely unattainable due to the lack ofavailable techniques. It is therefore paramount to develop new methods toprobe physical and mechanical properties at the single-cell level with relevanttemporal and nanometer-scale spatial resolution. On a fundamental level, wehave observed for the first time how bacterial cell surface physiology evolvesover long periods of time (�7 continuous days). In addition, the combinationof AFM and optical fluorescence microscopy provides precise spatial and tem-poral measures of protein localization, cell surface morphology and mechan-ical properties. We highlight how long-term time-lapse AFM reveals novelprinciples governing division, division site selection and growth inMycobacteria.

1831-PlatNovel Architecture and Composition of a Bacterial Flagellum in the Spiro-chete Leptospira biflexaKimberley H. Gibson1, Elsio A. Wunder Jr,2, Jun Liu3, Felipe Trajtenberg4,Alejandro Buschiazzo4, Albert I. Ko2, Charles V. Sindelar1.1Molecular Biophysics and Biochemistry, Yale University, New Haven, CT,USA, 2Department of Epidemiology of Microbial Disease, Yale University,New Haven, CT, USA, 3Department of Microbial Pathogenesis, YaleUniversity, New Haven, CT, USA, 4Laboratory of Molecular & StructuralMicrobiology, Institut Pasteur de Montevideo, Montevideo, Uruguay.Spirochetes are helix-shaped bacteria whose flagella filaments extendthrough the periplasmic space from a motor embedded in the inner mem-brane, and rotate therein. Leptospira spp. have two terminally opposingendo-flagella, whose curvature and rotation within the periplasm propels

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them in a corkscrew-like motion. Given its peculiar means of locomotion,we know very little about the architecture and assembly of these flagella.Current models of bacterial flagella are based on that of Salmonella enterica,which is composed of a single repeating FliC subunit. Meanwhile, Lepto-spira flagella filaments are assembled from several isoforms of flagellinFlaA and FlaB, and a recently discovered flagellar component unique to Lep-tospira spp. called flagella coiling protein A (FcpA) that is required forflagella curvature and motility. The precise architecture of the flagella andlocation of each flagellin and FcpA within the filament is as of yet undeter-mined. To this end, we performed cryo-electron tomography and subvolumeaveraging on flagella purified from the saprophytic Leptospira biflexa,revealing a novel structure resolved to 10.4 A. In contrast to the eleven radi-ally symmetric and identical protofilaments seen in Salmonella, our map in-dicates that individual protofilaments in Leptospira differ markedly fromeach other in composition and structure. Additionally, we solved an X-raycrystal structure of FcpA in its dimeric quaternary form. Using rigid fitting,we docked the FcpA dimer into an outer pseudo-helical protofilament withinour cryo-EM map of the flagellum, thereby providing a potential structuralbasis for modulating the curvature. The novel flagellar architecture describedhere for Leptospira establishes a new paradigm for flagellar structure in bac-teria, with profound implications for motility in this organism and otherSpirochetes.

1832-PlatStress Fiber Network Organization During Cell Spreading on Micropat-terned SubstratesDimitri Probst1, Julia J€ager1, Elena Kassianidou2, Anne-Lou Roguet3,Sanjay Kumar2, Ulrich S. Schwarz1.1Institute for Theoretical Physics & BioQuant, Ruprecht-Karls-Universit€atHeidelberg, Heidelberg, Germany, 2Department of Bioengineering,University of California, Berkeley, CA, USA, 3Ecole Polytechnique,Palaiseau, France.Cell spreading, adhesion and migration are strongly modulated by bothbiochemical and physical cues from the environment. This is especiallytrue for the actomyosin system, whose organization is strongly determinedby adhesive geometry, stiffness and topography of the extracellular environ-ment. Its organization in turn influences cellular behavior, e.g. differentia-tion, division and fate decisions. In order to understand how theactomyosin system dynamically responds to the adhesive geometry of itsenvironment, we have studied cell spreading onto rectangular fibronectinframes with varying gap locations that determine final cell shape. We findthat the global spreading dynamics onto a given pattern can be well pre-dicted with a Cellular Potts Model (CPM) modeling the interplay betweenadhesion and tension, and that the distribution of stress fiber (SF) orienta-tions can be predicted by assuming that discrete SFs form tangentiallybehind the advancing lamellipodium. We also find that adhesions and SFsare assembled in a periodic manner, similar to earlier findings for spreadingon homogeneous substrates. Exploiting the regularity of the spreading pro-cess on the rectangular frames and using quantitative image processing,we establish that new SFs are formed at average spatial intervals of approx-imately 2.0 mm and average temporal intervals of approximately 15 minutes.Because these times are comparable with the overall spreading times, cellshave a memory of their spreading history through the organization of theSF network, despite the fact that their final shape is mainly dictated bythe pattern geometry.

1833-PlatInterrogating Cell-Mediated Remodeling of the Extracellular Matrix byDynamic Light Scattering MicrorheologyBrad A. Krajina1, Audrey Zhu1, Sarah C. Heilshorn2,Andrew J. Spakowitz1,2.1Chemical Engineering, Stanford University, Stanford, CA, USA, 2MaterialsScience and Engineering, Stanford University, Stanford, CA, USA.Mounting evidence indicates that tissue mechanics and tumor-stroma inter-actions play an intimate role in breast cancer progression. Although in vitromodelsusing tissue mimetic materials with defined mechanical propertieshave provided valuable insights into the interplay between tissue mechanicsand malignant behavior, these studies typically do not address dynamicchanges in mechanics that occur as a consequence of cell-matrix interac-tions. The ability to non-destructively probe changes in matrix rheologyover time is essential to understanding the reciprocal interactions betweentumors and their mechanical environment and developing in vitro modelsof the temporal evolution of breast cancer. To this end, we develop a non-destructive and non-invasive microrheology technique based on dynamic

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light scattering that enables in situ measurements of matrix rheology in tis-sue mimetic in vitro 3-dimensional cell culture systems. Our technique en-ables rheological measurements of materials over a broad range ofbiologically-relevant mechanics (10 Pa to 10 kPa), encompassing tissuesas soft as healthy breast tissue and as stiff as highly invasive tumors. Weleverage these capabilities to study matrix remodeling in 3-dimensionalmodels of stromal and epithelial breast cancer tissue. Our measurementsreveal matrix stiffening mediated by mammary fibroblasts and simulta-neously capture the active dynamics in the surrounding matrix induced bycontractile forces generated by the actin cytoskeleton. We compare these re-sults to measurements on breast cancer cell lines with increasing degrees ofmalignancy and find striking differences in matrix remodeling and active dy-namics. Our results highlight the dynamic nature of cell-matrix interactionsin in vitro breast cancer models and suggest that this approach may lead tonew insights into the mechanobiology of breast cancer progression. Morebroadly, this approach provides a readily accessible method for non-destructively interrogating reciprocal interactions between mammalian cellsand their mechanical environment.

1834-PlatA Catch-Bond Drives Stator Mechanosensitivity in the Bacterial FlagellarMotorAshley L. Nord1, Emilie Gachon1, Ruben Perez-Carrasco2, Jasmine Nirody3,Alessandro Barducci1, Richard M. Berry4, Francesco Pedaci1.1Single Molecule Biophysics Dept., Centre de Biochimie Structurale,Montpellier, France, 2Dept of Mathematics, University College London,London, United Kingdom, 3Biophysics Graduate Group, University ofCalifornia, Berkeley, Berkeley, CA, USA, 4Dept of Biophysics, University ofOxford, Oxford, United Kingdom.The bacterial flagellar motor (BFM) is the rotary motor which rotates eachbacterial flagellum, powering the swimming and swarming of many motilebacteria. The torque is provided by stator units, ion motive force poweredion channels known to assemble and disassemble dynamically in theBFM. This turnover is mechanosensitive, with the number of engaged unitsdependent upon the viscous load experienced by the motor through the fla-gellum. However, the molecular mechanism driving BFM mechanosensitiv-ity is unknown. Here we directly measure the kinetics of arrival anddeparture of the stator units in individual wild-type motors via analysis ofhigh-resolution recordings of motor speed, while dynamically varying theload on the motor via external magnetic torque. The kinetic rates obtained,robust with respect to the details of the applied adsorption model, indicatethat the lifetime of an assembled stator unit increases when a higher forceis applied to its anchoring point in the cell wall. This provides strong evi-dence that a catch-bond (a bond counter-intuitively strengthened by force)drives mechanosensitivity of the flagellar motor complex. These resultsadd the BFM to a short but growing list of systems demonstrating catch-bonds, suggesting that this�molecular strategy’ is a widespread mechanismto sense and respond to mechanical stress. We propose that force-enhanced stator adhesion allows the cell to adapt to a heterogeneous envi-ronmental viscosity, and may ultimately play a role in surface-sensing dur-ing swarming and biofilm formation.

1835-PlatA Molecular Rack and Pinion Actuates a Cell-Surface Adhesin and En-ables Bacterial Gliding MotilityAbhishek Shrivastava, Howard C. Berg.Molecular and Cellular Biology, Harvard University, Cambridge, MA,USA.Motile but non-swimming bacteria achieve self-propulsion over surfaces usingmechanisms that are barely understood. Flavobacterium johnsoniae, which ex-hibits some of the fastest motion over a surface of all known non-flagellatedbacteria, has a mobile cell-surface adhesin SprB, and a powerful rotary motorthat is fueled by a proton motive force. The attachment of SprB to an externalsurface is required for ‘bacterial gliding’. We coated gold nanoparticles with anSprB antibody and tracked them in three-dimensional space in an evanescentfield where the nanoparticles appeared brighter when they were closer to theglass surface. The nanoparticles bound to SprB followed a spiral trajectoryon the surface of the cell. We generated elongated F. johnsoniae cells withirregular shapes, followed the displacement of asymmetric cells in three dimen-sions, and found that cells rolled about their long axes as they moved forward,following a right-handed trajectory. A fluorescent tag attached to a putative mo-tor protein GldL localized close to the track that SprB followed on the cell-surface. Based on our results, we propose a model where a molecular rackand pinion-like assembly actuates the motion of SprB on the cellsurface. Via

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its adhesin domains, SprB adheres to the surface and the cell moves forwardalong a right-handed trajectory, as observed, but in a direction opposite tothat of SprB.

1836-PlatNon-Uniform Mechanical Stress Promotes Metal Efflux Pump Disas-semblyMelanie F. Roberts, Lauren A. Genova, Lucy M. Wang, Peng Chen,Christopher J. Hernandez.Cornell University, Ithaca, NY, USA.Multicomponent efflux pumps are a clinically relevant drug resistance mech-anism in Gram-negative bacteria. CusCBA is the efflux pump system respon-sible for removal of toxins Cuþ and Agþ and shifts between an assembledand disassembled form. When assembled, CusCBA creates a rigid link be-tween the inner and outer membranes to facilitate efflux. Here, we test if me-chanical deformation of the cell envelope impairs the ability of CusCBA toassemble. Mechanical deformation was performed on Escherichia coli usinga microfluidic device. Bacteria were flowed into the device and then trappedin tapered channels with nanoscale features (250 nm smallest size). Differ-ences in fluid pressure across the cell generated mechanical stress and strainand deformed the cell envelope. The device design allowed examination ofhundreds of bacteria at different levels of mechanical stress magnitude. Ac-tivity of CusCBA in deformed cells was observed using single-molecule su-per-resolution imaging. When the pressure differential across the trappedbacteria was increased, a smaller proportion of CusCBA complexes wereassembled. Since similar multicomponent efflux complexes participate inantibiotic efflux, this result suggests that mechanical stress may also inter-fere with antibiotic resistance.

Platform: Protein Assemblies

1837-PlatStructure and Functional Anatomy of the Nuclear Pore ComplexSeung Joong Kim1, Javier Fernandez-Martinez2, Ilona Nudelman2, Yi Shi3,Wenzhu Zhang3, Barak Raveh1, Paula Upla4, Ilan E. Chemmama1,Riccardo Pellarin1, Ignacia Echeverria1, Steven J. Ludtke5,Christopher W. Akey6, Brian T. Chait3, Andrej Sali1, Michael P. Rout2.1Bioengineering and Therapeutic Sciences, University of California, SanFrancisco (UCSF), San Francisco, CA, USA, 2Laboratory of Cellular andStructural Biology, Rockefeller University, New York, NY, USA,3Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, RockefellerUniversity, New York, NY, USA, 4Simons Electron Microscopy Center, NewYork Structural Biology Center, New York, NY, USA, 5National Center forMacromolecular Imaging, Department of Biochemistry and MolecularBiology, Baylor College of Medicine, Houston, TX, USA, 6Department ofPhysiology and Biophysics, Boston University School of Medicine, Boston,MA, USA.Nuclear pore complexes (NPCs) act as gatekeepers of the nucleus by selec-tively transporting proteins and RNAs between the cytoplasm and nucleoplasm.However, the large size and dynamic nature of NPCs have impeded efforts tocomprehensively understand their functional organization. By integratingdiverse structural data (including 2,725 chemical cross-links, a Cryo-electrontomography map, 147 SAXS profiles of 18 Nups, and quantitative mass spec-trometry data), we determined a sub-nanometer precision structure for theentire 552-protein yeast NPC, mapping its functional elements in unprece-dented detail. The NPC is built of sturdy diagonal columns to which areattached connector cables, imbuing it with both strength and flexibility andtying together all other elements of the NPC, including the membrane-interacting regions and the RNA processing platforms. Inwardly-directed an-chors create a high density of transport factor-docking Phe-Gly repeats in thecentral channel, organized in distinct functional units that define acytoplasmically-biased permeability barrier. Taken together, this integrativestructure allows us to rationalize the architecture, transport mechanism, andevolutionary origins of the NPC.

1838-PlatInvestigating Ph-Induced Changes of the Influenza a Virus Matrix LayerLisa Selzer, Jasmine Moshiri, Karla Kirkegaard.Stanford University, Stanford, CA, USA.Viral matrix layers are important structural components of many enveloped vi-ruses that connect the viral envelope with the viral genome on the inside of thevirus. Within influenza A virus (IAV) the viral matrix layer is formed by thematrix protein M1, which oligomerizes to form a single matrix layer under-neath the viral envelope. M1 is a 252 amino acid protein composed of an

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alpha-helical N-terminal domain (NTD) and a flexible, intrinsically disorderedC-terminal domain (CTD). Upon viral entry endosomal acidification causesuncoating of the virus particle and release of the viral genome into the cyto-plasm. Interestingly, cryo-electron microscopy studies have identified asolenoid-like structure of the matrix layer within low pH-treated virions, sug-gesting the matrix layer arranges into an alternate oligomeric conformationupon acidification.We have purified recombinant M1 matrix protein and shown that it oligomer-izes into structures highly reminiscent of the solenoids observed in virions un-der acidic conditions. Biochemical investigation of these oligomers showedthat the CTD of M1 plays an important role during this oligomerization byfolding onto the NTD. Interestingly, folding of the CTD also increases inlow pH, suggesting that this structural change is driving the formation of thesolenoid structures within virions at low pH. We are further investigatingthis by analyzing the molecular interactions of M1 within virions under neutraland acidic conditions.

1839-PlatVirion Capsid Dynamics and Quaternary Conformational Changes UponHost EntryRanita Ramesh, Xin-Xiang Lim, Ganesh S. Anand.Biological Sciences, National University of Singapore, Singapore,Singapore.Viruses are metastable macromolecular entities that are assembled intocompact particles. Entry into a target host cell initiates quaternary conforma-tional changes in response to distinct host-specific environmental changesincluding temperature, pH, osmolality, divalent cation concentrations, whichpromote disassembly and release of the virion’s genome into the host cell.This host stimulus-triggered disassembly is critical for initiating the infectiousphase of the particle. How viruses use their quaternary coat protein structures asthermodynamic sensors for detecting entry into a conducive host cell environ-ment is largely unknown. The process of viral disassembly is largely imper-vious to static structural methods as these only capture end-stateconformations. Amide Hydrogen Deuterium Exchange Mass Spectrometry(HDXMS) can be used to monitor whole viral particle dynamics in solution.The disassembling virion represents the virus at its most vulnerable state andis consequently a transient dynamic intermediate. A map of the quaternarystructural changes in this state offers critical insights into cryptic epitopes asnovel vaccine targets. Our research has pioneered structural mass spectrometrycharacterization of temperature and divalent cation-dependent conformationalchanges on Dengue virus and Turnip Crinkle Virus (TCV) and mapped the spe-cific disassembly processes in these two model virus systems. We haveextended these studies to deconstruct the whole viral particle-antibody(DENV2-2D22) complex. Our results reveal the high intrinsic dynamics ofthe RNA packing capsid C-protein which underpins viral transition fromcompact to expanded states in the process of disassembly. We have also uncov-ered new disassembly intermediates triggered by varying monovalent and diva-lent cation concentrations. Viral disassembly processes in TCV and Denguereveal conformations of disassembly intermediates which represent powerfulnew targets for therapeutic antibody design as well as offer fundamental in-sights into strategically timed viral disassembly processes which can beextended to all viruses.

1840-PlatStructural Studies that Define Regulatory Interactions Within the Mito-chondrial Fission MachineryJason A. Mears, Christopher A. Francy, Ryan W. Clinton, Serena Lee.Department of Pharmacology, Case Western Reserve University, Cleveland,OH, USA.Mitochondrial fission is essential for distributing cellular energy throughoutcells and for isolating damaged regions of the organelle that are targeted fordegradation. This multistep process is initiated by the enhanced recruitmentand oligomerization of dynamin-related protein 1 (Drp1) at the surface ofmitochondria. In fact, Drp1 is essential for inducing mitochondrial divisionin mammalian cells and homologous proteins are found in all eukaryotes.Drp1 localization within cells is largely cytosolic, but it assembles on mito-chondria at sites of ensuing fission. Interactions with specific lipids at themitochondrial outer membrane (MOM) coordinate assembly of functionalcomplexes that mediate membrane scission. The presence of a mitochon-drial specific lipid, cardiolipin, in membrane templates stimulates Drp1 ac-tivity by promoting self-assembly into larger helical polymers that we aimto study using electron microscopy (EM) and complementary methods. Toachieve this goal, our lab has examined Drp1 assemblies on various lipidtemplates using structural and functional studies. There are similarities

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with other mechanochemical dynamins, but we continue to find novelattributes within the mammalian mitochondrial fission machinery. Specif-ically, Drp1 polymers assemble exclusively through stalk and G-domaindimer interfaces, which expand the helical symmetry when compared toother dynamins. Interestingly, we found that a previously defined gapbetween Drp1 and the lipid bilayer was lost when the mitochondrial spe-cific lipid cardiolipin was present, as Drp1 directly interacted with themembrane. Moreover, this interaction leads to a change in the helicalstructure, which alters G-domain interactions to enhance Drp1 activity.These results demonstrate how lipid cues at the surface of mitochondriacan regulate the structure and function of the mitochondrial fissionmachinery.

1841-PlatDirect Evidence of APLP1 Trans Interactions in Cell-Cell Adhesion Plat-forms Investigated via Fluorescence Fluctuation SpectroscopyValentin Dunsing1, Mayer Magnus2, Filip Liebsch3, Gerhard Multhaup3,Salvatore Chiantia1.1Institute of Biochemistry and Biology, University of Potsdam, Potsdam-Golm, Germany, 2Institute of Chemistry and Biochemistry, Free Universityof Berlin, Berlin, Germany, 3Department of Pharmacology and Therapeutics,McGill University, Montreal, QC, Canada.The Amyloid-precursor-like protein 1 (APLP1) is a neuronal type I trans-membrane protein which plays a role in synaptic adhesion and synaptogen-esis. Past investigations indicated that APLP1 is involved in the formation ofprotein-protein complexes that bridge the junctions between neighboringcells. Nevertheless, APLP1-APLP1 trans interactions have never beendirectly observed in higher eukaryotic cells. Here, we investigate APLP1 in-teractions and dynamics directly in living human embryonic kidney (HEK)cells, using fluorescence fluctuation spectroscopy techniques, namelycross-correlation scanning fluorescence correlation spectroscopy (sFCS)and Number&Brightness (N&B). Our results show that APLP1 forms homo-typic trans complexes at cell-cell contacts. In the presence of zinc ions, theprotein forms macroscopic clusters, exhibiting an even higher degree of transbinding and strongly reduced dynamics. Further evidence from Giant PlasmaMembrane Vesicles and live cell actin staining suggests that the presence ofan intact cortical cytoskeleton is required for zinc-induced cis multimeriza-tion. Subsequently, large adhesion platforms bridging interacting cells areformed through APLP1-APLP1 direct trans interactions. Taken together,our results provide direct evidence that APLP1 functions as a neuronalzinc-dependent adhesion protein and provide a more detailed understandingof the molecular mechanisms driving the formation of APLP1 adhesion plat-forms. Further, they show that fluorescence fluctuation spectroscopy tech-niques are useful tools for the investigation of protein-protein interactionsat cell-cell adhesion sites.

1842-PlatUnravelling the Contrasting Phase Behavior of Wheat Storage Proteins:How to Store Storage Proteins?Adeline Boire1, Christian Sanchez2, Marie-H�elene Morel2,M. Paul Lettinga3,4, Paul Menut2,5.1INRA, Nantes, France, 2UMR IATE, Montpellier, France, 3J€ulichForschungscentrum, J€ulich, Germany, 4KU Leuven, Leuven, Belgium,5UMR GENIAL, Massy, France.Plant seeds have the unique ability to naturally store large reservoirs of pro-teins in stable and compact environment for extended periods. Specific or-ganelles are dedicated to this storage: plant protein bodies (PBs). Inwheat, PBs form dense spherical accretions of about 0.5 to 2 mm in thelumen of endoplasmic reticulum once protein accumulation starts. PBsgrow by continuous protein synthesis and by fusion/coalescence with otherPBs. The mechanisms of proteins retention in endoplasmic reticulum andthe subsequent formation of protein bodies remain unclear. Among other hy-potheses, it has been suggested that wheat protein spontaneously aggregatein PB after their synthesis. To tackle this question, we choose an in vitroapproach which consists in probing the phase diagram of a wheat proteinisolate to infer their interaction properties. We show that gliadins undergoa phase separation upon temperature decrease. Using time-resolved smallangle light scattering, we show that gliadins undergo liquid-liquid phase sep-aration through two mechanisms: Nucleation and Growth and SpinodalDecomposition depending on temperature quench. The absence of arrestedphase separation suggested at first sight weak attraction in gliadins disper-sion. Interestingly, we show that the interaction potential is in the same orderof magnitude of other protein system but is more sensitive to temperature.Gliadins assemble therefore upon a minute-change of solvent quality but

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remain in a liquid-like state over an extended range of concentration andtemperature. We suggest the existence of clusters that prevent percolationand question the respective role of gliadin intrinsic flexibility, hydrationand polydispersity in this behavior.This liquid-like behavior, despite ofhigh concentrations and attraction, can be seen as a strategy for wheat seedsto store as much amino-acids as possible while being easily dispensablewhen needed.

1843-PlatIdentifying the Factors that Control the Size of Bacterial Microcompart-mentsFarzaneh Mohajerani, Michael F. Hagan.Physics, Brandeis University, Waltham, MA, USA.Bacterial microcompartments (BMCs) are icosahedral protein-based organ-elles that assemble around enzymes and reactants to compartmentalizecertain metabolic pathways. BMCs are found in at least 20% of bacterialspecies, and enable functions such as growth, pathogenesis, and carbon fix-ation. While experiments have visualized BMCs assembling around theirenzymatic cargo, the role of the cargo in determining the size of a BMCis hard to interpret from experiments. In some BMC systems, empty shellsare smaller and more monodisperse than full shells, whereas in other sys-tems empty shells are larger than full ones. Thus, experimental resultsimply that the cargo may increase or decrease shell size. This presentationwill describe coarse-grained theoretical and computational models for theassembly of icosahedral shells around a fluid cargo containing many mole-cules. We study the shell size distribution as a function of factors such asthe cargo and shell subunit concentrations, interaction strengths, and theshell protein spontaneous curvature. We find that in certain parameter re-gimes the presence of a cargo can increase the typical shell size (in com-parison to the size of empty shells), whereas in other regimes the cargodecreases the shell size. We compare assembly pathways, and the probabil-ity of shell completion, in each of these regimes. Understanding how BMCproteins assemble around a fluid cargo consisting of thousands of moleculeswould provide information for developing novel antibiotics that block BMCformation.

1844-PlatProbing Peptide Domains Implicated in Amyloid Fibril Formation DuringAmelogenin Nanoribbon AssemblySarah A. Engelberth1, Susrut Akkineni2, Chun-Long Chen3,Margot Bacino1, Shaiba Sandhu1, Ksenia Bubukina1, Jeremy Horst4,Johan Bonde5, Jim De Yoreo3, Stefan Habelitz1.1PRDS, UCSF School of Dentistry, San Francisco, CA, USA, 2MaterialsScience and Engineering, University of Washington, Seattle, WA, USA,3Physical Sciences Division, Pacific Northwest National Laboratory,Richland, WA, USA, 4Department of Biochemistry and Biophysics, UCSF,San Francisco, CA, USA, 5Center for Applied Life Sciences, LundUniversity, Lund, Sweden.The supramolecular structure of the hydrophobic protein amelogenin pro-vides an organic template for biomineralization of inorganic nanofibrousapatite during dental enamel formation. Work in our laboratory indicatesthat amelogenin (recombinant human rH174), rich in proline and histidine,self-assembles into 17nm wide nanoribbons that can grow up to microns inlength. Previous X-ray diffraction (XRD) and FTIR analysis indicated thesenanoribbons contain the b-sheet structure (4.7A lattice). To elucidate thisstructural formation, in silico studies (Zipper-DB, TANGO, PASTA2 andWALTZ) were used to predict four different domains of interest withinrH174 that have a high propensity for b-sheet or amyloid formation. Pep-tides were synthesized corresponding to each domain of interest. This studyprobed the self-assembly behavior of these domains using atomic force mi-croscopy (AFM), transmission electron microscopy (TEM), absorbance-based amyloid assays, and XRD to observe evidence for b-sheet and aggre-gated b-sheet formation. Peptide self-assembly was conducted at 37�C inunbuffered simulated enamel fluid (SEF) for up to 2 weeks at slightly acidicand alkaline pH ranges. AFM analysis revealed that all four domainsassembled into nanoribbon-like structures within one week at pH8.450.3 or two weeks at pH 6.850.6. Quantification of fibril width revealspeptide fibrils are smaller (7-12nm) and more coiled than the nanoribbonsassembled in similar conditions using rH174 (16-18nm). The self-assembly of the domains in solution upholds our calculated models andrecent XRD of rH174 nanoribbons revealed the presence of both a 4.6and 10A band, indicating the formation of a structural amyloid. Given thesefindings, we are working to understand how intermolecular interactions be-tween these domains relate to form the cross b-sheet aggregation observed

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in the developing enamel matrix and in the nanoribbons of recombinantamelogenin proteins in vitro.

Platform: Voltage-gated K Channels I

1845-PlatThe hERG PAS Domain Facilitates Gating Charge Deactivation at Physi-ological TemperatureDavid K. Jones1, Carol Harley2, Anthony Amolo3, Joao Morais-Cabral2,Gail A. Robertson1.1Neuroscience, University of Wisconsin, Madison, WI, USA, 2Instituto deBiologia Molecular e Celular, Universidade do Porto, Porto, Portugal,3Kenyon College, Gambier, OH, USA.The hERG PAS domain serves to suppress current, and disruption of thePAS domain enhances current in a manner that may have therapeutic poten-tial in treating cardiac arrhythmias. We have developed two anti-PAS anti-bodies that interfere with PAS action and enhance current amplitude. Tounderstand the underlying mechanism of the increased current amplitude,we tested the effect of the anti-PAS antibodies on movements of the voltagesensor domain (VSD). Working at physiological temperature (36 5 1�C),we measured VSD activation and deactivation by recording gating currentsfrom �120 mV and þ50 mV holding potentials, respectively, from HEK293cells stably expressing hERG. In controls, VSD deactivation displayed adouble Boltzmann and a Vmedian that was 16 mV more negative than theVmedian of activation, a reflection of VSD ‘‘relaxation’’ at positive voltages.The antibodies converted VSD deactivation to a single Boltzmann and hy-perpolarized the Vmedian compared to controls by approximately 20 mV.Conversely VSD activation was unaffected by either antibody. Thus, the ef-fect of the antibodies on the PAS domain is to stabilize the VSD relaxedstate and in this way promote channel open probability, consistent with theireffects on cardiac IKr amplitude (Harley et al., 2016). These effects on theVSD are not reflected in the conductance-voltage relationship of the ioniccurrent, suggesting that the antibody reduces coupling of VSD movementduring closure of the pore gate. Our findings support a conclusion from aprevious study, done at room temperature, that the PAS domain promotesVSD and pore gate coupling (Tan et al., 2012). Our findings also explainthe mechanism of action by which manipulation of the PAS domain mayprovide therapeutic action in the setting of prolonged action potential dura-tion and arrhythmia.

1846-PlatExploiting P-Stacking Interactions to Improve Inhibition of the Hv1Channel by Aromatic Guanidine DerivativesChang Zhao1, Liang Hong1, Jason D. Galpin2, Christopher A. Ahern2,Francesco Tombola1.1Department of Physiology & Biophysics, University of California Irvine,Irvine, CA, USA, 2Department of Molecular Physiology & Biophysics,University of Iowa, Iowa City, IA, USA.The human Hv1 voltage-gated proton channel is an emerging drug target forcancer and stroke. It resides on the cell plasma membrane and endocyticcompartments where it mediates outward proton movement and regulatesthe activity of NOX enzymes. The channel consists of two identical subunitswhich gate cooperatively. Each subunit contains a proton-conductingvoltage-sensing domain (VSD) which can be blocked by aromatic guanidinederivatives such as 2-guanidinobenzimidazole (2GBI). We have previouslyfound that mutating the Hv1 residue F150 to an alanine increases the2GBI binding affinity more than two orders of magnitude. Understandinghow 2GBI and its analogs interact with Hv1 F150A is critical to the designof more effective inhibitors for the wild type channel. We hypothesized thatthe binding of 2GBI to the mutant channel is stabilized by p-stacking inter-actions between the inhibitor and aromatic residues located in the core of theVSD. We tested this hypothesis using a combination of electrophysiologicalrecordings, classic mutagenesis, and site-specific incorporation of fluorinatedaromatic amino acids via nonsense suppression methodology. Consideringtheir proximity to the 2GBI binding site, we investigated residues F149and F182. We found that F182 does contribute to 2GBI binding via p-stack-ing interactions while F149 does not. To test whether strengthening the p-stacking interactions can lead to more potent inhibitors, we measured thebinding affinities of a series of aromatic guanidine derivatives carrying anincreasing number of fluorine substituents. We discuss the results based onour understanding of the mechanism of channel block by these compounds.Possible alterations of the inhibitor structure to improve binding to the wildtype channel are also discussed.

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1847-PlatIdentification of the C-Linker and CNBD Residues Accounting for theHigh Efficacy of Camp Activation in HCN2 ChannelsClaudia P. Alvarez Baron1, Vadim A. Klenchin1, Baron Chanda2.1Neuroscience, University of Wisconsin, Madison, Madison, WI, USA,2Neuroscience and Biomolecular Chemistry, University of Wisconsin,Madison, Madison, WI, USA.Hyperpolarization-activated, Cyclic Nucleotide–gated (HCN) channels are ma-jor determinants of the firing rate of pacemaker centers in the heart and brain.Direct binding of cAMP to the cyclic nucleotide binding domain (CNBD) ac-tivates HCN channels by increasing the maximum open probability and shiftingthe voltage-dependence of activation to less hyperpolarized potentials. HCNisoforms differ greatly in kinetics, voltage gating, and response to cyclic nucle-otides. HCN2 responds strongly to cAMP despite having similar ligand affinityto HCN1, an isoform that responds very poorly to cAMP. To identify the keymolecular determinants responsible for cAMP activation, we progressivelymutated sites in the C-linker and CNBD of the mHCN2 to HCN1. Our studiesidentified two clusters of mutations that determine the differences in voltagedependent activation between these two isoforms. One mapped to the C-linkerregion (M485F, G497D, and S514T), far from the cAMP-binding site. Anotherwas found in proximity to the binding site (V562A/S563G, L565I, and S575T).Concurrent substitutions of five sites (M485F, G497D, S514T, and V562A/S563G) is sufficient to confer HCN1 phenotype on the mutated HCN2 chan-nels. To understand the role of these residues on various allosteric parametersthat determine the gating of these channels, we built an allosteric model con-sisting of four separate modules: pore gates, four voltage-sensors, ligand bind-ing domain, and C-linker. Our models suggest that these substitutions mustalter at least the oligomerization state of the linker and two coupling parame-ters: one between the linker and the pore and the other between the linkerand the voltage sensors. These findings will be discussed in light of the high-resolution structures of HCN channels.

1848-PlatAtom-by-Atom Tuning of an Electrostatic Potassium-Channel ModulatorMalin Silvera Ejneby1, Xiongyu Wu2, Nina E. Ottosson1, E Peter M€unger2,Ingemar Lundstrom2, Peter Konradsson2, Fredrik Elinder1.1IKE, Linkoping University, Linkoping, Sweden, 2IFM, LinkopingUniversity, Linkoping, Sweden.Dehydroabetic acid (DHAA), naturally occurring in pine resin, opens voltage-gated potassium (Kv) channels by altering the movement of the channel’svoltage sensor. The hydrophobic part of DHAA (a three-ringed motif) anchorsthe compound near the extracellular end of the voltage sensor, in a pocket be-tween the channe�ls transmembrane segments S3 and S4, and the lipid membrane.The negatively charged carboxyl group of DHAA executes an electrostatic effecton the positively charged voltage sensor S4, to open the channel. Here, we aimedto increase the channel-opening effect by altering the charge of the effector andby introducing an atomic stalk between the hydrophobic anchor and the chargedeffector, to allow the compound charge to come close to the voltage-sensorcharge. The Shaker Kv channel was expressed in Xenopus laevis oocytes andion currents were measured by a two-electrode voltage-clamp technique.Altering the partially charged carboxyl group to a permanently chargedsulfonic-acid group increased the channel opening effect. Introduction of a car-bon stalk between the anchor and the charged effector increased the channel-opening effect further, until a critical stalk length was reached. Longer stalksrendered the compounds without effect. This is consistent with a simple electro-static model, where the charge location depends on the stalk length. To increaseto effect of the compound further we increased the affinity by altering the halo-genation of the anchor. This compound significantly opened the human Kv7.2/7.3 (M-type) potassium channel at 1 mM. These results suggest that a stalk be-tween the anchor and the effector is a powerful method to increase thechannel-opening effect for electrostatically active compounds.

1849-PlatAn Allosteric Action Mechanism of a KD Pore Blocker Revealed at theAtomic LevelIzhar Karbat1, Hagit Altman-Gueta2, G. Tibor Szanto3,Shelly Hamer-Rogotner4, Orly Dym5, Felix Frolow6, Dalia Gordon1,Gyorgy Panyi3, Michael Gurevitz2, Eitan Reuveny1.1Biomolecular Sciences, Weizmann Institute, Rehovot, Israel, 2MolecularBiology and Ecology of Plants, Tel Aviv University, Tel Aviv, Israel,3Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary,4Israel Structural Proteomics Center (ISPC), Weizmann Institute, Rehovot,Israel, 5Structural Proteomics Unit (SPU), Weizmann Institute, Rehovot,Israel, 6Molecular Microbiology and Biotechnology, Tel Aviv University,Tel Aviv, Israel.

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Voltage gated ion channels gate in response to changes in the electrical mem-brane potential by the coupling of a voltage sensing module with an ion-selective pore. Toxins that target these channels are traditionally classified aseither pore-blockers or gating-modifiers, the former bind and physicallyocclude the channel pore, while the later bind the voltage sensing moduleand restrict its movement in response to alterations in the membrane potential.Here we present Cs1, a kunitz-fold cone-snail toxin that blocks the DrosophilaShaker isoform with high affinity and seem to defy the traditional classification.We first obtained high resolution crystal structures of Cs1 and several of its mu-tants. Then, using site directed mutagenesis followed by double-mutant cycleanalysis we identified key residue pairs in contact at the toxin-channel inter-face, which was clearly confined to the channel pore-module. Unconstrainedrigid docking followed by a whole-atom molecular dynamics simulationsyielded a model that was consistent with the experimental results, in whichthe toxin was bound off the pore axis and allowed the access of water moleculesto the pore. Electrophysiological assays established that Cs1 does not dissociatefrom its binding site upon depolarization, and that its affinity for the channel isindependent of the external Kþ concentration, further setting it apart from theclassical pore-blockers. Analysis of our MD simulations suggest that Cs1blocks ion conductance using a novel allosteric mechanism that directly in-volves structural water molecules buried behind the selectivity filter of thechannel.

1850-PlatVoltage-Gated Channel Regulation by an Amino Acid TransporterVictoria A. Baronas1, Runying Yang2, Harley T. Kurata2.1University of British Columbia, Vancouver, BC, Canada, 2University ofAlberta, Edmonton, AB, Canada.Kv1.2 is a prominently expressed voltage-gated potassium channel in the cen-tral nervous system, and influences action potential generation and propagation.Genetic deletion of Kv1.2 leads to death within two weeks of birth in mice, andthe identification of Kv1.2 mutations in severe human neurological diseaseslike epilepsy is accelerating. Kv1.2 was the first eukaryotic voltage-gated chan-nel described by an atomic resolution structure, and has thus served as a tem-plate for interpreting structure-function studies. In contrast to our detailedunderstanding of Kv1.2, its interactions with accessory proteins and lipidshas been less investigated. To this end, we heterologously expressed Kv1.2in HEK cells and used bifunctional crosslinkers and immunoprecipitation toisolate nearby proteins. We identified crosslinked proteins via mass spectrom-etry, followed by screening for effects on Kv1.2 expression and function. Sur-prisingly, we found that Slc7a5, a neutral amino acid transporter, has aprofound impact on Kv1.2 expression and gating. Co-transfection withSlc7a5 reduces Kv1.2 expression and hyperpolarizes the activation curve by48 5 4 mV. Addition of Slc3a2, a previously identified binding partner ofSlc7a5, attenuates the effects of Slc7a5 on Kv1.2 gating and expression. Usinga bioluminescence resonance energy transfer approach, we demonstrate thatKv1.2 and Slc7a5 are in close proximity, and we have identified segments ofboth Kv1.2 and Slc7a5 that are critical for the observed functional outcomes.Interestingly, two recently reported Slc7a5 mutations linked to neurodevelop-mental delay either abolish (Ala246Val) or markedly attenuate (Pro375Leu)Slc7a5 regulation of Kv1.2. Taken together, we have identified and character-ized a novel interaction between Slc7a5 and Kv1.2 which may contribute tonormal neuronal function and disease pathogenesis in patients with Slc7a5mutations.

1851-PlatMolecular Mechanism Underlying a Traditional Anticonvulsant: Syner-gistic KCNQ2/3 Potassium Channel Activation by DUAL Components ofMallotus Oppositifolius ExtractRıan Manville, Maria Papanikolaou, Geoffrey W. Abbott.Physiology and Biophysics, University of California, Irvine, Irvine, CA,USA.Voltage-gated potassium channels formed by KCNQ2 and KCNQ3 generatethe phosphatidylinositol 4,5-bisphosphate (PIP2)-augmented M-current, whichregulates neuronal excitability. Hyperexcitability of neuronal cells is associatedwith benign familial neonatal seizures (BFNS), a disease linked to mutations inthe gene encoding KCNQ2. In the developing world, an estimated 80% of ep-ilepsy patients use herbal remedies for primary healthcare, such as the Ghana-ian shrubMallotus oppositifolius.M. oppositifolius extract has been previouslyshown to delay the onset of seizures as well as reduce their frequency and dura-tion in mouse models of epilepsy. However, the active components and the mo-lecular basis for these anticonvulsant properties were unclear. Here, we reportthat KCNQ2/3 channels are activated by mallotoxin (MTX), a natural productisolated from the Ghanaian shrubM. oppositifolius. Application of MTX to oo-cytes expressing KCNQ2/3 channels shifted the voltage for half-activation

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(V0.5) in the hyperpolarizing direction, leading to an increase in current ampli-tude at test potentials between �80 mV and �40 mV. MTX also had a markedeffect on KCNQ2/3 channel kinetics, increasing the rate of activation but slow-ing deactivation. Similar effects of MTX were observed on KCNQ2 or KCNQ3alone, with KCNQ2 exhibiting greater sensitivity, suggesting KCNQ2 may bethe primary molecular target of MTX. Additionally, isovaleric acid (IVA),another component ofM. oppositifolius extract, also activated KCNQ2/3 chan-nels, albeit less potently than MTX. Strikingly, dual application of MTX andIVA to KCNQ2/3 channels produced a highly effective, synergistic KCNQ2/3 activation. Finally, MTX and IVA were also more effective in combination,versus alone, in suppressing pentylenetetrazole-induced tonic seizures in mice.Our results suggest that KCNQ2/3 activation by both MTX and IVA is the mo-lecular basis for the anticonvulsant effects of M. oppositifolius extract.

1852-PlatThe Morbidity of Epilepsy and Cardiac Arrhythmia is Attributed to Com-mon Channelopathy of Genetic Mutants of Slack ChannelsYun Xu, Fei-Fei Zhang, Jie Xu, Wen Sun, Xiao-Yun Zhao, Qiong-Yao Tang,Zhe Zhang.Jiangsu Key Laborotary of Anesthesiology, Xuzhou Medical University,Xuzhou, Jiangsu, China.In recent years, more and more genetic mutants of Slack channels that are asso-ciated with epilepsy, Brugada syndrome and cardiac arrhythmia have beenidentified from patients with epilepsy. However, whether the mutants relatedto cardiac arrhythmia share similar biophysical properties with mutants associ-ated with epilepsy remains elusive. In this abstract, we characterized the bio-physical properties of 13 epilepsy associated genetic mutants of Slackchannels, including two mutants associated with cardiac arrhythmia. We foundthat 12 of total 13 mutants possess enhanced maximum Po while some of themalso have enhanced sodium sensitivity. Furthermore, the patient who carriedthe only one mutant that has decreased Po and lower maximum Po actuallyhas other factor that may induce epilepsy. Thus, the enhanced maximum Poand increased sodium sensitivity could be a standard to judge if a Slack channelmutant is really associated with epilepsy. In addition, two functional alternatedsplicing isoforms of human Slack channels have been cloned and characterized,which we name hSlackB1 and hSlackB2. When expressed in Xenopus ooctyes,robust sodium dependent potassium current were recorded from hSlackB1 andhSlackB2 constructs while no potassium current could be recorded fromhSlackA construct. In the meantime, rundown of hSlackB1 activity can beobserved but no rundown of hSlackB2 activity can be observed. Takentogether, we concluded that Brugada syndrome, cardiac arrhythmia and epi-lepsy led by Slack channel mutants is common channelopathy caused byenhanced maximum Po, increased sodium sensitivity and suitable splicing.

Platform: Membrane Active Peptides and Toxins

1853-PlatMembranes Matter: Predicting Drug ToxicityR. Lea Sanford1, Jeanne Chiaravalli-Giganti2, Wesley Chao1,J. Fraser Glickman2, Olaf S. Andersen1.1Biophysics and Physiology, Weill Cornell Medicine, New York, NY, USA,2The Rockefeller University, New York, NY, USA.It remains a challenge to predict whether a new drug candidate will have unde-sirable side effects. Many biologically active molecules, including drugs anddrug-leads, are amphiphiles that partition into lipid bilayers, which may alterbilayer physical properties, thereby modulating membrane protein function.Such bilayer-modifying molecules may be promiscuous modifiers of mem-brane protein function, raising the possibility that they will have off-target ef-fects. Thus, it may be possible to predict whether a compound will haveimportant off-target effects based on quantitative studies of the compound’sbilayer-modifying potential. We developed an assay to quantify the bilayer-modifying potential of large numbers of compounds. Using a gramicidin-based fluorescence assay (GBFA), which reports how a compound alters theconformational equilibrium of a membrane embedded reporter protein andthus indicates global changes in bilayer properties. We have shown thatmany drugs and drug-leads alter lipid bilayer properties at the concentrationswhere these compounds become indiscriminate modifiers of membrane proteinfunction. We pursued this question in a blinded study on a library of 504 com-pounds (204 non-toxic, 300 toxic) that had been tested for cytotoxicity in‘‘high-content’’ screening assays using the GBFA and found that the changesin fluorescence quench rate can be used to predict cellular toxicity, with a falsepositive rate of only 5%. These results support a mechanism by which amphi-philes exert their toxicity, namely by altering lipid bilayer physical propertiesand that such an in vitro measurement could be used as a warning sign for off-target biological effects in drug discovery efforts. We are currently employing

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machine-learning techniques to better understand which physicochemical pa-rameters influence a small molecule’s bilayer perturbing potency.

1854-PlatMechanism of Action of pH-Triggered, Membrane Active Peptides: Effectof Negative ChargeSarah Y. Kim1, William C. Wimley2, Kalina Hristova3.1Program in Molecular Biophysics, Johns Hopkins University, Baltimore,MD, USA, 2Department of Biochemistry and Molecular Biology, TulaneUniversity School of Medicine, New Orleans, LA, USA, 3Department ofMaterials Science and Engineering, Johns Hopkins University, Baltimore,MD, USA.The pH-dependent delivery (pHD) peptides form large pores in membranesonly at acidic pH. These peptides, which have 5-6 acidic residues, are nega-tively charged when inactive at pH 7 in the presence of neutral, POPC mem-branes. Consequently, we expected that the peptides would decrease inpotency on negatively charged membranes. To explore the mechanism of ac-tion, we measured the peptides’ circular dichroism, binding, and macromolec-ular leakage. Surprisingly we found that the peptides become more potent atreleasing macromolecules from vesicles upon the incorporation of charge.

1855-PlatAssessing the Translocation of Cell Penetrating Peptides using Force Mea-surements, Electrophysiology and EmulsionsSimon Kulifaj, Sophie Cribier, Vincent Vivier, Nicolas Rodriguez,Kieu Ngo.UMR 7203, University Pierre et Marie Curie, Paris, France.Cell Penetrating Peptides (CPPs) are known to be able to cross cell membraneswith a cargo through two different mechanisms: endocytosis and direct trans-location. However, the molecular mechanism of the translocation is largely un-known. Our objectives are to find the intermediate structures formed by CPPand protein, sugar, and lipid partners, to quantify the strength of the interactionsand to measure the kinetics of the steps of the interaction of a CPP with a mem-brane. This is achieved by using two approaches: (i) coupled BioMembraneForce and electrophysiology, (ii) model membrane in inverse emulsions.(i) Biomembrane Force Probe is a technique for measuring the adhesion forcebetween few molecules grafted on a micrometric bead and a target cell or aliposome. This technique has been used to identify partners of CPP at thecell surface. Electrophysiological measurements on the target cell enable todetect a possible perturbation of a membrane interacting with CPPs.(ii) Inverse emulsions are aqueous droplets into oil covered by lipids. At theinterface between two adhering droplets a bilayer is formed. We monitor thetranslocation of fluorescently labeled CPP through this bilayer. The transloca-tion of several CPPs through negatively charged bilayer within tens of minuteshas been detected. The nature of the lipids and the asymmetry of the bilayerseem to impact the translocation. We are currently improving the formationof the pairs of droplets with microfluidic devices for statistical analysis.

1856-PlatMelittin-Induced Permeabilization, Re-Sealing, and Re-Permeabilizationof E. coli MembranesZhilin Yang1, Heejun Choi2, James Weisshaar1.1Chemistry, Univ Wisconsin-Madison, Madison, WI, USA, 2JaneliaResearch Campus, Ashburn, VA, USA.The permeabilization of pure lipid bilayers by cationic peptides has been stud-ied extensively over decades, with the bee-sting toxin melittin perhaps servingas the canonical example. However, the relevance of these studies to the per-meabilization of real bacterial membranes by antimicrobial peptides remainsuncertain. Here we employ single-cell fluorescence microscopy in a detailedstudy of the interactions of melittin with the outer membrane (OM) and thecytoplasmic membrane (CM) of live E. coli. Using periplasmic GFP as probe,we find that melittin at 2X MIC first induces abrupt cell shrinkage and perme-abilization of the OM to GFP. Within �4 s of OM permeabilization, the CMinvaginates to form inward facing ‘‘periplasmic bubbles’’. Seconds later thebubbles begin to leak periplasmic GFP into the cytoplasm. Permeabilizationis localized, consistent with possible formation of toroidal pores. Within �20s, first the OM and then the CM re-seals to GFP. Some 2-20 min later, bothCM and OM are re-permeabilized to GFP. We invoke a mechanism based oncurvature stress concepts derived from model membrane studies. The permea-bilization and re-sealing events involve sequential, time-dependent build-up ofmelittin density within the outer and inner leaflets of each bilayer. We also pro-pose a mechanical explanation for the early cell shrinkage event induced bymelittin and a variety of other cationic peptides. As peptides gain access tothe periplasm, they bind to the anionic peptido-crosslinks of the lipopolysac-charide layer, increasing its longitudinal elastic modulus. The cell wall shrinksbecause it can withstand the same turgor pressure with smaller overall

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extension. Shrinkage in turn induces invagination of the CM, preserving its sur-face area. We conclude proposing experiments on GUVs that might furtherstrengthen the connections between studies of model lipid bilayers and realbacteria.

1857-PlatThe Antimicrobial Peptide Piscidin P1 Uses Weak Spots in Membranes asSites of ActionLaura Lucas1, Roderico Acevedo2, Myriam Cotten3, Ella Mihailescu2.1The Catholic University of America, Washington, DC, USA, 2Institute forBioscience and Biotechnology Research, Rockville, MD, USA, 3Departmentof Applied Science, College of William and Mary, Williamsburg, VA, USA.Antimicrobial peptides (AMPs) play an integral role in the fight againstinvading pathogens. To fully exploit their potential as prototypes for novel an-timicrobials the molecular basis of their mechanism of action, which includesdisrupting plasma membranes, must be understood. The host-defence peptidepiscidin P1, an AMP isolated from the mast cells of striped bass, has potentantimicrobial activity against a large number of Gram-positive and -negativebacteria, including methicillin-resistant S. aureus (MRSA), viruses such asHIV-1, fungi, yeasts, and cancer cells. Solid state NMR showed that P1 adoptsa helical structure in fluid 3:1 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholi-ne(DMPC)/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) and1:1 1-palmitoyl-2-oleoylsn- glycero-phosphatidylethanolamine (POPE)/1-pal-mitoyl-2-oleoyl-sn-glycero-phosphoglycerol (POPG) . In this work, we inves-tigated the structural interactions of this 22-residue helical, cationic peptidewith lipid membranes in order to gain insights into its mechanisms of action.By using a combination of deuterium labeling methods, neutron and Xraydiffraction, and differential scanning calorimetry we examined the organiza-tional preferences of the P1 in membranes of compositions mimicking eitherbacterial (POPE/POPG and POPC/POPG) or the outer leaflet of mammalian(POPC/cholesterol) plasma membranes. We employed neutron diffraction todetermine the P1’s depth of insertion and the magnitude of bilayer structuralperturbations that correlate with its potency. We used Differential ScanningCalorimetry and Xray scattering to show that, on the one hand, P1 causes reor-ganization of Cholesterol in membranes by segregating with a cholesterol-depleted fluid phase, and on the other hand, they relieve curvature strainscaused by nonlamellar-forming lipids such as PE, in PG/PE mixture. Thesefindings are contrasted to the behaviors of other peptides in similar lipid mix-tures, emphasizing the ability of membrane-active peptides to uniquely modifythe local membrane environment, that can ultimately serve for their entry intothe cell.

1858-PlatAb-Initio Prediction of Antimicrobial Peptides Channels in MembranesJakob Ulmschneider.Shanghai Jiao Tong University, Shanghai, China.AMPs are a key component of the immune defense of all forms of life, but themechanisms driving their antimicrobial activity is only poorly understood.AMPs preferentially bind to microbial membranes and form pores that arebelieved to kill by depleting the cellular electrochemical gradient. Up tonow, determining AMP pore architectures and the mechanisms of membranebinding, channel formation, and conduction remained difficult.We can now directly predict the insertion mechanism, native state structuresand conduction of membrane active peptides at atomic resolution. We showthat atomic detail equilibrium molecular dynamics simulations can accuratelyreproduce experimental ensemble averages and partitioning data, revealinghow AMPs target microbial membranes and spontaneously assemble into chan-nels, and how these channels conduct. Obtaining detailed channel structuresgreatly enhances our understanding of the role of AMPs in innate immune de-fense and will ultimately enable rational design and optimization of new AMPsfor clinical, bioengineering, and agricultural applications.

1859-PlatAntimicrobial Selectivity and Membrane Leakage Mechanisms: The Roleof LipidsAnja Stulz1, Larissa Akil1, Karen Lienkamp2,3, Maria Hoernke1,4.1Pharmaceutical Sciences, Albert-Ludwigs-Universit€at, Freiburg i.Br.,Germany, 2IMTEK, Albert-Ludwigs-Universit€at, Freiburg i.Br., Germany,3Fit, Albert-Ludwigs-Universit€at, Freiburg i. Br., Germany, 4BIOSS Centrefor Biological Signalling Studies, Albert-Ludwigs-Universit€at, Freiburg i.Br., Germany.Synthetic mimics of antimicrobial peptides (smAMPs) are promising alterna-tives to classical antibiotics because they are less prone to resistance. Like nat-ural antimicrobial peptides (AMPs), smAMPs are thought to act on the cellmembrane, but offer the advantage of better stability and easier production.Common to all antimicrobial treatment is the need for selectivity.

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Two series of short polymeric smAMPs with differing backbone chemistry andantifungal or antibacterial gram-selectivity are studied by fluorescent dyerelease, monolayer binding, and differential scanning calorimetry to determinelipid clustering. Systematically varied lipid compositions mimic membranes ofdifferent types of cells and microbes and are found to reflect differences in anti-microbial action.Sophisticated analysis of vesicle leakage mechanisms and kinetics reveals howthe combination of smAMP design and lipid mixture can determine the mode ofaction and thus selectivity. Activity and selectivity relate to molecular mecha-nisms like asymmetry stress, stabilization of local curvature (as in toroidalpores), and strong leakage events (possibly involving lipid clustering or oligo-meric pores). We also discuss that vesicle leakage experiments do not reflect allaspects of antimicrobial action by comparison to MIC values.We find that antimicrobials acting by asymmetric binding, leakage uponcracking-in followed by annealing might be less selective and more haemolytic.Asymmetry stress tends to affect lipid membranes faster than other leakagemechanisms. Conversely, an appropriate balance of electrostatically and hydro-phobically mediated smAMP binding is crucial and allows for leakageinvolving stabilization of local curvature (as in disordered toroidal pores).Selectivity can be increased by additional rare and strong leakage events, espe-cially in lipid compositions containing cardiolipin being more prone to electro-static clustering. A systematic view on membrane leakage, antimicrobialactivity and selectivity will aid future design of antimicrobials and improvemembrane models for in vitro studies.

1860-PlatThe Cell Cycle Dependence of the Activity of Antimicrobial Peptides Re-sults in a Higher Resistance of Starving Cells to the PeptidesMehdi Snoussi, Mehrnaz Siavoshi, Paul Talledo, Sattar Taheri-Araghi.CalState Northridge, Northridge, CA, USA.Antimicrobial peptides (AMPs) are broad spectrum antibiotics that utilize elec-trostatics to selectively target bacteria. Despite the recent progresses uncover-ing the time course of the action of AMPs, the correlations of the action with thephysiological state of the target cells are still illusive. In this talk, I present ourrecent findings that AMPs’ activity is dependent on the progression of the cellcycle in individual Escherichia coli cells. Specifically, the formation of septum,prior to the cell division, increases the cells’ vulnerability against AMPs. As aresults, the the minimum inhibitory concentration (MIC) of the AMPs not onlydepend on the characteristics of the peptides and the cell density, but also on thegrowth rate of the cells in a culture. Utilizing a high throughput single-cell im-aging platform, we demonstrate that rapidly growing cells have lower survivalrate due to a higher frequency of cell divisions. On the other hand, slowgrowing cells, in which the duration of the septum formation constitutes a smallfraction of the cell cycle, are more resistant against AMPs.

Platform: Channel Regulation

1861-PlatCodon Usage Influences Gating of Small KD ChannelsKerri Kukovetz1, Anja Engel1, Sebastian Gutsfeld1, Marina Kithil1,Oliver Rauh1, Anna Moroni2, Gerhard Thiel1.1TU Darmstadt, Darmstadt, Germany, 2University of Milano, Milano, Italy.Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes.It has been proposed that synonymous codons can affect the velocity of proteintranslation and as a consequence translation accuracy and protein folding. Herewe used small Kþ channels to examine the impact of codon usage on basic pa-rameters of ion channel function. The model channel KcvPBCV1 was thereforeexpressed in a cell-free translation system and reconstituted in planar lipid bi-layers for single channel recordings. We find that preferred codons enhance therate of translation elongation. This has no impact on the unitary conductance ofthe channel but eliminates one voltage dependent gating component. The dataunderscore that the same amino acid sequence can generate proteins withdistinctly different functional features. The dynamics of protein synthesishence provides an additional layer for the modulation of structure/function cor-relates in ion channels. This could be important for an understanding of socalled ‘‘silent mutations’’, which are so far generally ignored in the contextof an analysis of channelopathies.

1862-PlatGgamma Assists Gbeta to Activate GIRK1 by Relaxing InhibitoryConstraintGalit Tabak, Tal Keren Raifman, Vladimir Tsemakhovich, Nathan Dascal.Tel Aviv University School of Medicine, Tel Aviv, Israel.G protein-sensitive inwardly rectifying potassium (GIRK) channels mediateinhibitory effects of neurotransmitters acting via G protein-coupled receptors.Following receptor activation and dissociation of the Gabg heterotrimer,

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Gbg subunits bind to GIRK’s cytosolic domain, promoting channel opening.The main activating moiety is Gb which harbors most of GIRK-interacting res-idues within its Ga-binding interface. Gg is not considered important for GIRKactivation, although one work challenged this view [1]. We found that expres-sion of Gg in Xenopus oocytes activates GIRK1* (a homotetramericGIRK1F137S channel), enhancing both basal and agonist-evoked currents (Ibasaland Ievoked). YFP-tagged Gg, which expressed as a fairly stable protein in E.coli or mammalian cell lysate, activated GIRK1* better than untagged Gg.Gg activated GIRK1/3 and GIRK1/4 to variable extent, but, importantly, notthe homotetrameric GIRK2. The presence of the unique, long distal C-terminusof GIRK1 (dCT) was necessary but not sufficient for GIRK1* activation by Gg.Expression of Gg did not change plasma membrane levels of GIRK1*, thus Ggacted by affecting the gating of the channel. Activation by Gg depended on thepresence of ambient (endogenous) Gb. Interestingly, coexpression of low dosesof Gb’s RNA enhanced the effect of Gg on Ibasal but high doses reduced it andsuppressed Ievoked, indicating that excess Gb intercepts both free Gg andpossibly Ga and obstructs their action(s). The observed phenomena are inline with the hypothesis that dCT of GIRK1 subunit is part of a ‘‘lock’’ mech-anism that reduces channel’s open probability, helping to keep it closed at rest[2]. We propose that Gg relieves the inhibitory constraint imposed by the‘‘lock’’, helping Gb to activate the GIRK channels.1. Kawano et al. FEBS Lett 463, 355-359 (1999).2. Rubinstein et al. J Physiol 587, 3473-3491 (2009).

1863-PlatControl of AMPA Receptor Activity by the Extracellular Loops of Auxil-iary ProteinsClarissa Eibl1,2, Irene Riva1,2, Rudolf Volkmer3, Anna L. Carbone1,2,Andrew J.R. Plested1,2.1Molecular and Cellular Physiology, Leibniz-Forschungsinstitut f€urMolekulare Pharmakologie, Berlin, Germany, 2Cellular Biophysics,Humboldt Universit€at zu Berlin, Berlin, Germany, 3Chemical Biology,Leibniz-Forschungsinstitut f€ur Molekulare Pharmakologie, Berlin, Germany.The ligand gated AMPA type glutamate receptors (AMPARs) mediate the ma-jority of fast excitatory neurotransmission throughout the mammalian brainwhere they play a central role in synaptic plasticity and cognition. At thepost-synapses, AMPA receptors form complexes with auxiliary subunits,including the transmembrane AMPA receptor regulating proteins (TARPs)that are known to increase receptor trafficking and modulate AMPAR proper-ties. However, the mechanism for AMPAR gating modulation by TARPs re-mains poorly understood.Here, we sought to identify those sites in TARPs g2 and g8 responsible formodulation of channel gating. Therefore, we built structural models ofTARP-AMPA receptor complexes for TARPs g2 and g8, combining recentstructural studies and de novo structure predictions. These models, combinedwith peptide binding assays, provide evidence for multiple interactions be-tween GluA2 and the variable extracellular loops of both TARPs. Substitutionsand deletions of these loops had surprisingly rich effects on the kinetics ofglutamate-activated currents, without any effect on assembly. Critically, substi-tutions in the linker domains of GluA2 completely removed any effect of g2 onreceptor kinetics, indicating a dominant role for this previously overlooked siteproximal to the AMPA receptor channel gate. Likewise, by altering the two in-teracting loops of g2 and g8, we could entirely remove all allosteric modulationof GluA2, without affecting formation of AMPA receptor-TARP complexes.These findings indicate a complex set of modulatory interactions, and providetools for future studies of TARP action in the brain.

1864-PlatProtonation State of Glutamate 73 Regulates the Formation of a UniqueDimeric Association of VDAC1Lucie A. Bergdoll1, Michael T. Lerch2, John W. Patrick3,Christian Altenbach4, Paola Bisignano5, Arthur Laganowsky6,Michael Grabe7, Wayne Hubbell4, Jeff Abramson1.1Physiology, UCLA, Los Angeles, CA, USA, 2Biophysics, Medical Collegeof Wisconsin, Milwaukee, CA, USA, 3Chemistry, Texas A&M University,College Station, TX, USA, 4Chemistry, UCLA, Los Angeles, CA, USA,5UCSF, San Francisco, CA, USA, 6Chemistry, Texas A&M University,College Station, TX, USA, 7UCSF, San Francsisco, CA, USA.The Voltage-Dependent Anion Channel (VDAC) is the most abundant proteinin the outer mitochondrial membrane and constitutes the primary pathway forthe exchange of ions and metabolites between the cytosol and the mitochondria.There is accumulating evidence supporting VDAC’s role in mitochondrialmetabolic regulation and apoptosis, where VDAC oligomerization has beenimplicated with these processes. We report a specific pH-dependent dimeriza-tion of murine VDAC1(mVDAC1) identified by double electron-electron reso-

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nance (DEER) and native mass spectrometry (MS). Inter-molecular distanceson 4 singly spin-labeled mVDAC1 mutants were used to generate a model ofthe low-pH dimer, establishing the presence of the membrane-facing residueE73 at the interface. This dimer arrangement is different from any oligomericstate previously described, and it forms as a steep function of pH with anapparent pKa of 7.4. Moreover, the monomer-dimer equilibrium affinity con-stant was determined using Native MS, revealing a nearly 8-fold enhancementin dimerization affinity at low pH. Mutation of E73 to either alanine or gluta-mine severely reduces oligomerization, demonstrating the role of protonatedE73 in enhancing dimer formation. Based on these results, and the knownimportance of E73 in VDAC-physiology, VDAC dimerization likely plays asignificant role in mitochondrial metabolic regulation and apoptosis in responseto cytosolic acidification during cellular stress.

1865-PlatVoltage-Dependent Conformational Changes of KV1.3 Potassium Chan-nels are an Essential Element for KV1.3-induced cell proliferationM. Teresa P�erez-Garcıa1,2, Pilar Cidad1,2, Esperanza Alonso1,2,Pablo Fernandez-Velasco1, Miguel A. de la Fuente2,3,Jos�e R. Lopez-Lopez1,2.1Department of Physiology, Universidad de Valladolid, Valladolid, Spain,2Ibgm, CSIC, Valladolid, Spain, 3Department of Cell Biology, Universidadde Valladolid, Valladolid, Spain.Kþ channels have been shown to regulate cell proliferation. Kþ channel func-tion, by modulating membrane potential (EM), cell volume and/or Ca2þ influx,is an important element in cell cycle regulation. However, non-canonical func-tions can influence cell proliferation in the absence of (or in addition to) Kþ

efflux. Kv1.3 channels are implicated in the control of cell cycle in differentcells and in different ways. The specific requirement of Kv1.3 for proliferationin several cell types supports the involvement of molecule-specific interactionsvia mechanisms that are just starting to be identified.We have previously found that heterologous expression of Kv1.3 channelsincreased HEK cells proliferation, while Kv1.5 expression decreased it. Inthis system, Kv1.3-induced proliferation does not require Kþ fluxes. We iden-tified the molecular determinants of Kv1.3-induced proliferation at the C-termi-nal domain. In this region, individual point mutations of two phosphorylationsites (Y447A and S459A) abolished proliferation. Here we explore the hypoth-esis that voltage-dependent transitions of Kv1.3 are required for channel phos-phorylation, activating signaling pathways leading to proliferation.We explore the effects of EM variations on Kv1.3-induced HEK cells prolifer-ation. Kv1.3 channels were co-transfected with WT-KATP channels (composedof SUR1þKir6.2) or gain of function (GOF), ATP-insensitive channels(SUR1þKir6.2G334D).Resting EM was significantly hyperpolarized in cells expressing GOF-KATP

channels. Kv1.3-induced proliferation was unchanged by co-expression ofKv1.3þWT-KATP, but was abolished in the Kv1.3þGOF-KATP transfectedcells. Increasing [Kþ]e was able to restore Kv1.3-induced proliferation in cellsexpressing Kv1.3þGOF-KATP channels. Moreover, both Kv1.3 phosphoryla-tion and Kv1.3 interaction with scaffold proteins involved in cell proliferationare facilitated by membrane depolarizations in the range of Kv1.3 channel acti-vation threshold. Altogether, these data indicate that close to open transitions ofKv1.3 channels are required for Kv1.3 signaling in proliferation. Supported byBFU2016-75360-R.

1866-PlatAssociation of HERG and SCN5A Transcripts Regulates Ion ChannelExpression and Function in Stem Cell Derived CardiomyocytesCatherine A. Eichel, Erick Rios-Perez, Fang Liu, David K. Jones,Gail A. Robertson.Neurosciences, University of Wisconsin–Madison, Madison, WI, USA.The balance of ion currents that generate the cardiac electrical impulse iscrucial for proper heart function. Perturbation in the expression of either therapid delayed rectifying potassium channel gene (KCNH2) or the sodium chan-nel gene (SCN5A) can result in dramatic cardiac arrhythmias and sudden death,but how cardiac cells coordinate expression of these channels is unknown. Werecently demonstrated that alternate transcripts from theKCNH2 gene encodinghERG1a and 1b subunits are physically associated. This allows a proximity thatpromotes co-translational assembly and ensures proper subunit constituency.Here, we report that hERG transcripts are also associated with SCN5AmRNAs.Using single-molecule fluorescence in-situ hybridization (smFISH) in cardio-myocytes derived from induced pluripotentent stem cells (iPSC-CMs), wefound that 45% of the actively translated hERG1a transcripts are associatedwith SCN5A mRNAs. Of those complexes, 70% were located within 10 mmfrom the nucleus, consistent with a distribution in active protein synthesis do-mains. RT-PCR after immunoprecipitation of either hERG or NaV1.5 channels

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revealed association of hERG1a, hERG1b, and SCN5A mRNAs and their cor-responding proteins hERG1a, hERG1b and NaV1.5, indicative of a co-translational complex. Furthermore, the transcripts can be co-regulated iniPSC-CMs as indicated by a coordinate decrease of SCN5A, hERG1a andhERG1b transcript levels upon hERG1b-specific silencing. Whole-cell patchclamp revealed a corresponding reduction in the magnitude of the potassiumIKr and sodium INa currents. The co-translational association and co-regulation of transcripts may represent a general mechanism by which cardiaccells coordinate expression and thus activity of different ion channel types. Thismechanism may shed new light on cardiac rhythmmaintenance and arrhythmo-genic disorders, as well as other ion channel-related diseases.

1867-PlatA Hyperpolarization-Activated Proton Channel in Zebrafish SpermReinhard Seifert1, Lea Wobig1, Therese Wolfenstetter1, Sylvia Fechner2,Wolfgang Bonigk1, Heinz-Gerd Korschen1, U. Benjamin Kaupp1,Thomas Berger1.1MNS, Caesar Research Center, Bonn, Germany, 2Department of Molecularand Cellular Physiology, Stanford University, Stanford, CA, USA.Most fish reproduce by external fertilization. Gametes are released into theaquatic habitat and the motile sperm need to find the egg. Ion channels in theplasma membrane of sperm are important players in the fertilization process.We have identified in sperm of the zebrafish Danio rerio ahyperpolarization-activated ion channel highly similar to HCN channels foundin heart and brain. Strikingly, the channel is highly selective for protons that arecarried into the cell during hyperpolarization. The selectivity of the channel isabout as high as that of the Hv1 proton channel. We are currently investigatingthe molecular determinants of this proton selectivity by targeted mutagenesisand electrophysiological characterization.The change in selectivity from Naþ/Kþ as in HCN channels to Hþ as in thezebrafish channel investigated here is probably an evolutionary adaptation toenable fertilization in freshwater, where ion concentrations, especially that ofNaþ, are particularly low.

1868-PlatInsights on Gating Functions of Cytosolic Domains of Connexin26 Hemi-channels Revealed by a Human Pathogenic Mutation (N14K)Juan M. Valdez Capuccino1, Payal Chatterjee2, Isaac Garcia3,Andrew L. Harris1, Yun Luo2, Jorge E. Contreras1.1Pharmacology, Physiology, and Neuroscience, Rutgers, Newark, NJ, USA,2Western University, Los Angeles, CA, USA, 3Universidad de Valparaiso,Valparaiso, Chile.Connexin 26 (Cx26) is a hexameric, transmembrane protein. At the cell mem-brane, it can be found forming hemichannels or gap junction channels (GJCs)with opposing cells. The N-terminal domain (NT) of these channels is foldedinto the pore, playing an important role in permeability and gating. A groupof mutations within the NT that produce aberrant hemichannels with increasedbasal activity is responsible for Keratitis-Ichthyosis-Deafness (KID) syndrome.In this study we focus on N14K, an NT KID mutant. Structural data reveals aninteraction between the NT and cytoplasmic loop (CT) of the channel, whereposition 14 is located. Here, we explored how the N14K mutant affects the in-teractions between these two regions and consequently, promotes gain in func-tion. Assessing macroscopic and single-channel recordings, we observed thatthe N14K mutant shows an increase in the energy barrier for the transition be-tween open and closed states, shifting calcium sensitivity, voltage sensitivity,and deactivation time constants. Correlation analysis of Cx26 WT moleculardynamics (MD) simulations identified several sites of NT-CL interaction:These include interactions between N14 and residues H100 and Y97, and be-tween K15 and E101. Interestingly, the same analysis performed on theCx26 N14K MD simulations showed that the insertion of a Lys at position14 completely disrupted the NT-CL interactions. To test this, we used doublemutant cycle analysis, which showed that the NT-CL interaction does occurin the WT channel and is disrupted in the N14K mutant. Our data suggestthat disruption of NT-CL interaction facilitates hemichannel opening and sta-bilizes the open state. In addition, it provides a mechanistic understanding ofhow mutations at position 14 cause human disease.

Platform: Membrane Structure

1869-PlatLipid Organization in Simulations of Cell MembranesSvetlana Baoukina1, Helgi I. Ingolfsson2, Siewert J. Marrink3,D. Peter Tieleman1.1University of Calgary, Calgary, AB, Canada, 2Lawrence Livermore NationalLaboratory, Livermore, CA, USA, 3University of Groningen, Groningen,Netherlands.

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Cell membranes contain multiple lipid and protein components, that are orga-nized in the membrane plane. The membrane lateral organization is currentlyviewed as dynamic nano-scale clusters/assemblies of lipids and proteins, butthe nature of these clusters remains elusive. They are described by a numberof theories, the most common being the "raft hypothesis". According to thesetheories, the clusters/rafts could represent domains of an Lo-like phase, orcomposition (critical) fluctuations, or a micro-emulsion. The clusters lie belowthe spatio-temporal resolution of the most experimental techniques, but are sup-ported by a large number of indirect studies. These studies have shown that cellmembrane extracts separate into Lo and Ld phases at lower (room) tempera-tures, but the phase state at higher (physiological) temperatures is to beuncovered.Here we investigate the temperature-dependent phase behaviour of a modelmembrane with a realistic lipid composition. The model is based on an ideal-ized plasma membrane, with an asymmetric distribution of components be-tween the leaflets [JACS, 2014, 136, 14554]. We use molecular dynamicssimulations with the coarse-grained Martini force field. We investigate thechanges in the membrane lateral structure in a wide temperature intervalof 260-330 K, and observe dynamic nano-scale lipid clusters. We charac-terize the properties of these clusters, including their local compositionand structure, sizes and lifetimes, coupling between the leaflets, and lipidflip-flop.

1870-PlatCholesterol-Induced Membrane Organization Promotes Influenza VirusBindingIsabel Nadine Goronzy1, Robert Rawle2, Steven Boxer1, Peter Kasson2.1Department of Chemistry, Stanford University, Stanford, CA, USA,2Department of Molecular Physiology and Biological Physics, University ofVirginia, Charlottesville, VA, USA.The influenza virus attaches to a cell surface by binding sialic-acid contain-ing receptors with the viral ligand hemagglutinin. Stable viral binding re-quires more than one viral ligand- host receptor interaction tosimultaneously occur. Thus, binding can be modulated by the spatial distri-bution of target receptors in the host membrane. We show that membranesterols critically control viral attachment and propose that sterols alter thenanoscale clustering of viral receptors to facilitate binding. Using single vi-rus fluorescence microscopy, we demonstrated that viral binding is depen-dent on the cholesterol content of the target membrane. Fluorescentlylabeled viral particles preferentially bound synthetic membranes supple-mented with increasing amounts of cholesterol (0-40 mol%). Other sterolsexhibited a similar effect on binding, independent of their ability to supportliquid-liquid phase separation. To develop a molecular explanation for cho-lesterol’s effect, we ran a series of course grained molecular dynamics sim-ulations of lipid bilayers containing the viral receptor, disialogangliosideGD1a. While simulated GD1a molecules self-associated independent ofcholesterol, the dissociation rate between pairs of GD1a molecules was afunction of bilayer cholesterol concentration. Additionally, cholesterolincreased the order parameter of simulated GD1a lipid tails. We suggestthat, by preordering the viral receptor in its monomeric state, cholesterollowers the entropic penalty of receptor association. This in turn promotesthe formation of GD1a multimers and increases the influenza virus bindingavidity of the lipid bilayer. Our findings assign a critical role to the hostcellular membrane in viral infectivity and reveal sterol-dependent membraneorganization not associated with phase separation.

1871-PlatNanoscale Membrane Curvature Generated by Cholera Toxin Subunit B:The Effects of Lipid Cross-Linking and Lipid PhaseAbir Maarouf Kabbani, Xinxin Woodward, Christopher V. Kelly.Physics and Astronomy, Wayne State University, Detroit, MI, USA.With polarized localization microscopy (PLM), we have resolved the inherentmembrane bending capability of cholera toxin subunit B (CTxB) in supportedlipid bilayers. PLM is single-molecule localization microscopy techniquecapable of revealing membrane orientation with super-resolution. Membranebuds of <50 nm radius were observed to grow into >200 nm radius andextended tubules with dependence on the membrane tension, CTxB concentra-tion, and the number of GM1 bound per CTxB. However, the membranebending induced by CTxB was apparently independent of the lipid phase char-acteristics of the GM1 or the surrounding lipids. The CTxB was (12 5 4)xmore concentrated on the positive curvature top and (26 5 11)x more concen-trated on the negative Gaussian curvature neck of the nanoscale membranebuds compared to the surrounding planar supported lipid bilayer. WhereasCTxB is frequently used as a marker for liquid-ordered lipid phases, thecoupling between CTxB and membrane bending provides an alternate

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understanding of CTxB-induced membrane reorganization. Single-moleculetracking was performed on lipids and CTxB to reveal the correlations betweendiffusion rates, CTxB clustering, and membrane topography. Slowed lipid andCTxB diffusion was observed at the nanoscale buds locations, suggesting alocal increase in the effective membrane viscosity or molecular crowdingupon membrane bending. These results suggest inherent CTxB-induced mem-brane bending as a mechanism for initiating CTxB internalization in cells thatcould be independent of clathrin, caveolin, actin, and lipid phase separation.

1872-PlatA Step Forward in the Design of Liposomes: Symmetric & AsymmetricVesicles from Lipid ExtractsLaura Paulowski, Thomas Gutsmann, Tonio Kutscher.Research Center Borstel, Borstel, Germany.Nature’s complexity in the design of cellular boundaries represent a challengein monitoring processes that are located on or associated with the membrane.Liposomes are the method of choice when studying membrane-peptide or -pro-tein interactions due to the good control regarding composition and the reducednumber of component variables. One major drawback in their design is thesymmetric architecture and the related lack of controlling different lipid com-positions between inner (IL) and outer leaflet (OL). Since biological mem-branes are highly asymmetric, we compare different techniques forengineering lipid vesicles and point out challenges, opportunities and potentiallimitations: Symmetric vesicles are prepared by electroformation whereasasymmetric vesicles are generated from a double-emulsion phase. The differentfashioned vesicles are either reconstituted from pure DOPC or lipid mixturessuch as DOPC:SM:Chol as host cell mimicry and model for lipid rafts aswell as POPE:POPG as inner membrane model of Gram-negative bacteria.An extension from these synthetic lipid species to natural lipid extracts, e.g.S. cerevisiae, C. albicans, E. coli, and bovine liver and heart build a bridge be-tween the pure model and native cellular systems. Asymmetric vesicles providea perfect tool in modelling the bacterial outer leaflet by using lipopolysaccha-rides (LPS) as OL-mimicry and a phospholipid mixture composed ofPE:PG:CL for the IL. Both vesicle design concepts are compared regardingsize distribution, reconstitution potential in terms of the applied lipid spectrum,introduction of fluorophores, the ability to undergo phase separation and thedominating domain-sizes as investigated by fluorescence and confocal micro-scopy. Additionally, asymmetric vesicles are used for the determination of lipidflip-flop with respect to the OL-composition. To which extent lipid specificityand interleaflet coupling play a role in phase separation and lipid flip-flopshould be elucidated.

1873-PlatStructural Determinants and Functional Consequences of Protein Associ-ation with Membrane DomainsJoseph Lorent, Blanca Barbara Diaz-Rohrer, Xubo Lin, Alex Gorfe,Kandice R. Levental, Ilya Levental.Integrative Biology and Pharmacology, University of Texas Medical Schoolat Houston, Houston, TX, USA.Eukaryotic plasma membranes (PMs) are compartmentalized into functionallateral domains, including lipid rafts. Such domains are involved in most PMfunctions by selective recruitment and retention of specific proteins. Howev-er, the features that determine transmembrane protein partitioning to raft do-mains are unknown. We have used Giant Plasma Membrane Vesicles(GPMVs) isolated directly from live cells to directly quantify the structuraldeterminants and functional consequences of protein association with mem-brane domains. Using quantitative raft affinity measurements for >100 pro-teins, we identified three physical features of transmembrane domains(TMDs) - surface area, length, and palmitoylation - that independently affectraft partitioning. These observations were rationalized with a mechanistic,physical model that could be used to correctly predict raft affinity directlyfrom protein sequence. Application of these predictions to the human prote-ome revealed that PM proteins have higher raft affinity than those of intra-cellular membranes, consistent with the hypothesis of raft-mediated proteinsorting to the PM. We validated this prediction by showing that PM locali-zation was indeed dependent on raft partitioning across a large panel of un-related constructs, demonstrating that raft association is necessary andsufficient for PM sorting in the absence of other trafficking signals. Finally,we showed that abrogation of raft partitioning led to mis-targeting of pro-teins to late endosomes / lysosomes. Thus, our experimental observationsand physical model establish general rules for raft partitioning of TMDsand support the central role of lipid-driven domains in membrane traffic.Current work is designed to identify the pathways and effectors responsiblefor specific trafficking of raft-associated proteins and lipids to the plasma

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membrane using unbiased high-throughput screening and geneticmodification.

1874-PlatCurvature-Mediated Transmembrane Coupling in Asymmetric LipidsVesiclesBarbara Eicher1, Drew Marquardt2, Frederick A. Heberle3,Ilse Letofsky-Papst4, John Katsaras3, Georg Pabst1.1Institute of Molecular Biosciences/Biophysics division, University of Graz,Graz, Austria, 2Department of Chemistry and Biochemistry, University ofWindsor, Windsor, ON, Canada, 3Shull Wollan Center, Oak Ridge NationalLaboratory, Oak Ridge, TN, USA, 4Institute for Electron Microscopy &Nanoanalysis and Center for Electron Microscopy, Graz University ofTechnology, Graz, Austria.We studied the effect of intrinsic lipid curvature (J0) on structural properties ofasymmetric vesicles composed of palmitoyl-oleoyl-phosphatidylethanolamine(POPE; J0< 0), and palmitoyl-oleoyl-phosphatidylcholine (POPC; J0 �0).Cryo-electron microscopy and dynamic light scattering were used to evaluatevesicle size and morphology, and X-ray and neutron scattering combinedwith calorimetric measurements yielded insights into leaflet-specific lipid pack-ing and melting processes. Strong transmembrane coupling in asymmetric ves-icles with an inner leaflet composed of POPE and an outer leaflet enriched inPOPC was found below the lipid melting temperatures. This was demonstratedby lipids melting cooperatively in both leaflets and a rearrangement of lipidpacking in both leaflets. In contrast, no coupling was observed in vesicleswith POPC inner bilayer leaflets and enriched POPE outer leaflets. In thiscase, the leaflets melted independently and did not affect each other’s acylchain packing. In addition, no evidence for transbilayer structural couplingwas found above the melting temperature of both systems regardless of theleaflet distribution of POPE. These findings are consistent with the energeti-cally preferred location of POPE residing in the inner leaflet, where it is alsolocated in natural membranes, most likely causing the coupling of both leaflets.The loss of this coupling in the fluid bilayers is probably the result of entropiccontributions.This work is supported by the Austrian Science Fund FWF, Project No.P27083-B20 (to G.P.).

1875-PlatBeta-1 Integrin Association with Ordered Membrane Domains is Depen-dent on their Activation StateJulia T. Bourg, Sarah L. Veatch.Biophysics, University of Michigan, Ann Arbor, MI, USA.Integrins are transmembrane adhesion receptors that partner with ligands inthe extracellular matrix and cytoskeletal proteins to form multi-protein adhe-sive complexes known as focal adhesions. These complexes provide a linkbetween the cell and its environment while contributing to the regulationof cell viability, growth, and motility. Integrins have been shown undergolarge conformational change in their ectodomains, from a resting ‘‘bent’’state to an active ‘‘extended’’ state, which helps drive the formation andbreakdown of focal adhesions. This functionality is hypothesized to be sen-sitive to cell membrane composition, driven at least in part by the tendencyfor the plasma membrane to separate into ordered and disordered domainsresembling liquid phases observed in model membranes. In this work, weexplore how the conformational state of the b1 integrin receptor impactsits local membrane composition and how perturbations of membrane do-mains alter b1 integrin activity. We use two-color super resolution micro-scopy to quantify the local membrane environment surrounding b1integrins by tabulating cross-correlations between antibody labeled b1 integ-rins and expressed peptide markers of ordered and disordered phase-like do-mains expressed in intact cells. Through the use of antibodies with wellcharacterized specificity to different integrin activation states, we are ableto compare how integrin activity impacts their local membrane environment.In separate measurements, we are exploring how membrane perturbationsthat alter domain stability in isolated plasma membrane vesicles (GPMVs)impact the activity of b1 integrins as well their assembly within focaladhesions.

1876-PlatSupported Lipid Bilayers on Silica Nanoparticles as a Platform for Study-ing Lipid-Protein Interactions at Highly Curved SurfacesHyeondo (Luke) Hwang, Peter Chung, Alessandra Leong, Ka Yee C. Lee.Chemistry, University of Chicago, Chicago, IL, USA.Nanoparticle-supported lipid bilayers present a robust platform for drug deliv-ery and the study of curvature sensing proteins due to the monodisperse

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control of membrane curvature that cannot be realized with conventional lipo-some preparation methods. Most studies on supported lipid bilayer (SLB) for-mation on solid substrates have focused on planar surfaces and lipid chargeeffects— limited in scope with regard to lipid composition and constrainour understanding for the potentially versatile use of nanoparticle-SLBs. Inthis work, we have examined the mechanism of lipid adsorption onto highlycurved silica nanoparticles, using particles with different surface functionalgroups and the lipids of different compositions. Our work reveals a varietyof morphological features (e.g. single lipid bilayers, lipid ‘‘sheaths’’ aroundnanoparticle aggregates and fused, unruptured vesicles on silica surface)based on composition and aqueous milieu. We have successfully coated singleSLBs of compositions containing highly charged lipids (up to 50 mol % of1,2-dioleoyl-sn-glycero-3-phosphate; DOPA) as well as lipids with negativecurvatures (up to 50 mol % of 1,2-dioleoyl-sn-glycero-3-phosphoethanol-amine; DOPE) onto monodispersed spherical nanoparticle substrates at orbelow 50 nm in diameter using a generalizable osmotic stress technique.SLB formation has been validated with cryo-electron microscopy, conven-tional TEM, and quantitative phosphate analysis of lipids on silica. Ourstudies provide general insights into SLB formation on high-curvature,three-dimensional surfaces and significantly extend the use of this modelsystem.

Platform: Cytoskeletal Assemblies and Dynamics

1877-PlatLabel-Free Visualisation of Actin Nucleation and Polymerisation at theSingle-Molecule Level using Interferometric Scattering MicroscopyNikolas Hundt, Andrew Tyler, Gavin Young, Daniel Cole,Adam J. Fineberg, Joanna Andrecka, Philipp Kukura.Department of Chemistry, University of Oxford, Oxford, United Kingdom.Actin filaments are a major component of the cytoskeleton involved inmany basic processes such as cell migration, cell adhesion, cell divisionand muscle contraction. The most important property of actin is that itforms filaments (F-actin) from globular subunits (G-actin). In the 1960s,Oosawa and co-workers developed a model for the G- to F-actin transitionwhere the rate-limiting step is the formation of a stable actin nucleus,later determined to consist of 2-4 actin monomers. Nuclei are elongatedby the addition of monomers to the filament ends. This model is widelyaccepted, although the molecular details could never be visualisedexperimentally.Here, we used interferometric scattering microscopy (iSCAT) to monitornucleation and elongation of actin filaments at the single-molecule level.Building on recent improvements in label-free single molecule sensitivityand mass accuracy, we could reveal the polydispersity of G-actin at concen-trations relevant to nucleation and filament growth, showing signatures ofsmall protein oligomers in line with the nuclei sizes discussed above. Usingthe same approach, we could monitor the nanoscopic arrival of individual sub-units dynamically attaching to and detaching from the filament tip duringpolymerisation. Contrary to the standard model, where actin grows exclu-sively from monomers, we also found signatures of larger oligomers beingadded to the filament. Together with our polydispersity measurements, theseresults point towards a nucleation and growth mechanism for actin filamentsbased on monomers and small oligomers, rather than exclusively monomers.These results demonstrate the potential of iSCAT for label-free single-mole-cule imaging and for investigating the mechanisms of mesoscopic dynamicsin solution.

1878-PlatF-Form Actin Crystal Structures: Mechanisms of Actin Assembly andF-Actin ATP-HydrolysisShuichi Takeda1, Akihiro Narita1, Toshiro Oda2, Kotaro Tanaka1,Ryotaro Koike3, Motonori Ota3, Ikuko Fujiwara4, Nobuhisa Watanabe5,Yuichiro Maeda1.1Struct. Biol. Res. Ctr. Nagoya University, Nagoya, Japan, 2TokaigakuinUniversity, Kakamigahara, Japan, 3Grad. Sch. Info. Sci. Nagoya University,Nagoya, Japan, 4FRIMS, Nagoya Inst. Tech., Nagoya, Japan, 5SynchrotronRad. Res. Ctr. Nagoya University, Nagoya, Japan.Monomeric G-actin polymerizes into filamentous F-actin. The polymeriza-tion is associated with a conformational transition of actin from G-form toF-form (1), which triggers the hydrolysis of one ATP molecule bound toeach actin subunit within F-actin. Atomic details of G-form actin are welldocumented with more than one hundred G-actin-containing crystal struc-tures. In contrast, the atomic details of F-form actin are obscure (cf. 1, 2),because crystal of F-form actin was not available. To understand mecha-nisms of actin assembly, F-actin ATPase and dynamic properties of F-actin,

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F-actin structures at atomic resolutions are indispensable. Here we have ob-tained crystal structures of actin in complexed with fragmin, a gelsolin fam-ily protein. In one type of crystals, four actin molecules are assembled in thesame manner as in the double stranded F-actin, with the conformation of thetwo bared-end subunits in F-form. These crystal structures reveal details ofintra- and inter-strand actin-actin contacts. Furthermore, with the aid of afragmin sub-domain, we have obtained crystal structures of F-form actinwith bound Mg-AMPPNP (as a Mg-ATP analogue), Mg-ADPPi andMg-ADP, all at �1.3 A resolution. Intriguingly, Mg-AMPPNP-F-form actinand Mg-ADP-F-form actin appear to be in the identical structure, withdistinct structural stability. Based on these new structures, we will discussthe mechanism of ATP hydrolysis that is fundamental to the dynamic natureof actin filaments. (1) Oda et al (2009) Nature, 457: 441. (2) von der Eckenet al (2015) Nature, 519: 114.

1879-PlatConformational Twisting of MREB Double Protofilament in SimulationPredicts Filament Length In VivoHanduo Shi1, KC Huang1,2.1Bioengineering, Stanford University, Stanford, CA, USA, 2Microbiologyand Immunology, Stanford University, Stanford, CA, USA.The actin homolog MreB directs cell wall insertion and maintains rod shapein bacteria, but it remains unclear how MreB structural changes affect itsphysiological function. To bridge this gap, we performed molecular dy-namics simulations for Caulobacter crescentus MreB, the first crystal struc-ture revealing an anti-parallel double protofilament MreB conformation. Inour simulations, we observed a consistent left-handed twisting that only ex-isted in MreB double protofilaments bound to ATP, which was suppressedwhen the double protofilament was in proximity to a membrane patch.The binding to membrane also induced an inward membrane curvaturethat was physiologically relevant compared to typical curvatures in bacterialcell body. Twisting was also affected by MreB point mutations. To furtherprobe the effect of twisting, we developed a course-grain model based onthe mechanical parameters obtained in simulation to predict MreB filamentproperties in vivo. We found that the degree of left-handed twisting set thelimit length of MreB filaments in bacteria, which was further verified in vivofor wild-type and MreB mutant strains of Escherichia coli using structuralillumination microscopy. Together, our approach empowers the predictionof MreB filament conformations in vivo from molecular dynamics simulationresults, and this work provides a paradigm of connecting protein structure toits cellular function.

1880-PlatA Computational Investigation of Asymmetric Emergent Structures inActomyosin Dynamics During ChemotaxisCallie J. Miller1, Sreeja Asokan2, Jason Haugh3, James E. Bear2,Timothy C. Elston2.1Engineering, James Madison University, Harrisonburg, VA, USA,2University of North Carolina, Chapel Hill, NC, USA, 3North Carolina StateUniversity, Raleigh, NC, USA.We have developed a particle-based computer simulation to study emergentproperties of the actomyosin cytoskeleton. In particular, our model ac-counts for biophysical interactions between filamentous actin (f-actin) andnon-muscle myosin II (NM II). Our investigations were motivated byrecent studies that demonstrate regulation of myosin activity is criticalfor directed migration of fibroblasts responding to gradients of plateletderived growth factor so we have incorporated the dynamics for NM II for-mation. Individual NM II transition from a folded inactive state to anactive unfolded state. Once active, two NM II bundle together to createa processive NM II mini-filament capable of binding to f-actin. We per-formed a parametric analysis that led to the identification of biophysicalparameters that control the formation of f-actin asters. We identified thataster formation was sensitive to filament length and the ability of motorsto exert a spring-like force via changes in the spring constant for motors,or the maximum stretch allowed. When we considered the steps for NM IIassembly, we found that inhibiting motor-filament binding and not motoractivation or motor bundling was responsible for disrupting the actinmorphology. Extending the bulk parameter analysis, we simulated chemo-taxis by introducing the parameters to the computational simulation in aspatial gradient. We found that spatially regulating the ability of NM IIto bind to f-actin resulted in a significant variation in actomyosinmorphology in space. Additionally, we were able to generate a dynamicpulsatile aster structure through spatially regulating motor stiffness. Ouridentification of spatial regulators with the computational simulation willhelp guide future experimentation.

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1881-PlatC. Elegans Chromosomes Connect to Centrosomes by Anchoring into theSpindle NetworkStefanie Redemann1,2, Johannes Baumgart3, Norbert Lindow4,Michael Shelley5, Ehssan Nazockdast6, Andrea Kratz4, Steffen Prohaska4,Jan Brugues7, Sebastian F€urthauer5, Thomas M€uller-Reichert8.1Center of Membrane and Cell Physiology and Department of MolecularPhysiology and Biological Physics, University of Virginia School ofMedicine, Charlottesville, VA, USA, 2Experimental Center, Medical FacultyCarl Gustav Carus, Technische Universit€at Dresden, Dresden, Germany,3Max Planck Institute for the Physics of Complex Systems, Dresden,Germany, 4Zuse Institute Berlin, Berlin, Germany, 5The Courant Institute ofMathematical Sciences and Flatiron Institute, Center for ComputationalBiology, New York, NY, USA, 6Department of Applied Sciences, Universityof North Carolina, Chapel Hill, NC, USA, 7Max Planck Institute for thePhysics of Complex Systems and Max Planck Institute of Molecular CellBiology and Genetics and Centre for Systems Biology, Dresden, Germany,8Technische Universit€at Dresden, Experimental Center, Medical Faculty CarlGustav Carus, Dresden, Germany.The mitotic spindle is a three-dimensional, highly dynamic microtubule basedapparatus that ensures the faithful segregation of chromosomes. The dy-namics properties are regulated by a number of factors, such as polymerases,depolymerases, motor proteins, cross-linkers and other microtubule-associated proteins. Remarkably, despite the high turnover of microtubulesthroughout mitosis, the spindle maintains its bipolar structure over longertime periods. We set out to identify the detailed arrangement of microtubulesin C. elegans mitotic spindles, and to understand how this arrangement isgenerated, using a combination of large-scale electron tomography, light mi-croscopy and mathematical modeling. Based on our 3D reconstructions weclassified the microtubules composing mitotic spindles in three classes, kinet-ochore (KMTs), spindle (SMTs) or astral microtubules (AMTs) according totheir positions, and quantified distinct properties of each class. While our lightmicroscopy and mutant studies show that microtubules are nucleated from thecentrosomes, we find only a few KMTs directly connected to the centrosomes.Indeed, by quantitatively analyzing several models of microtubule growth, weconclude that minus-ends of KMTs have selectively detached and depolymer-ized from the centrosome. Our results show that the connection between cen-trosomes and chromosomes is mediated by an anchoring into the entirespindle network and that any direct connections through KMTs are few andlikely very transient.

1882-PlatCryo-Em Insight into Microtubule-Doublecortin (MT-DCX) Interactionand the Stages of MT Dynamic Instability Harnessed by DCXSzymon W. Manka, Carolyn A. Moores.Birkbeck College London, Institute of Structural and Molecular Biology,London, United Kingdom.Microtubule (MT) switching between phases of growth and shrinkage, known asMT dynamic instability, is fundamental for cell function. Its structural basis is notclear due to the difficulty to capture in solution the distinct structural states of thetubulinGTPase cycle that drives this behavior. To address this issuewedesigned afast sample preparation protocol involving MT stabilization by a neuronal MT-associated protein doublecortin (DCX), which allowed us to gain insight intothe long sought GDP-Pi transition state and the otherwise unstable GDP state.We used cryo-EM to reconstruct these lattices alongside the GMPCPP latticeimitating the GTP state. Our 3.8-4.5 A resolution structures reveal two conforma-tional transitions corresponding to the distinct steps of the tubulin GTPase cycle:1) GTP hydrolysis to GDP-Pi, and 2) Pi release. These transitions renderMTs un-stable through longitudinal compactionof theMT lattice.DCXbinds all the latticestates in the vertex of 4 tubulin dimers, but shows marked preference for the post-hydrolysis compacted lattice. Since it has two ubiquitin-like N- and C-terminalDC domains separated by a 42-residue linker, it has long been an enigma whichof themmediates this interaction. Our reconstructions showed DCX density con-taining overlapping signals from both domains, indicating that they bind to MTssimultaneously, and improved mapping of the overall DCX footprint on an MT.These results further our understanding of the MT dynamic instability and theindispensability of DCX for normal brain development.

1883-PlatUltrafast Force-Clamp Spectroscopy Reveals ‘‘Sliding’’ Catch-BondBehavior of the Microtubule-Binding NdC80 ProteinVladimir M. Demidov1,2, Suvranta K. Tripathy2, Fazly I. Ataullakhanov1,Ekaterina L. Grishchuk2.1Center for Theoretical Problems of Physicochemical Pharmacology, RussianAcademy of Sciences, Moscow, Russian Federation, 2Department of

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Physiology, Perelman School of Medicine, University of Pennsylvania,Philadelphia, PA, USA.The microtubule wall-binding Ndc80 complex serves as the primary molecularglue connecting mitotic kinetochores to spindle microtubules. During metaphasechromosome oscillations, the kinetochore-localized Ndc80 is thought to glidealong microtubules by forming mobile diffusive bonds, while the kinetochoresmove repeatedly toward the plus- andminus-ends of themicrotubules without de-taching. To investigate the biophysical underpinning of this striking behavior, weadopted a highly sensitive ultrafast force-clamp spectroscopy in vitro. A ‘‘dumb-bell’’ was formed by stretching a taxol-stabilizedmicrotubule between two beads,held in a dual-beam optical trap. Dumbbell beads were oscillated synchronouslyvia an acousto-optic deflector under the force-clamp feedback ran on the FPGA-equipped I/O board at 33 kHz, resulting in sub-millisecond temporal resolution.Two detection beams and quadrant photo-detectors monitored one dumbbellbead and the coverslip-immobilized pedestal with the conjugated Ndc80 protein,achieving nm-scale position sensing. Using this approach, we examined bi-directional Ndc80 gliding along the microtubule under constant force, imitatingthe forces and motions Ndc80 experiences during chromosome oscillations. Un-expectedly, we found that Ndc80 motility depends strongly on the direction offorce.Whenpulled toward themicrotubuleminus-end,Ndc80obeys a typical pre-diction for a diffusiveMAP,moving smoothly andwith littlemolecular friction, incorrespondence with its relatively fast unloaded diffusion. However, Ndc80strongly resists pulling toward the microtubule plus-end, exhibiting frequentpauses that are indicative of the catch-bond-like behavior. Strikingly, the pausingNdc80 retains the ability to glide continuously, while translocating 10-timesslower than in the opposite direction. Such sliding catch-bond behavior has notbeen previously seen for any microtubule-binding protein. We propose thatNdc80 serves as an intrinsic regulator ofmolecular frictionat humankinetochores,ensuring theirmobility simultaneouslywith themicrotubule-wall adhesion in cor-respondence with the direction of kinetochore gliding.

1884-PlatOptical Control of EB1 Reveals Local Functions of the MicrotubuleDTIPComplex During Cell Migration and DivisionJeffrey van Haren1, Rabab Charafeddine1, Andreas Ettinger2, Hui Wang3,Klaus M. Hahn3, Torsten Wittmann1.1Department of Cell and Tissue Biology, University of California, SanFrancisco, San Francisco, CA, USA, 2Institute of Epigenetics and Stem Cells,Helmholtz Center Munich, Munich, Germany, 3Department ofPharmacology, The University of North Carolina at Chapel Hill, Chapel Hill,NC, USA.Microtubules (MTs) form a highly dynamic network of polarized intracellulartracks that enable directional transport, facilitate chromosome segregation andhelp to establish cell polarity. Although the MT network is polarized towardsthe leading edge of migrating cells, due to a lack of tools to locally perturb MTdynamics, it has remained unclear if MT network polarity is a cause or conse-quence of directional cell movement. Similarly, the function of MT subpopula-tions within mitotic spindles has been difficult to study. We therefore developedan optogenetics approach to manipulate MTs with high spatial and temporal pre-cision in live cells.We targeteda key regulator ofMTdynamics, EB1,which func-tions as an adaptor to recruit otherþTIPs to the growing ends ofMTs, by insertinga light-sensitive protein interactionmodule betweenEB1 functional domains.Weshow that blue light rapidly and reversibly dissociates all tested þTIPs fromMTends. In cells inwhichwe replaceendogenousEB1with this photo-inactivated (pi)variant, blue light acutely and reversibly inhibited MT polymerization andinduced depolymerization of cell edge associated MTs. Rapid reversibility ofpi-EB1allowedus to spatially controlMTpolymerization and analyze cell biolog-ical consequences. In migrating cells, local pi-EB1 inactivation resulted in an im-mediate aversive turning response, indicating that EB1-mediated þTIPinteractions are essential to maintain migration direction. In dividing cells, pi-EB1 also allowed us to investigate the consequences of instant EB1 inactivationon mitotic spindle organization and positioning, and we found that the EB re-cruited þTIP complex is required for spindle size homeostasis.

Platform: Cardiac, Smooth, and Skeletal MuscleElectrophysiology and Regulation

1885-PlatMechanism and Regulation of JPH2/PM AssociationJunping Hu1, Min Jiang2, Tseng Gea-Ny1.1Physiology & Biophysics, Virginia Commonwealth University, Richmond,VA, USA, 2Chinese Academy of Medical Sciences and Peking UnionMedical College, Beijing, China.Background: Junctophilins (JPH1-4) tether SER (sarco-endoplasmic reticu-lum) to PM (plasma membrane) in excitable cells. SER/PM junctions are

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critical for Ca homeostasis. Junctophilins are anchored in the SER membraneby their C-terminal transmembrane domain (TMD), while using their N-termi-nal membrane-organization-recognition-nexus ‘MORN’ motifs (MORN1-MORN8) to associate with PM. However, it is not clear: (1) The specific mech-anism by which the MORN motifs associate with PM, or (2) Whether/how thisMORN-PM association is modulated by cellular context or junctophilins’ post-translational modifications. Objective: We tackle these questions, focusing onJPH2 (human isoform). Methods: We use mutagenesis, immunoprecipitation/immunoblotting, click reaction/proximity ligation assay, confocal and TIRFimaging on COS-7 cells for proof-of-concept experiments. We further testthe concepts in adult ventricular myocytes, Results: Palmitoylation is criticalfor JPH2-PM association. Palmitoylated JPH2 binds to lipid-ordered domains(LOD) on PM, manifested as distinct puncta in TIRF imaging. DisruptingLOD by depleting cholesterol with methoxy-beta-cyclodextrin reduces JPH2-PM association. JPH2 has 4 cysteine residues (C15 and C29 close toMORN1, C328 close to MORN8, and C678 adjacent to the SER TMD), allof which are engaged in palmitoylation. Mutating all 4 Cys to Ala markedlyreduces JPH2 protein level and alters its subcellular distribution pattern, sug-gesting that palmitoylation promotes JPH2 protein stability and stabilizesJPH2-PM association. Coexpressing selected palmitoyl transferases (DHHCenzymes) increases JPH2 palmitoylation, among which DHHC1-DHHC5 arehighly expressed in cardiac myocytes. In cardiac myocytes, native JPH2 phys-ically interacts with Ca/calmodulin-dependent kinase II (CaMKII). CaMKIImediates JPH2 phosphorylation, which increases the degree of JPH2 palmitoy-lation. Conclusion: We propose that JPH2 and likely other junctophilins usepalmitoylation to stabilize SER/PM tethering. Reducing JPH2 palmitoylationdestabilizes SER/PM junctions by decreasing the overall JPH2 protein leveland by weakening the JPH2-PM association.

1886-PlatMechanisms of Atrial Electrical Remodeling in Obese HeartUjala Srivastava1,2, Aparajita Bhattacharya1,2, Mohamed Boutjdir1,3,Ademuyiwa S. Aromolaran1,2.1Cardiovascular Research, VA HCS, Brooklyn, NY, USA, 2Cell Biology,SUNY Downstate, Brooklyn, NY, USA, 3Medicine, Cell Biology andPharmacology, SUNY Downstate, Brooklyn, NY, USA.Obesity is associated with increased risk of supraventricular arrhythmias yet themolecular mechanisms that underlie altered atrial electrical activity in obesityare unknown. Ca current (ICa,L) through L-type voltage-gated Ca channels (a-1D and a-1C) and the outward delayed rectifier K current (IK) composed of therapidly (IKr) and the slowly (IKs) activating components, are critical contribu-tors to atrial repolarization; however, there is a paucity of studies on the rolesfor ICa,L and IK in obesity. Therefore, we assessed the functional expression ofICa,L and IK in high-fat diet (HFD) fed guinea pig heart. HFD (45kcal% fat)-fedguinea pigs displayed significant weight gain, and increased total cholesteroland triglycerides compared to low-fat diet (LFD, 10kcal% fat) controls after50 days. Obese hearts displayed shorter atrial action potential duration, anddecreased ICa,L density compared LFD-fed non-obese controls. We furtherintroduce the novel finding that a-1D mRNA level is significantly down-regulated in obese atria while a-1C levels remained essentially unchanged.The electrical abnormalities in obese atria were also associated with increasedmRNA level of IK channel subunits (hERG & KCNQ1) as well as increased IKdensity. Exposure to palmitic acid increased heterologously expressed IKr andIKs densities, while oleic acid, severely reduced IKr and had no effect on IKs. Thedata are first to show a surprising heterogeneity of free-fatty acid mechanismsthat underlie increased IK in obese heart. These findings provide novel insightsinto the mechanism leading to atrial electrical dysfunction in obese heart. Suchinformation is essential for developing potential targeted therapies forarrhythmic events in obese patients.

1887-PlatStructural and Biochemical Mechanisms of Myosin-Induced DilatedCardiomyopathyKaren H. Hsu1, Adriana Trujillo1, Thomas C. Irving2, Sanford I. Bernstein1.1Biology, San Diego State University, San Diego, CA, USA, 2Biology,Illinois Institute of Technology, Chicago, IL, USA.Dilated cardiomyopathy (DCM), the most common cardiomyopathy form,causes pathological heart failure and wall thinning, leading to severe contractiledeficits. We generatedDrosophilamodels for myosin-based DCM to determinethe mechanistic basis whereby myosin mutations cause tissue-level dysfunc-tion. For this, we investigated the biochemicaland structural effects of twohMHC-b DCM mutations (S532P and R369Q) located within the actin-binding region of the motor domain. 2D X-ray diffraction patterns were ob-tained using the BioCAT beamline 18ID at the Advanced Photon Source forindirect flight muscles (IFMs) of young flies. The lattice spacings and intensity

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ratios of I20/I10 decreased in S532P IFMs but not R369Q IFMs compared tocontrols. Our data suggest that the S532P mutation reduces the space availablefor myosin head movement within the compressed lattice, causing myosinheads to be positioned more towards the thick filament backbone comparedto controls and R369Q mutants. Additionally, thick filament axial repeatsand troponin axial repeats are decreased in S532P IFMs but not R369QIFMs. Overall, the S532P mutation causes an abnormal and disordered thickfilament array. The R369Q mutants do not show structural changes in X-raydiffraction studies. However, both mutations decrease affinity for F-actin in vi-tro in co-sedimentation assays. Flight and jump tests were performed to deter-mine tissue-level skeletal muscle defects induced by the R369Q or S532Pmutations. Severe and progressive defects were observed in mutant skeletalmuscle function, indicating that the myosin forms produced by our transgenicmodels have compromised functionality in vivo. We conclude that the R369Qor S532P mutations cause pathogenic decreases in muscle function by disrupt-ing actin affinity. For S532P mutants, alterations in thick and thin filamentstructure and packing may compensate for weakened actin affinity byincreasing the probability and number of heads available to bind actin.

1888-PlatMechanism of Protection against Myocardial Ischemia-ReperfusionInjury in Mice Resistant to CaMKII OxidationYuejin Wu, Ning Feng, Qinchuan Wang, Mark E. Anderson.Johns Hopkins University Department of Medicine, Baltimore, MD, USA.Ca2þ/calmodulin-dependent protein kinase II (CaMKII) is a maladaptive medi-ator of myocardial ischemia-reperfusion (I/R) injury. CaMKII is activated byboth Ca2þ and oxidative stress (ox-CaMKII). We first found mice lacking reg-ulatory methionines required for ox-CaMKII by knockin replacement with va-lines (MMVV) showed significantly protected fractional shortening 24 or 72 hafter I/R compared to WT controls. We next performed simulated I/R in iso-lated ventricular myocytes. These studies confirmed that ventricular myocytesfrom MMVV mice have significantly improved viability compared to WT ven-tricular myocytes. ATP sensitive Kþ channels (IK-ATP) play an important rolein protection from I/R injury. CaMKII regulates IK-ATP in cardiac myocytes,at least in part, by a post-translational process that rapidly reduces sarcolemmalIK-ATP channel expression, thereby contributing to excessive action potentialduration and cellular Ca2þ overload. The IK-ATP opener pinacidil significantlyincreased cell viability of WT but not MMVV myocytes after simulated I/Rinjury. On the other hand, the IK-ATP blocker glibenclamide decreased cellviability in both WT and MMVV myocytes to a similar extent after simulatedI/R injury. Lastly, we discovered that whole cell IK-ATP current densities weresignificantly reduced by treatment of H2O2 (400 mM) in WT ventricular myo-cytes, but not in MMVV myocytes. In cell on patch recordings the number ofchannels responsive to IK-ATP activators pinacidil or 2, 4 dinitrophenol (DNP)was significantly reduced after H2O2 (1 mM) in WT myocytes, but not inMMVVmyocytes. Taken together, these findings support a view that I/R injurytriggers an ox-CaMKII mediated reduction in available IK-ATP current, andthat this pathway is a critical factor promoting I/R injury in heart.

1889-PlatFacilitation of SK Channel Activity via Inhibition OF PYK2-DependentTyrosine Phosphorylation Alleviates Ventricular Tachyarrhythmia inCardiac HypertrophyShanna Hamilton1, Iuliia Polina1, Radmila Terentyeva2, Karim Roder1,Tae Yun Kim1, Jin O-Uchi1, Gideon Koren1, Bum-Rak Choi1,Dmitry Terentyev1.1Cardiovascular Research Center, Brown University/Rhode Island Hospital,Providence, RI, USA, 2Cardiovascular Research Center, Rhode IslandHospital, Providence, RI, USA.Small conductance calcium (Ca2þ)-activated K channels (SK) expressed inventricular myocytes (VMs) are thought to play an antiarrhythmic role in car-diac disease by mitigating reduced repolarization reserve. Using the rat modelof hypertrophy induced by thoracic aortic banding (TAB), we thought to testthe antiarrhythmic potential of enhanced SK channel activity by inhibiting aputative negative regulator of SK activity, tyrosine kinase Pyk2. Immunopre-cipitated SK channels (SK2) from TAB VMs demonstrated increased levelsof Tyr phosphorylation reversible by incubation with PF431396 (10 mM,n=3), a specific Pyk2 inhibitor. Western blot analysis showed �2 fold increasein expression levels of Tyr kinase Pyk2 but not Src/Fyn in TABVMs. In ex vivooptically mapped TAB hearts stained with voltage sensitive dye di-4 ANNEPS,VT/VF was observed in 8/8 hearts challenged with b-adrenergic agonist isopro-terenol (50 nM). PF431496 (2 mM, 30 min.) alleviated VT/VF and shortenedAPD under isoproterenol (n=3). To unravel the mechanism of Pyk2-dependent modulation of SK activity, we used cultured adult rat VMs overex-pressing SK2. Simultaneous whole-cell patch clamp and confocal Ca2þ

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imaging revealed that Ca2þ-voltage(Vm) dependence of SK channels isbiphasic with inhibition at higher [Ca2þ]/Vm and this can be enhanced by stim-ulation of Pyk2 (phenylephrine, 100 mM and propranolol, 1 mM). Furthermore,N-terminus de-phosphomimetic SK2mutant Y138F was completely insensitiveto phenylephrine, suggesting phosphorylation of this Tyr residue confers Pyk2-mediated enhancement of Ca2þ-Vm dependent inhibition of SK2. Coexpressionof specific Pyk2 inhibiting peptide, CRNK, fully restored SK2 responsivenessto Ca2þ in phenylephrine-treated cells. In conclusion, SK channel activity inTABs can be further increased by inhibiting Pyk2. Additional facilitation ofSK channels via Pyk2 inhibition may be a rational antiarrhythmic target,increasing repolarization reserve that is diminished in cardiac disease.

1890-PlatHigh-Throughput Investigation of Contractile and ElectrophysiologicalProperties of Optically Stimulated hiPSC-CM MonolayersShan Parikh1, Nikhil Chavali1, Andrew Glazer2, Christian Shaffer1,Marcia Blair3, Dan Roden3, Bjorn Knollmann2.1Vanderbilt University, Nashville, TN, USA, 2Clinical Pharmacology,Vanderbilt University Medical Center, Nashville, TN, USA, 3VanderbiltUniversity Medical Center, Nashville, TN, USA.The accelerated utilization of human induced pluripotent stem cell derived car-diomyocytes (hiPSC-CM) for drug screening and disease modeling requiresadaptation of high-throughput technology for more efficient evaluation. Multi-ple technologies have emerged including single/multi-electrode array for extra-cellular field potential (EFP) detection, and voltage sensitive dyes andautomated patch-clamp for action potential measurement. EFP assessment todate predominantly is performed on spontaneously beating hiPSC-CM, howev-er, as rate significantly influences EFP morphology at baseline and in responseto pharmacological stimulation, our work focused on developing the use of op-tical pacing in academically generated patient specific hiPSC-CM. In thisstudy, we provide a physiological and pharmacological assessment of opticallystimulated hiPSC-CM using the CardioExcyte96 (Nanion), which allows forrapid 96-well evaluation of action potential and contractility correlates.HiPSC-CM were transduced with an adeno-associated virus (AAV) encodinga non-selective cation channel (channel rhodopsin2) and seeded on a matrigelcoated sensor plate for ten days. An efficient workflow with criteria for the gen-eration of high quality hiPSC-CMs, data conversion tools, and criteria for EFPand IMP analysis were developed. Using this workflow, our study provides adetailed characterization of the use of AAV-ChR2 infected hiPSC-CM formeasuring field potential duration (a correlate of action potential duration)and impedance (contractility correlate). Optical pacing increased reliabilityof waveform detection, enhanced synchronization of cardiomyocyte depolari-zation, and allowed for frequency dependent assessment of pharmacologicalIkr blocker (moxifloxacin) between 0.5 Hz and 3.0 Hz. High-throughput assess-ment of EFP and IMP using optical stimulation will greatly enhance our abilityto model cardiomyopathy and drug response using hiPSC-CM.

1891-PlatSimulating Drug-Induced Arrhythmia Sensitivity using an Expression-Based Theoretical Model of Human IPSC-Derived CardiomyocytesXin Gao1, Yue Yin2, Tyler Engel2, Neil J. Daily2, Li Pang3,Brian E. Carlson1, Tetsuro Wakatsuki2.1Molecular and Integrative Physiology, University of Michigan, Ann Arbor,MI, USA, 2InvivoSciences, Inc., Madison, WI, USA, 3Division ofBiochemical Toxicology, FDA National Center for Toxicological Research,Jefferson, AR, USA.The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, spon-sored by an international consortium, promotes the coupling of in vitro exper-imental assays with theoretical models of cardiomyocyte electrophysiologyand ion handling to assist in the prediction of cardiac torsadogenic drugrisk. Currently, there is a gap between current in vitro experimental models

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and computational models used to simulate these experimental observations.CiPA proposes the use of human induced pluripotent stem cell derived cardi-omyocytes (hiPSC-CMs) for experiments however the recommended modelof cardiomyocyte electrophysiology and ion handling, the O’Hara-Rudymodel (ORd), was developed to simulate electrophysiology of human adultventricular cardiomyocytes (hAVCMs). To reconcile this mismatch, wehave developed an electrophysiology model of the hiPSC-CM by incorpo-rating differences in gene expressions of selected ion channels, exchangers,receptors and pumps in iPSC-CMs against those found in hAVCMs. ThehiPSC-CM model developed here recapitulates a ventricular-like action po-tential along with an elevated resting membrane potential (�65 to �70mV) when compared to an hAVCM (�80 mV). The elevated resting mem-brane potential matches experimental data and the hiPSC-CM model showsNaþ channels in an inactivated state with the overexpression of L-TypeCa2þ channel current driving AP depolarization. This approach can be usedto model any hiPSC-CM cell line where expression data has been obtainedand replaces background currents for Kþ and Naþ in the ORd model withthe ultrarapid Kþ channel and hyperpolarization activated Kþ/Naþ channelcurrents. With this hiPSC-CM model variation in three batches from twodifferent hiPSC-CM cell lines are matched to experimental data. In addition,in silico combination blocking of Naþ, L-type Ca2þ and rapid delayed rectifierKþ channels shows that variability across lines must be accounted for whenusing these in vitro assays for testing of proarrhythmic risk.

1892-PlatWhole Heart Cytoarchitecture at Micron-Scale ResolutionErica Lazzeri1, Irene Costantini1, Samantha Cannazzaro2,Cecilia Ferrantini3, Giacomo Mazzamuto1, Claudia Crocini1,Raffaele Coppini3, Silvia Guerini1, Francesco Giardini3, Leonardo Bocchi3,Elisabetta Cerbai3, Corrado Poggesi3, Francesco Saverio Pavone1,Leonardo Sacconi1.1LENS, Florence, Italy, 2National Research Council, Florence, Italy,3University of Florence, Florence, Italy.Remodeling processes associated with genetic and non-genetic cardiac diseasescan cause alterations of electrical conduction and electro-mechanical dysfunc-tion, eventually leading to arrhythmias. These alterations consist mainly incollagen deposition (fibrosis) and cellular disorganization (myofilament align-ment), and their predictive models are often based on non-integrated and low-resolution information. Here, we combine advances in tissue clearing, stainingand high-resolution optical microscopy to reconstruct the three-dimensional or-ganization of cardiac conduction in the whole mouse heart. We developed apassive Clarity protocol for clearing the heart and for achieving fluorescentprobe penetration into the whole tissue. A fluorescent staining protocol is opti-mized to visualize the whole heart cytoarchitecture at sub-cellular level. We ex-ploited the high contrast and resolution of non-linear microscopy tosimultaneously image cellular organization by staining and collagen distribu-tion by second-harmonic generation deep in the cardiac tissue. A cytoarchitec-tonic analysis is applied to map cells alignment in three dimensions, definingthe conduction pathway of action potential propagation at intercellular level.We investigated the three-dimensional cytoarchitectonic remodeling in trans-genic mouse models of hypertrophic cardiomyopathy characterized by a severedegree of left ventricle hypertrophy and interstitial fibrosis. First, using arecently-developed ultra-fast optical system we mapped the propagation ofelectrical activity in whole diseased hearts. Then, we correlated the propagationmaps with the pathological disorganization of myofilaments and collagen depo-sition using the three-dimensional high-resolution optical reconstruction. Thisinnovative experimental approach will allow to dissect the morphologicalcauses leading to alterations of electrical conduction and to electro-mechanical dysfunction, and, more generally, will represent a whole new para-digm for diagnostic and therapeutic investigations.

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Platform: Force Spectroscopy and ScanningProbe Microscopy

1893-PlatMultimodal Measurements of Single-Molecule Dynamics using FlouRBTIvan E. Ivanov1, Paul Lebel2, Florian C. Oberstrass3, Charles Starr4,Angelica Parente4, Athena Ierokomos4, Zev Bryant3,5.1Department of Chemical Engineering, Stanford University, Stanford, CA,USA, 2Department of Applied Physics, Stanford University, Stanford, CA,USA, 3Department of Bioengineering, Stanford University, Stanford, CA,USA, 4Program in Biophysics, Stanford University, Stanford, CA, USA,5Department of Structural Biology, Stanford University, Stanford, CA, USA.Single-molecule methods provide direct measurements of macromolecular dy-namics, but are limited by the number of degrees of freedom that can be fol-lowed at one time. High-resolution rotor bead tracking (RBT) measuresDNA torque, twist, and extension, and can be used to characterize the structuraldynamics of DNA and diverse nucleoprotein complexes (1). Here, we extendRBT to enable simultaneous monitoring of additional degrees of freedom.Fluorescence-RBT (FluoRBT) combines magnetic tweezers, infrared evanes-cent scattering, and single-molecule FRET imaging, providing real-time multi-parameter measurements of complex molecular processes. We demonstrate thecapabilities of FluoRBT by conducting simultaneous measurements of exten-sion and FRET during opening and closing of a DNA hairpin under tension,and by observing simultaneous changes in FRET and torque during a transitionbetween right-handed B-form and left-handed Z-form DNA under controlledsupercoiling. We discover unanticipated continuous changes in FRET withapplied torque, and also show how FluoRBT can facilitate high-resolutionFRET measurements of molecular states, by using a mechanical signal as anindependent temporal reference for aligning and averaging noisy fluorescencedata. By combining mechanical measurements of global DNA deformationswith FRET measurements of local conformational changes, FluoRBT willenable multidimensional investigations of systems ranging from DNA struc-tures to large macromolecular machines.1. Lebel, P., Basu, A., Oberstrass, F.C., Tretter, E.M. & Bryant, Z. Gold rotorbead tracking for high-speed measurements of DNA twist, torque and exten-sion. Nat Meth 11, 456-462 (2014).

1894-PlatVideo Rate Atomic Force Microscopy of Biological SamplesSophia V. Hohlbauch.Asylum Research an Oxford Instruments Company, Santa Barbara, CA,USA.The atomic force microscope (AFM) has found broad use in the biological sci-ences largely due to its ability toacquire three-dimensional, high resolution imagesof native structures (molecules, cells, tissues) under fluid in near-physiologicalconditions. Since AFM is relatively non-destructive and requires minimal samplepreparation (i.e. no coating/fixing/labeling), it is frequently used to monitor dy-namic events. One of the challenges of traditional AFMs has been temporal reso-lution – acquiring scans at high enough speeds to capture biological processes inreal-time.With traditional AFMs we can easily observe the ‘‘before’’ and ‘‘after’’structures but very rarely canwe capture the kinetics. Nowwecan observe the pro-gression of these reactions and visualize their intermediate forms.With the arrival of video rate AFMs, we have a new set of standards enablinghigh resolution imaging of dynamic events at high speeds, up to 625 lines/sec-ond (10 frames per second). This is about 300x faster than typical AFMs and10x faster than current ‘‘fast scanning’’ AFMs. Video rate AFMs are idealfor researching dynamic events and have enabled a new set of experimentsincluding biochemical reactions, membrane studies, conformational changes,self-assembly and degradation. In most cases the spatial resolution is notcompromised enabling us to locate the target or active site while tracking theprogression of the reaction.We will present a set of data to illustrate the potential of this new capability.Examples include DNA digestion and cleavage, molecular structure and rear-rangement in the bacteriorhodopsin membrane, assembly of Type I collageninto fibrils and dynamic motion of CTAB hemi-micelles at the solid(HOPG) – liquid (aqueous buffer) interface.

1895-PlatA Simple and Fast Drift Correction Method for High-throughput Micro-scopyArin Marchesi1, Ignacio Casuso1, Simon Scheuring2, Felix Rico1.1Inserm U1006, Marseille, France, 2Dept Anesthesiology, Weill CornellMedical College, New York, NY, USA.High-speed atomic force microscopy (HS-AFM) and high-throughput fluores-cence microscopy quickly generate large amounts of digital image data that

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needs specialized software for handling and processing. For instance, HS-AFM produces large data sets of image sequences that are affected by a num-ber of instrument-specific issues, such as mechanical and/or piezoelectric el-ements drift that in many instances need to be corrected manually, in a frame-by-frame fashion. Therefore, automated image treatment and analysis iscurrently one of the biggest challenges in this field. The normalized cross-correlation (NCC) is regarded as the gold standard method for image match-ing, and therefore is currently the most adopted method for X- and Y-driftcompensation in time-lapse imaging series. However, NCC is computationallyintense and not very robust when dealing with noisy experimental data. Infact, the occurrence of false matches and the computational cost are so highthat in many instances a manual rough alignment needs to be performedbefore NCC is attempted. While in previous studies template matching wasbased on NCC of 2-D images, here we propose a template matching algorithmusing data projection and decomposition in 1-D. Our results shown that theproposed algorithm is more efficient and outperforms NCC in synthetic,experimental, and ‘‘real world’’ situations. It corrects temporal drift intime-lapse microscopy data in diverse imaging conditions and thus improvesingle-molecule quantification of dynamics, structure, and function. Finally,our method is also expected to generalize to many other imaging applications(e.g. medical imaging) that use NCC template matching to find imagecorrespondences.

1896-PlatAn Electromagnetic Tweezers for Studying Fast Protein Folding DynamicsRafael Tapia-Rojo, Jaime Andres RIvas-Pardo, Julio M. Fernandez.Columbia University, New York, NY, USA.Protein folding under force does mechanical work, however, for that work tobe physiologically relevant, it must be delivered fast enough to compete withthe speed of molecular motors. For instance, it is not known if titin domainscan fold fast enough to follow the myosin motors, thus contributing to the me-chanical power during muscle contraction. Accurate measurement of theinitial folding velocity requires applying fast force changes in order to resolvethe initial folding events. Yet, most force-spectroscopy instruments change theforce by moving mechanical components, introducing a time lag-at least 100ms in the case of magnetic tweezers-where these early molecular events arelost. Here, we present a newly developed electromagnetic tweezers instru-ment, where the magnetic field is generated by an electromagnetic head.The force acting on the superparamagnetic beads depends on the electric cur-rent flowing through the head, and can therefore be changed as fast as currentcan be supplied. Based on Karlqvist approximation of the field created byan electromagnetic head, we work out a closed analytical expression that re-lates the force on the superparamagnetic beads with the electric current andthe vertical distance of the head to the bead. We calibrate our instrumentby relating this force expression with the step-sizes of protein L unfolding/re-folding events, allowing to reach forces of 50 pN in 5 ms, when working at adistance of 200 mm and an electric current of 600 mA. This force range canpotentially be increased up to 500 pN by using thinner fluid chambers, ap-proaching the AFM force range, but with the exquisite stability of magnetictweezers. Our instrument development offers a unique tool for studyinghow proteins respond mechanically to force perturbations as those experi-enced in vivo.

1897-PlatDirect and Indirect Magnetic Force Microscopy in HistologyGunjan Agarwal1, Brooke Ollander1, Joshua Sifford1, Kevin J. Walsh1,Angela R. Blissett1, Ping Wei2, Dana M. McTigue2.1Biomedical Engineering, Ohio State University, Columbus, OH, USA,2Neuroscience, Ohio State University, Columbus, OH, USA.Iron (Fe) is an essential metal involved in a wide spectrum of physiologicalfunctions. Sub-cellular characterization of the size, composition, and distribu-tion of ferritin(iron) can provide valuable information on iron storage and trans-port in health and disease. While histochemical stains (e.g. Perls stain) canprovide a rapid evaluation of iron, their spatial resolution is limited. Ultra-structural characterization of the distribution and composition of iron depositscan be accomplished using transmission electron microscopy (TEM) basedtechniques. However, TEM involves stringent sample preparation protocols,which could alter the state of iron. Magnetism-based microscopy can serveas an attractive approach to characterize iron deposits in situ in biological tissuesections and serve as a bridge between histochemical staining, TEM studies andMRI analysis. In this study we employ the scanning probe based technique,namely magnetic force microscopy (MFM) to map sub-micron iron depositsin tissue sections. We demonstrate howMFM can identify ferritin(iron) rich ly-sosomes in tissue sections compatible with histochemical staining. Quantitativeanalysis of MFM data can reveal differences in lysosomal ferritin(iron) content.

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We further present a novel indirect MFM technique, which preserves the highspatial resolution and sensitivity of the conventional or direct MFM but enableshigh-throughput detection of magnetic domains. In indirect MFM an ultrathinmembrane separates the sample and the MFM probe, thus minimizing the effectof sample topography in MFM imaging. Samples prepared for indirect MFMare compatible with multimodal imaging using light and electron microscopy.We thus elucidate the potential of direct and indirect MFM in analyzing ironcontent in histology.

1898-PlatLive Cell STED-AFM Analysis Correlates Cytoskeletal Structure Remod-elling and Membrane Physical Properties during Polarized Migration inAstrocytesNathan Curry1, Gregory Ghezali1,2, Gabriele S. Kaminski Schierle1,Nathalie Rouach1,2, Clemens Kaminski1.1Chemical Engineering and Biotechnology, University of Cambridge,Cambridge, United Kingdom, 2Center for Interdisciplinary Research inBiology, College de France, Paris, France.The establishment of cell polarity, migration and adhesion is a complex inter-play between cytoskeletal organisation and cell physical properties. Measure-ment of cell physical properties, such as mechanical stiffness, isconventionally performed by scanning probe microscopy – such as atomicforce microscopy (AFM). AFM does not allow the specific imaging of subcel-lular cytoskeletal elements. To overcome this live cell mechanical propertymaps (AFM) are correlated with STED super-resolution images of the cytoskel-eton by mounting the AFM on the optical microscope. Live cell organic dyes(SiR-actin/tubulin) label cytoskeletal elements allowing STED imaging.STED images are correlated with AFM topography and force maps.This technique is used to investigate polarised migration [1].A scratch assay isused to induce polarised migration in astrocytes and it is found that both theactin and tubulin cytoskeleton rearrange during migration. In the leadingedge of migration an increase in actin stress fibers is observed. This corre-sponds to an increase in astrocyte stiffness compared to the basis. Expressionof the gap junction protein connexin30, which is known to colocalise with actinand inhibit migration, is found to reduce stress fiber formation and astrocytestiffness in the leading edge.Overall combining STED and AFM allows investigations into the relation ofchanges in cell topography to functional mechanical properties with subcellularresolution and will be important when investigating cell migration, adhesionand division in physiological and pathological conditions.[1] Curry N, et al., Frontiers in Cellular Neurscience, 11:104 (2017)

1899-PlatMoving Beyond the Mechanical Clamp: An Exploration into DifferentialMechanical Stability of Ubiquitin Family ProteinsMona Gupta, Ravindra Venkatramani, Sri Rama Koti Ainavarapu.Department of Chemical Sciences, Tata Institute of Fundamental Research,Mumbai, India.Mechanical properties of proteins with a b-grasp/b-sandwich fold topologyhave been studied extensively by both single-molecule force spectroscopyexperiments and steered molecular dynamics simulations (SMD), owing totheir endurance towards high rupture forces. Previous studies have shownthat the backbone hydrogen bonding network holding the terminal b-strands(b-clamp) in these proteins contributes prominently to their mechanical sta-bility. However, the role of interactions beyond the b-clamp in providing me-chanical resistance is not well understood. Ubiquitin family proteins, whichshare a common b-grasp fold including a b-clamp, exhibit distinct mechan-ical stability. The low sequence homology shared by these proteins implies adifferential interaction network constituting the protein fold and this couldserve as a paradigm to explore the role of collective structural contributionstowards mechanical stability of a protein. Here, we explore the structuralorigin behind differences in the mechanical properties of three members ofthe ubiquitin family (ubiquitin, SUMO1 and SUMO2). SMD simulationshave been employed to reveal a high resolution atomistic view of proteinstructural changes as they unravel under the influence of a mechanical forceapplied along their termini. The simulations corroborate experimentallyobserved differences in mechanical stability of these three proteins. Wemonitor collective structural changes to reveal the major secondary structuralelements that contribute to their mechanical stability. Differential couplingbetween various b-strands and the a-helix results in differences in themechanical stability of the ubiquitin family proteins. Our study broadensthe outlook of mechanical stability in proteins with a b-grasp fold to gobeyond the ‘b-clamp’, hitherto considered as the sole contributor to theirmechanical stability. Our study highlights the coupling of b-strands with

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a-helix as the next major contributor to mechanical stability of proteins ofubiquitin fold.

1900-PlatDeconstructing the Single Molecule Mechanics of an Ultrastable PathogenAdhesinLukas F. Milles1, Rafael C. Bernardi2, Klaus Schulten2, Hermann E. Gaub1.1Chair of Applied Physics/Biophysics, LMU Munich, Munich, Germany,2Theoretical and Computational Biophysics Group, University of Illinois atUrbana-Champaign, Urbana, IL, USA.Pathogenic Staphylococci invade their human hosts by targeting adhesive ma-trix proteins. Withstanding high forces is a prerequisite to efficiently adhere to atarget under demanding mechanical conditions. Using AFM based single mole-cule force spectroscopy we investigate the mechanical stability of an archetypalstaphylococcal adhesin, which targets a short fibrinogen peptide. A single ad-hesin:peptide complex can withstand immense forces, well into the nN regime,approaching the strength of a covalent bond. All atom steered molecular dy-namics simulations supported by a number of mutants deciphered the molecu-lar mechanism of this highly unusual mechanical strength. Through minimizingthe target peptide, we make the nN regime routinely accessible in forcespectroscopy.

Platform: Protein Structure andConformation III

1901-PlatTime-Resolved Fluorescence Spectroscopy Captures Excited States of aMembrane Associated ProteinJakub Kubiak, Thomas Peulen, Claus A.M. Seidel.Molecular Physical Chemistry, Heinrich-Heine-Universit€at D€usseldorf,D€usseldorf, Germany.For a mechanistic understanding of the function of proteins it is imperativeto capture time-resolved structures. Fast dynamics is typically studied byMD or NMR, the techniques that have high temporal resolution and yieldper-residue information, but inadequate force fields, high energy barriersand low populated states render excited states invisible and prohibit a deepunderstanding of proteins in action. We demonstrate how fluorescence spec-troscopy captures of large scale conformational transitions and dynamics us-ing the g-aminobutyrate type A receptor-associated protein (GABARAP) asan example. Here, anisotropy decays of cystein-conjugated BodipyFL informon the local flexibility and the effect of charged residues on stability of sec-ondary structure. FRET spectroscopy on the other hand revealed importantstructural features of GABARAP in solution, not captured by X-ray crystal-lography and NMR derived models, with possible functional impact. Wemodeled possible conformational dynamics of GABARAP with FRET-guided MC simulations.

1902-PlatA Glimpse into the Sequence of Structural Changes in the Orange Carot-enoid Protein Which Switch on the Photoprotection Mechanism in Cyano-bacteriaSayan Gupta1, Maria A. Dominguez-Martin2, Han Bao2, Markus Sutter1,Jun Feng1, Leanne-Jade G. Chan1, Christopher J. Petzold1,Cheryl A. Kerfeld2, Corie Y. Ralston1.1Molecular Biology and Integrated Bioimaging, LBNL, Berkeley, CA, USA,2MSU-DOE Plant Research Laboratory, Michigan State University, EastLansing, MI, USA.Photoprotective mechanisms are key to the survival of photosynthetic organ-isms. In cyanobacteria, the Orange Carotenoid Protein (OCP) senses excesslight and binds to the light-harvesting antenna, triggering the dissipationof captured light energy. Using time-resolved X-ray footprinting with massspectrometry (XFMS), we traced the time-evolution of both the local andglobal structural changes in the OCP photoactivation and relaxation processes.Activation is accompanied by a fast decrease in the solvent accessibilityaround conserved carotenoid binding residues, and a slow increase inthe solvent accessibility at the inter-domain interface. Accessibility changesin the relaxation process follow a reverse trend and, in addition, passthrough a compact intermediate state. This study identified the driving factorsfor structural changes transmitted from the carotenoid binding pocketto the protein surface upon light activation of the OCP as well as theback-conversion process with unprecedented mechanistic detail. The physio-logical relevance of the kinetic mechanism is explained in terms of themode of interaction between the Fluorescence Recovery Protein (FRP) andthe OCP.

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1903-PlatRefining Peptide Conformational Landscape by Amide I Infrared Spec-troscopy and MD SimulationsChi-Jui Feng, Balamurugan Dhayalan, Xinxing Zhang, Andrei Tokmakoff.Unveristy of Chicago, Chicago, IL, USA.Amide I spectroscopy has growing capabilities of probing complex proteinstructures including intrinsically disordered regions. Amide I spectroscopicmaps allow predicting amide I spectra from simulated conformational distri-butions, providing a route to ensemble refinement against experiments. As aproof of principle study, we perform ensemble refinement of Ala–Ala–Ala(AAA) against experimental amide I spectra including site-specific isotope la-bels. By comparing experimental spectra with simulations drawn from multi-ple force fields and water models, we show that the spectra of AAA aresensitive to the underlying conformational distribution. Decomposing simu-lated ensemble spectra into individual conformers suggests that the conforma-tional distribution of AAA is dominated by ppII conformer, with a minorpopulation of b conformer. Also, Bayesian ensemble refinement allows usto provide quantitative descriptions of conformational ensemble consistentwith IR spectroscopy, which can be a potential tool for further force fielddevelopment. This ensemble refinement scheme is being developed to applyon more complicated system as human insulin, specifically the conformationalchanges of the insulin monomer during monomer association. The associationprocess involves folding of the disordered monomer B chains and formationof inter-monomer b-sheet, recognized as coupled-folding and bindingprocess. To understand such process, we utilize site-specific isotope labelsto study conformational ensembles of the insulin monomer and the dimer,with the focus on dissociation of the b-sheet, and disordering of the monomerB chain.

1904-PlatMagnetic Alignment of a Protein with Two Spin-Labels: 1 D 1 s 2?James M. Gruschus1, Madeleine Strickland1, Marie-Paule Strub1,Charles Schwieters2, Nico Tjandra1.1NHLBI, National Institutes of Health, Bethesda, MD, USA, 2CIT, NationalInstitutes of Health, Bethesda, MD, USA.The use of weak alignment in solution-state NMR to yield residual dipolarcoupling (RDC) information has been one of the most important advances inthe field, providing more accurate structure determination and deeper insightsinto long-range motions and ensemble populations in biomolecules. Becausethe net alignments are weak, typically differing only 0.1-1% from isotropic mo-lecular tumbling, alignments brought about by multiple, simultaneous meansmight be assumed to have simple additive effects, that is, higher order contri-butions could be negligible. Here, we explore this assumption using the proteinubiquitin labeled with one or two lanthanide-containing compounds via ligationto surface cysteine residues. The magnetic anisotropy of the unpaired electronsof the lanthanide causes it to have a preferred orientation in the magnetic field,causing the protein to weakly align, giving rise to RDCs as well as pseudo-contact shifts (PCSs) in the NMR spectra. Comparing the alignment tensorsand PCSs for the two singly labeled proteins with the doubly labeled protein,significant deviations from the expected result are seen; the results for thedoubly labeled protein are not simply the sum of results for the two singlylabeled proteins. Energy minimization incorporating PCS-derived terms re-veals subtle changes in the average positions of the lanthanide atoms. In sum-mary, one should not assume that higher order terms for weakly alignedproteins are always negligible.

1905-PlatProtein Yoga: Conformational Flexibility of a Novel FoldAnne R. Kaplan.Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA.Bacillus cereus is an emerging human pathogen which has been shown tocause food poisoning, and life-threatening infections. In addition to multiplevirulence factors, B. cereus secretes the pore-forming toxin hemolysin II(HlyII), which has a unique 94 amino acid C-terminal domain (HlyIIC) thatshows no sequence or structural homology to any known proteins. HlyIICexhibits splitting of NMR resonances due to cis/trans isomerization of asingle proline near the C-terminus. To overcome heterogeneity, we solvedthe structure of P405M-HlyIIC, a mutant that exclusively stabilizes the transstate. The NMR structure of HlyIIC reveals a novel fold, consisting of twosubdomains aA-b1-b2 and b3-b4-aB-b5 which come together in a barrel-like structure held together through hydrophobic contacts. At the bottom ofthe barrel opposite the N-terminal linker connection to the HlyII-core is apositively charged patch that could potentially bind negatively charged sur-face moieties on cellular membranes for target-cell surface recognition, or

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to stabilize the heptameric pore complex. In the WT domain, the N-terminallinker and first a-helix show dynamic flexibility. In the destabilizing P405Mmutant, increased dynamics are evident throughout the first subdomain, sug-gesting that the HlyIIC structure may have arisen through gene fusion. TheHlyIIC domain exhibits unusually high conformational promiscuity such ascis/trans isomerization due to Pro405 in WT-HlyIIC, and a temperature andconcentration dependent monomer-dimer equilibrium in p405m-HlyIIC. Weare working on characterizing of all four states of this unusually flexibledomain.

1906-PlatStrucutural Basis of Reversible Amyloid-Like Interaction in MediatinghnRNP A1 Phase SeparationXinrui Gui1, Feng Luo1, Dan Li2, Cong Liu1.1Interdisciplinary Research Center on Biolocy and Chemistry, ChineseAcademy of Sciences, Shanghai, China, 2Bio-x, Shanghai JiaotongUniversity, Shanghai, China.Many RNA-binding proteins can undergo phase separation via their lowcomplexity (LC) domains to drive membraneless compartments formation.Some of them (e.g. FUS, hnRNPA1) form amyloid aggregates under patho-logical circumstances associated with neurodegenerative diseases such asALS and FTD. Whether the amyloid formation contributes to the assemblyof membraneless compartments has long been controversial. Here, we foundthat hnRNP A1 amyloid fibrils are highly reversible and encapsulated in theliquid-like droplets. We further identified three reversible-amyloid-formingcores (RACs) of hnRNP A1 that can form reversible fibrils/hydrogels similarto that of the full-length hnRNP A1. By using micro-electron diffraction, wedetermined the crystal structure of RAC1 at a resolution of 1 A. The structureof RAC1 reveals two features that determine the reversibility of fibrils/hydro-gels: hydrophilic zipper interface and p-p interactions via a kink structure.Moreover, we show that deletion, insertion or mutation of the three individualRAC significantly influences the phase separation of full-length hnRNP A1 invitro and the stress granule formation in vivo. Our work reveals the structuralbasis of reversible amyloid fibrils and highlights their important roles inhnRNP A1-mediated phase separation under physiological and pathologicalconditions.

1907-PlatConformational Dynamics of Human Prion Protein and Binding Sites ofZn CationsMaciej Gielnik1, Micha1 Nowakowski2, Micha1 Taube1, Igor Zhukov3,4,Wojciech M. Kwiatek2, Dmitry M. Lesovoy5, Maciej Kozak1,6.1Department of Macromolecular Physics, A. Mickiewicz University, Pozna�n,Poland, 2Institute of Nuclear Physics, Polish Academy of Sciences, Krakow,Poland, 3Institute of Biochemistry and Biophysics Polish Academy ofSciences, Warsaw, Poland, 4NanoBioMedical Centre, Pozna�n, Poland,5Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow,Russian Federation, 6Joint Laboratory for SAXS studies, A. MickiewiczUniversity, Pozna�n, Poland.Conformational dynamics within the unstructured N-terminal domain of hu-man prion protein (huPrP) as well as in the structurally ordered C-terminaldomain of this protein are closely related to the ability of this protein to un-dergo pathological conformational changes and form insoluble deposits in thebrain, which were observed in Creutzfeldt-Jakob disease (CJD) [1]. Theimportant feature of this protein, which also could be associated with patho-logical changes in PrP, is its ability to bind divalent metal ions (copper, zinc)as there are four binding sites (tandem octarepeats) within the N-terminal partof the prion protein molecule [2]. The present study was aimed at determina-tion of the conformational dynamics of cellular form of huPrP (23-231) atdifferent pH values and the presence of zinc ions (at pH 5.4). Relaxation pa-rameters (relaxation times of methyl groups within the polypeptide chain)characterizing this protein at different pH conditions (in neutral and mildacidic environment) were obtained on the basis of NMR and supported bythe Ensemble Optimization Method analysis using the previously recordedSAXS data. On the other hand, X-ray absorption spectroscopy (XAS)based on synchrotron radiation provided the information on the localstructure of zinc ion binding sites in various stoichiometries. Significant dif-ferences in huPrP-Zn(II) spectra related to changes in Zn concentrationwere observed. This work was supported by the funds from the National Sci-ence Centre (Poland) granted on the basis of decision no. 2014/15/B/ST4/04839. [1] Cobb N.J., Surewicz W.K. (2009) Biochemistry. 48(12), 2574-2585. [2] Walter, E. D., Stevens, D. J., Spevacek, A. R., Visconte, M. P.,Rossi, A. D., Millhauser, G. L. (2009). Current Protein & Peptide Science,10(5), 529-535.

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1908-PlatA Water-Soluble DSBB Variant that Catalyzes Disulfide-Bond FormationIn VivoDario Mizrachi1, Matthew DeLisa2.1Physiology and Developmental Biology, Brigham Young University, Provo,UT, USA, 2Cornell University, Ithaca, NY, USA.Escherichia coli DsbB is a transmembrane enzyme that catalyzes the reoxida-tion of the periplasmic oxidase DsbA by ubiquinone. Here, we soughtto convert membrane-bound DsbB into a water-soluble biocatalyst byleveraging a previously described method for in vivo solubilization of integralmembrane proteins (IMPs). When solubilized DsbB variants were coex-pressed with an export-defective copy of DsbA in the cytoplasm ofwild-type E. coli cells, artificial oxidation pathways were created that effi-ciently catalyzed de novo disulfide-bond formation in a range of substrate pro-teins, in a manner dependent on both DsbA and quinone. Hence, DsbBsolubilization was achieved with preservation of both catalytic activity andsubstrate specificity. Moreover, given the generality of the solubilization tech-nique, the results presented here should pave the way to unlocking the bio-catalytic potential of other membrane-bound enzymes whose utility hasbeen limited by poor stability of IMPs outside of their native lipid-bilayercontext.

Symposium: Modeling and Probing theCytoskeleton

1909-SympComputational Models of Individual and Collective Keratocyte MigrationAlex Mogilner.New York University, New York, NY, USA.Cell migration is a fundamental cell biological phenomenon that underliesmany physiological processes. To migrate, cells must polarize, sense adirection, and deploy a motile mechanical machinery. Computational modelingof simple motile fish keratocyte cells was an integral part of understandinggeneral mechanochemical mechanisms of these three aspects of cellmigration. I will demonstrate how a combination of data analysis anddetailed mechanistic modeling elucidated mechanics of self-polarization,steady migration and turning of single keratocytes. I will then describedata on directional migration of individual cells and cohesive groups ofcells in an electric field and show that a simple conceptual model shedslight on surprising properties of the directional sensing in collective cellmigration.

1910-SympHow Actin Polymerization Bends the Cell Membrane to Drive EndocytosisAnders E. Carlsson.Physics, Washington University, St. Louis, MO, USA.Polymerization of actin facilitates a broad range of membrane-bending phe-nomena in cells, including filopodial and lamellipodial protrusion, phagocy-tosis, and endocytosis. To demonstrate how actin polymerization generatespulling forces to drive endocytosis, we study a three-dimensional mechano-chemical model of clathrin-mediated endocytosis in yeast (CME) that treatsboth the distribution of forces and the endocytic protein dynamics. CME isdriven by complex of proteins appearing in a well-defined sequence, including5000-1000 copies of actin and much smaller numbers of other proteins,including nucleation-promoting factors (NPFs) that generate new actinfilaments. The key ingredients of the model are an NPF ring, an actin hemi-sphere that polymerizes near the NPF ring, and a negative-feedback interac-tion between polymerized actin and the NPFs. The actin network is grownexplicitly in three dimensions. The parameters in the model are fitted toknown properties of endocytosis in yeast. The actin network generates acombination of pushing forces in the outer region near the ring, and pullingforces inside the ring. The pulling forces result from retrograde flow in theouter, polymerizing regions of the actin hemisphere, which pull the innerpart of the network into the cell. This model explains several observationsin the literature, including a ‘‘comet-tail’’ phenotype resulting from deletionof proteins attaching polymerized actin the the membrane. It predicts thatthe NPF count will be increased by mutations that reduce NPF activity.This prediction is confirmed by quantitative fluorescence microscopymeasurements of protein counts in wild-type and mutant cells. It alsopredicts that even large reductions in NPF activity have relatively small im-pacts on the total amount of polymerized actin. This homeostatic mechanismcould be useful in stabilizing the the process against internal or external per-turbations.

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1911-SympA Minimal System for Microtubule-Based Cell PolarityMarileen Dogterom.Bionanoscience, Delft University of Technology, Delft, Netherlands.Establishment of cell polarity is essential for processes such as growth anddivision. In fission yeast, polarity factors travel at the tips of microtubulesto the cell ends where they associate with the membrane and subsequentlymaintain a polarized distribution. While many molecular components havebeen shown to play a role in this polarization process, it remains unknownwhich molecular functionalities are minimally required. We show that achimera protein that combines a membrane-binding motif with microtubuletip affinity is able to transiently concentrate at cell ends in wild type fissionyeast cells. However, unlike natural polarity factors, these chimera proteinsloose their polarized distribution as soon as microtubules disappear. In paral-lel, we established an in vitro system that allows us to verify the minimal re-quirements for microtubule-based cell polarity. We use micro-fabricationtechniques combined with surface functionalization to create rigid chamberswith affinity for proteins, and microfluidic techniques to create elongatedemulsion droplets with functionalized lipid boundaries. We demonstrate thatproteins that accumulate at the ends of growing microtubules can be trans-ferred to the boundaries of these confined spaces. Interestingly, only proteinsthat travel in clusters at microtubule ends remain associated with these bound-aries after microtubules disappear. This hints to a role of microtubule-endinduced protein-protein interactions in the establishment of robust cell polaritypatterns.

1912-SympTorques and Forces in the Mitotic SpindleKruno Vukusic1, Renata Buda1, Juraj Simunic1, Bruno Polak1, Maja Novak2,Zvonimir Boban2, Nenad Pavin2, Iva M. Toli�c1.1Ruder Boskovic Institute, Zagreb, Croatia, 2Department of Physics,University of Zagreb, Zagreb, Croatia.During cell division, microtubules of the mitotic spindle segregate chromo-somes by exerting forces on kinetochores, protein complexes on the chromo-somes. The central question is what forces drive chromosome segregation.The current model for anaphase in human cells includes shortening of kinet-ochore fibers and separation of spindle poles. Both processes require kineto-chores to be linked with the poles. By combining laser ablation,photoactivation and theoretical modeling, we show that kinetochores canseparate without any attachment to one spindle pole (Vukusi�c et al., DevCell 2017). This separation requires the bridging fiber, which connects sisterkinetochore fibers (Kajtez et al., Nat Commun 2016). Bridging microtubulesin intact spindles slide apart together with kinetochore fibers, indicating strongcrosslinks between them. Thus, sliding of microtubules in the bridging fibersdrives pole separation and pushes kinetochore fibers apart to segregatechromosomes.In addition to forces, torques may also exist in the spindle, yet they have notbeen investigated. We show that the spindle is chiral, based on our finding thatmicrotubule bundles follow a left-handed helical path, which cannot be ex-plained by forces but rather by torques acting in the bundles (Novak et al.,bioRxiv 167437). STED super-resolution microscopy, as well as confocal mi-croscopy, of human spindles shows that the bundles have complex curvedshapes. The average helicity of the bundles with respect to the spindleaxis is 1.5 degrees/mm. To explain the observed shapes, we introduce a theo-retical model for the balance of forces and torques acting in the spindle, andshow that torque is required to generate the helical shapes. We conclude thattorques, in addition to forces, exist in the spindle and determine itsarchitecture.

Symposium: Protein Dynamics, Folding, andAllostery I: How Do Proteins Fold and Misfold?

1913-SympPrinciples of Protein Structural Ensemble DeterminationMichele Vendruscolo.Department of Chemistry, University of Cambridge, Cambridge, UnitedKingdom.The biological functions of protein molecules are intimately dependent on theirconformational dynamics. This aspect is particularly evident for disorderedproteins, which constitute perhaps one-third of the human proteome. Therefore,structural ensembles often offer more useful representations of proteins than in-dividual conformations. Here, we describe how the well-established principlesof protein structure determination should be extended to the case of protein

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structural ensembles determination. These principles concern primarily how todeal with conformationally heterogeneous states, and with experimental mea-surements that are averaged over such states and affected by a variety of errors.We first review the growing literature of recent methods that combine experi-mental and computational information to model structural ensembles, high-lighting their similarities and differences. We then address some conceptualproblems in the determination of structural ensembles and define future goalstowards the establishment of objective criteria for the comparison, validation,visualization and dissemination of such ensembles.References[1] M. Bonomi, G. T. Heller, C. Camilloni and M. Vendruscolo. Principles ofprotein structural ensemble determination. Curr. Op. Struct. Biol. 42, 106-116(2017).[2] P. Sormanni, D. Piovesan, G. T. Heller, M. Bonomi, P. Kukic, C. Camilloni,M. Fuxreiter, Z. Dosztanyi, R. Pappu, M. M. Babu, S. Longhi, P. Tompa, A. K.Dunker, V. N. Uversky, S. C. E. Tosatto and M. Vendruscolo. Simultaneousquantification of order and disorder in proteins.Nat. Chem. Biol. 13, 339-342(2017)

1914-SympEvolutionary Couplings Reveal Alternative 3D StructuresDebora Marks.Systems Biology, Harvard University, Boston, MA, USA.The evolutionary trajectories of biological sequences are propelled by muta-tion and whittled away by selection to maintain function. In this talk Iwill computational methods that, when combined with recent growth insequence databases, quantify evolutionary constraints in terms of evolutionarycouplings between residues. We develop methods that not only predict accu-rate 3D structures of proteins, RNA and complexes but also their conforma-tional plasticity including alternative 3D states, and structural informationon ‘disordered’ proteins, propensity to form fibrils and polymerization Iwill introduce challenges and opportunities for extending these methods,including deep learning, to applications in molecular design and diseaseprediction.

1915-SympProtein Sequence Coevolution, Energy Landscapes and their Connectionsto Protein Structure, Folding and FunctionJose N. Onuchic1, Faruck Morcos2.1Center for Theoretical Biological Physics, Rice University, Houston, TX,USA, 2Department of Biological Sciences, University of Texas at Dallas,Dallas, TX, USA.Energy landscape theory has been a powerful approach to study protein foldingdynamics and function. The discovery that an accurate estimate of the jointprobability distribution of amino acid occupancies in protein families providesinsights about residue-residue coevolution and concrete details about proteinfolding landscapes and therefore has also advanced structural biophysics.Our realization that the collection of couplings and local fields as parametersof such distribution is inherently connected with the thermodynamics ofsequence selection towards folding and function demonstrates the importanceof coevolutionary methods to understand stability and function of biomole-cules. The synergy between structure based models and coevolutionary infor-mation has spearheaded the field of structure prediction, including proteinand RNA, as well as accelerating the discovery of functional structural statesand the prediction of protein complexes. Coevolution signals can also beused to create protein recognition metrics, which led to successful experimentalefforts, and the uncovering of novel molecular interactions. This idea hasopened the door to encode recognition in protein pairs. Coevolved interfacescan also be combined with small molecule hot spot estimation methods toimprove the discovery of druggable interfaces. Finally, we show that biophys-ical modeling via statistical inference approaches can shed light on structuralproperties of chromosomes. We demonstrate that it is possible to reconstructwith fidelity experimental maps of interacting genomic regions for particularchromosomal types by learning only generic properties of single chromosomes.We provide evidence of the depth and coverage of energy landscape theory ap-proaches linked with coevolutionary methods and their impact in our goal tounderstand biological function at the molecular level.

1916-SympThe Role of Ubiquitin in Chromatin Structural OrganizationGalia T. Debelouchina1,2.1University of California, San Diego, La Jolla, CA, USA, 2Department ofChemistry, Princeton University, Princeton, NJ, USA.In the nucleus, actively transcribed chromatin is separated into euchromatincompartments, while inactive, more compact regions can be found inside denseheterochromatic neighborhoods. The molecular basis for this separation is still

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not well understood but likely involves the interplay of transcription factors,chromatin architectural proteins, RNA and histone post-translational modifica-tions that work in a complex way to regulate access to genetic information inthe cell. In this presentation, I will focus on the role of histone mono-ubiquitination, a post-translational modification that can impact local andhigher order chromatin organization. I will contrast the functional and struc-tural impacts of histone H2B and H2A ubiquitination, and discuss the role ofubiquitin-histone electrostatic interactions in chromatin structure regulationand organization.

Platform: Optical Spectroscopy

1917-PlatHigh-throughput Rotation Tracking using DNA Origami RotorsPallav Kosuri1, Benjamin Altheimer1, Mingjie Dai2, Peng Yin2,Xiaowei Zhuang1.1Chemistry and Chemical Biology, Harvard University, Cambridge, MA,USA, 2Harvard University, Cambridge, MA, USA.We have invented a high-throughput single molecule method that enablesprecise rotational tracking of DNA with millisecond time resolution.ORBIT (Origami Rotational Beacon Image Tracking) uses fluorescentlylabeled nanoscale rotors to amplify the rotation of a DNA duplex of interest.Our method does not rely on an externally applied force but insteadachieves high spatiotemporal resolution due to the low drag of the origamirotor and the stiffness of the short DNA duplex. We used ORBIT tostudy the helicase activity of RecBCD and directly observed the sequenceof events during initiation at a double stranded break. Based on our resultswe propose a model in which the RecB subunit acts as a starter motor thatinitiates processive motion. The simplicity and customizable nature of ourmethod should enable high-resolution studies of a wide range of motorproteins.

1918-PlatHigh Speed Mechanical Measurements Based on DNA Origami TorqueSensorsDominik J. Kauert, Ralf Seidel.Peter Debye Institute for Soft Matter Physics, Leipzig University, Leipzig,Germany.Measurements of force and torque on single DNA molecules are widelyused to study the mechanical properties and the dynamics of DNA.While considerable advancements have been made to improve the spatio-temporal resolution in force-based measurements, fast twist and torquemeasurements remain challenging. Part of the problem arises hereby fromthe mechanical flexibility of DNA, which limits the accuracy of suchmeasurements.Here, we use rigid DNA origami nanostructures in magnetic tweezers-basedDNA twist and torque measurements to overcome previous limitations. DNAorigami is a technique that allows to fold DNA into three-dimensional objectsof a desired shape. We designed and fabricated an origami twist sensor consist-ing of a multihelical DNA stem and a DNA lever at which a gold nanoparticle isattached 50 nm off the rotation axis. DNA twist changes are seen as angulardisplacements of the gold nanoparticle. These are obtained by tracking the po-sition of the particle at kHz frequencies using light scattering. Both, DNA twistas well as DNA length changes can be monitored simultaneously in a fullycorrelated fashion thus enabling measurements of DNA structural changes athigh temporal resolution.

1919-PlatCharacterization of the Interaction of Liposomes and Gold Nanoparticlesusing Surface Enhanced Raman ScatteringVesna Zivanovic1,2, Christoph Arenz1,2, Janina Kneipp1,2.1School of Analytical Sciences Adlershof (SALSA), Humboldt-Universit€atzu Berlin, Berlin, Germany, 2Department of Chemistry, Humboldt-Universit€at zu Berlin, Berlin, Germany.Liposomes are important supramolecular structures with a wide range of ap-plications in biophysics and medicine, e.g., as carriers for therapeutic agents.In this context, the combination of liposomes with gold nanoparticles can pro-vide new composite systems for drug delivery. Here, we aim at the character-ization of potential liposomal carriers using surface enhanced Ramanscattering (SERS). SERS can provide detailed spectroscopic informationabout the structure and interaction of liposomes with gold nanoparticles.We investigated the influence of the liposomal composition on the interactionwith gold nanoparticles. The SERS data indicate that the interaction with thenanoparticles depends on the charge of the liposomes. As a potential applica-tion, the encapsulation of the tricyclic antidepressant desipramine and its

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interaction with the liposomes and nanoparticles are studied. This moleculewas shown to have an influence on lipid metabolism. The spectral signatureof desipramine is very stable and reveals that desipramine interacts with thegold nanostructures via the methyl-aminopropyl side chain and with partsof its ring system.1 In complementary experiments, the structure of the lipo-somal carriers was investigated using transmission electron microscopy. Thepossibilities of SERS to follow biochemical and physiological changes inthe endosomal system at the single cell level2 can in principle be used tomonitor the uptake of the liposomal carriers and of therapeutic effects ofdrugs on cells.1. Zivanovic V. et al. J. Phys. Chem. C, 2017, DOI:10.1021/acs.jpcc.7b080262. Kneipp J., ACS Nano, 2017, 2017, 11, 1136-1141

1920-PlatNon-Destructive Prediction of Transcriptomes from Single-Cell RamanMicroscopyKoseki J. Kobayashi-Kirschvink1, Hidenori Nakaoka1,Ken-ichiro F. Kamei1, Arisa Oda1, Kazuki Nosho1, Hiroko Fukushima1,Yu Kanesaki2, Shunsuke Yajima2, Haruhiko Masaki1, Kunihiro Ohta1,Yuichi Wakamoto1.1The University of Tokyo, Tokyo, Japan, 2Tokyo University of Agriculture,Tokyo, Japan.Raman micro-spectroscopy is a non-destructive imaging technique that canpotentially monitor whole-cell molecular compositions in vivo. However, dueto the complex molecular compositions of cells, it remains unknown whethercellular Raman spectra permit comprehensive quantification of biomoleculesin a useful fashion. To test this, we compared fingerprint Raman spectra inthe 700-1800 cm�1 fingerprint region with concurrently measured RNA-seqtranscriptomic data of Schizosaccharomyces pombe and Escherichia coligrown under a variety of culture conditions aimed at activating different por-tions of the transcriptome. Using partial least squares regression and machinelearning algorithms, we find that most of the variance of transcriptomes canbe represented by a small set of eigenvectors, and linearly linked with thechanges of Raman spectra. The estimated transformation parameters, andlow-dimensionality of transcriptomes, allowed us to predict reliably the expres-sion profiles of thousands of transcripts from Raman spectra. Interestingly, in S.pombe, ncRNAs contributed to the Raman-transcriptome linearity more signif-icantly than mRNAs, which supports their role in coordinating cellular molec-ular compositions. These results show that whole-cell Raman spectra canunravel cellular omics information in a non-destructive manner, and opensthe possibility of conducting live-cell omics.

1921-PlatMeasuring Structural Changes as a Function of Protein Environmentusing Infrared SpectroscopyCurtis W. Meuse1, Marco A. Blanco2.1Institute for Bioscience and Biotechnology Reseach and the BiomolecularMeasurement Division, NIST, Rockville, MD, USA, 2Institue for Bioscienceand Biotechnology Research and Biomolecular Measurement Division,NIST, Rockville, MD, USA.The formulation of biopharmaceutical protein drugs is based on the ability ofphysiochemical measurement techniques to characterize the structure, stabilityand dynamics of proteins in different chemical environments. The need toformulate these drugs at high concentrations and use them at much lower con-centrations makes it important to have techniques that can compare propertiesbetween different concentrations. Infrared spectroscopy has long been used todeduce concentration and structural descriptions of proteins. We are devel-oping new infrared methods to compare the structures, dynamics and functionsof nearly identical biopharmaceutical protein samples. We will present our re-sults describing structural changes in model polymers, proteins and the NISTreference monoclonal antibody across different pH, ionic strength and concen-tration conditions.

1922-PlatComprehensive Multivariate Analysis of Red Wine Phenolic Composition,Color and Quality Components With Simultaneous Absorbance and Fluo-rescence Excitation Emission MappingAdam Gilmore.Horiba Instruments Inc., Edison, NJ, USA.The color and phenolic composition of fermented red wines and unfermentedgrape juice are linked to a variety of quality components including theobvious visual, taste and ‘mouth-feel’ aesthetics. The phenolic compositionis a strong indicator of fruit ripeness and can be used to adjust harvestingpractices especially during the veraison; notably the phenolic compositioncan be followed throughout the wine making process to establish quality

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control and product variation guidelines. Conventional phenolic analysiecan be time- and labor intensive with discriminatory analyses usuallyrequiring HPLC or LC-MS instrumentation. Color analyses are typically car-ried out with conventional UV-VIS spectrophotometry. This study outlinesnew methodology for calibrating and validating phenolic analysis using apatented simultaneous absorbance and fluorescence excitation-emission map-ping (EEM) instrument. Compared to conventional column chromatography,the purely optical calibrated EEM method can provide comprehensivephenolic profiles in a few minutes while concomitantly providing all theinformation needed for International Commission on Illumination (CIE)tristimulus analyses as well as Hue and Intensity. The study compares theeffectiveness and pros and cons of different multivariate analysis techniquesof the EEM data, including Principal Components Analysis (PCA), ParallelFactor Analysis (PARAFAC) and Classical Least Squares (CLS). The com-parison is evaluated with respect to characterizing and classifying samplesas a function of ripening, varietal and process related parameters as well asto possibly screen for adulterants and storage issues such as oxidation ormicrobial spoilage. This study exemplifes the analysis of several varietiesof grape juice and fermented wine samples to illustrate the effectivenessof measuring common phenolic compounds as well as to show the effective-ness of the fingerprinting capabilities for several possible QC relatedmeasurements.

1923-PlatSelf-consistent Analysis of Large Fluorescence Data Sets for IntegrativeTime-Resolved Models of BiomoleculesThomas-Otavio Peulen, Hemmen Katherina, Claus A.M. Seidel.Physical Chemistry II, Heinrich Heine University, D€usseldorf, Germany.For a mechanistic understanding of large biomolecular molecular complexes itis imperative to (1) characterize individual components by molecular modelsincluding excited and transiently populated states, (2) monitor the time-evolution of the complexes performing their associated biological function,and (3) study the biological assembly in living cells.Here, methods and tools for the self-consistent analysis of large fluorescencedatasets for integrative molecular models of are presented to (a) generate mo-lecular models in a dynamic equilibrium, (b) describe time-ordered processes,e.g., oligomerization processes, and (c) characterize large molecular assembliesin living cells. An extendable Bayesian framework integrates these methods forensemble, single-molecule, and fluorescence image spectroscopy data.Forward modeling the multiple fluorescence intensity decays of a network ofFRET pairs resolves sample heterogeneities by coarse-grained structuralmodels. The framework, which considers the dye mobilities, their local envi-ronment, and FRET, is applied to a network of FRET pairs using T4 lysozymeas a test case. Moreover, this approach is validated by synthetic data and (a)promises structural models with Angstrom resolution, (b) monitors the oligo-merization in time-resolved experiments, and (c) recovers equilibrium con-stants and structural features from imaging data.

1924-PlatEndogenous Alpha-Synuclein Analysis using Single-Molecule Pull-DownAssayBenjamin Croop1, Goun Je2, Jialei Tang1, Yoon-Seong Kim2,Kyu Young Han1.1CREOL, University of Central Florida, Orlando, FL, USA, 2College ofMedicine, Burnett School of Biomedical Sciences, Orlando, FL, USA.Parkinson’s disease (PD) is the second most common neurodegenerativedisease, but molecular mechanisms underlying PD pathogenesis remain un-clear. Alpha-synuclein (a-SYN) and its oligomeric species are implicatedas a key player in Parkinson’s disease. Thus, the determination of a-SYNexpression levels and its oligomerization states is crucial to understand PD.However, it has been challenging to analyze them quantitatively becauseof the limited amount of specimens and the lack of techniques that are insen-sitive to the size and conformation of a-SYN oligomers. Here, we demon-strated quantitative analysis of endogenous a-SYN taken from postmortemhuman brain tissue using a single-molecule pull-down (SiMPull) assay. TheSiMPull assay probes immunoprecipitated proteins, which are then taggedwith a fluorescent molecule for imaging and detection. By utilizing in vivocrosslinking, we preserved the native oligomerization state of the a-SYN pro-teins. Our results showed that the tissue lysates from the substantia nigra of ahuman PD brain showed 3.3-fold higher number of a-SYN molecules and2.4-fold higher oligomeric population compared to a healthy brain. Ourtechnique is a powerful diagnostic tool for the detection of various neurode-generative diseases, such as Parkinson’s disease and Alzheimer’s disease, andcan be used to analyze various biospecimens not limited to post mortem braintissue.

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Platform: Membrane Dynamics and Fusion I

1925-PlatNanoscale Protein Interactions Determine the Mesoscale Dynamic Organi-sation of BiomembranesAnna L. Duncan1, Matthieu Chavent1,2, Patrice Rassam1, Jean H�elie1,3,Tyler Reddy1, Oliver Birkholz4, Dmitry Belyaev5, Ben Hambly5,Jacob Piehler6, Colin Kleanthous1, Mark S.P. Sansom1.1Department of Biochemistry, University of Oxford, Oxford, UnitedKingdom, 2IPBS, Universit�e de Toulouse, CNRS, Toulouse, France,3Semmle, Oxford, United Kingdom, 4Department of Biochemistry,University of Osnabr€uck, Osnabr€uck, Germany, 5Mathematical Institute,University of Oxford, Oxford, United Kingdom, 6University of Osnabr€uck,Osnabr€uck, United Kingdom.Recent advances in experimental biophysical techniques, such as super-resolution microscopy, have given new insights into the complex organizationof membrane proteins for length scales of hundreds of nanometers and time-scales of milliseconds. Likewise, progress has been made in term of compu-tational models, allowing the creation of biomembrane systems containingmolecular detail at the 100 nanometer lengthscale. Thus, it has recentlybeen shown that protein crowding underpins the turnover of bacterial outermembrane proteins, a process that is vital for the adaptation of certain bacteriato new environments1. Nevertheless, experiments as well as computationalmodels have been devoid of a full understanding of protein crowding inboth molecular detail and at experimentally observable time and length scales.I will describe how these clusters may be generated for the Outer MembraneProteins (OMPs) BtuB and OmpF using coarse- grain (CG) Molecular Dy-namics (MD) simulations. However, a mesoscale model is necessary to assessthe dynamic behaviour of these OMPs at the mesoscale (hundreds of nanome-ters and millisecond timescales) and to bridge MD simulations and singlemolecule fluorescence microscopy. From the dynamics and protein interac-tions observed in our CG-MD simulations, we generate such a mesoscalemodel, and are able to directly compare in vitro and in silico results by usingsingle molecule tracking analysis on both. Simulations using the mesoscalemodel reveal that bacterial outer membranes are comprised of protein clustersthat present a mesh of moving barriers that can act to confine newly insertedproteins into OMP ‘islands’. References 1. Rassam et al, Nature, 253, 333-6,2015

1926-PlatVolume and Surface Area Dynamics of Giant Unilamellar VesiclesMorgan Chabanon1, Wan-Chih Su2, Douglas L. Gettel3, James C.S. Ho4,Atul N. Parikh5, Padmini Rangamani1.1Mechanical and Aerospace Engineering, University of California San Diego,La Jolla, CA, USA, 2Department of Chemistry, University of CaliforniaDavis, Davis, CA, USA, 3Department of Chemical Engineering, Universityof California Davis, Davis, CA, USA, 4School of Materials Science andEngineering, Nanyang Technological University, Singapore, Singapore,5Departments of Chemistry, Chemical Engineering, Biomedical Engineering,and Materials Science, University of California Davis, Davis, CA, USA.Giant unilamellar vesicles (GUVs) have proven to be useful systems in thequest for artificially capturing the essential components of living cells. Herewe elucidate the mechanisms by which GUVs respond dynamically to twocommon environmental stressors: osmotic swelling by depletion of external os-molytes, and area reduction by exposure to surfactant. Strikingly, both of thesestressors produce a dynamic pulsatory behavior of the GUVs, characterized byrepeated sequences of large pore opening and closing.We first focus on how GUV volume change drives the dynamics of lipid ves-icles. We recently unraveled how hypotonic GUVs filled with sucrose solutionand bathed in water, exhibit characteristic swell-burst cycles. Here we ask,what is the effect of intra-vesicular crowding on the vesicle dynamics? Ourexperimental observations, supported by our model results, show that GUVsencapsulating PEG and/or Dextran polymers still exhibit swell-burst cycleseven at zero or negative concentration differential. Our model allows us toidentify the three main phenomena leading to this intriguing behavior: thenon-ideal osmotic pressure induced by the polymer solutions, the slowerpore dynamics due to higher solution viscosity, and the slower diffusive fluxthrough the pore.Second, we ask how the dynamics of GUVs is affected by surface stressors? Ithas been shown that lipid vesicles exposed to surfactants lose surface areathrough lipid solubilization, leading to two main possible outcomes: cyclesof pore opening/closing, or long-lived stable pores. We determine theparameter space related to these two behaviors, and solve numerically ourmodel, allowing us to predict the dynamics of GUVs exposed to varioussurfactants.

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1927-PlatThe Effect of pH on Single Virus Lipid Mixing KineticsElizabeth R. Webster1, Robert Rawle2, Peter Kasson2, Steven Boxer1.1Chemistry, Stanford, Stanford, CA, USA, 2Molecular Physiology andBiological Physics and Biomedical Engineering, University of Virginia,Charlottesville, VA, USA.Zika virus, an enveloped flavivirus, has emerged as a global health concern inrecent years due to its link to fetal microcephaly. The mechanism of entry andfusion for Zika is not yet well characterized and no key binding receptor hasbeen determined. To generate mechanistic insight into the fusion process ofZika Virus, this work uses a biophysical approach to study single virus lipidmixing events of Zika virus with a model membrane. (Rawle, Webster et al.,submitted) To bind the virus in the absence of a known receptor, syntheticDNA-lipids are utilized as a means of surrogate receptors (Biophysical Journal,111, 123 (2016)). We have determined the pH dependence of Zika virus hemi-fusion, and characterized the kinetics of lipid mixing. We find that the kineticsof lipid mixing are not strongly correlated to pH over a 100x fold change in pro-ton concentration. However, the efficiency of lipid mixing is directly dependenton pH. As pH is lowered, efficiency increases. Coupled together, the kinetic andefficiency data provide constraints for a kinetic model which is discussed incompanion work (poster/presentation R. Rawle, et al). These findings alsoshed light on the physiological environment at which fusion can occur in theendosome.

1928-PlatEffective Bending Rigidity of Membranes with Rigid InclusionsElizabeth Kelley1, Michihiro Nagao1,2, Paul Butler1,3.1Center for Neutron Scattering, NIST, Gaithersburg, MD, USA, 2IndianaUniversity, Bloomigton, IL, USA, 3Dept of Chemistry, University ofTennessee, Knoxville, TN, USA.The ability of biological membranes to bend and deform is essential to a widerange of cell processes. Significant advances in understanding the elasticity oflipid bilayers underlying these deformations have been made by studying ho-mogeneous fluid membranes. However, biological membranes are far from ho-mogeneous, and less is known about how the heterogeneity affects the materialproperties of the membrane. Here we use neutron spin echo spectroscopy(NSE) to study the effective bending rigidity (keff) of heterogeneous lipid mem-branes with coexisting rigid gel and soft fluid domains. Our data show that keffscales directly, but not linearly, with the area faction of gel phase and are inexcellent agreement with theoretical predictions for heterogeneous lipid mem-branes with rigid inclusions. While it is intuitively expected that keff should insome way depend on the fraction of gel phase, the functional form of the scalinghas important implications for understanding the effects of rigid inclusions,such as proteins or lipid domains, on the effective stiffness of biologicalmembranes.

1929-PlatEmergence of Undulations as 2-D Director Fluctuations in PhopholipidMembranesTrivikram R. Molugu1, Soohyun Lee1, Xiaolin Xu2, K.J. Mallikarjunaiah1,Constantin Job1, Michael F. Brown1,2.1Department of Chemistry and Biochemistry, University of Arizona, Tucson,AZ, USA, 2Department of Physics, University of Arizona, Tucson, AZ, USA.Hydrated lipids form lyotropic liquid crystals exhibiting long-range order inmolecular organization [1]. The statistical mean-torque model characterizesmembrane deformations on the order of the bilayer thickness [2]. Elastic defor-mations in such materials are manifested as director fluctuations, which are col-lective hydrodynamic phenomena with motional timescales spanning severaldecades (picoseconds to seconds). These fluctuations cause 2H NMR relaxationcorresponding to dynamic parameters of the mesogenic organization. Here weexamine the effect of hydration on liquid-crystalline properties of lipid mem-branes using NMR relaxation methods. Solid-state 2H longitudinal (R1Z) andtransverse quadrupolar-echo decay (R2

QE) rates were measured for acyl-chain-perdeuterated lipid multilamellar dispersions. Plots of the R1Z ratesversus squared segmental order parameters (S2CD) follow an empirical func-tional behavior showing the emergence of 3-D director fluctuations [3]. Slopesof these square-law plots are sensitive to the hydration level. Although ratesalso showed similar trends, at high hydration the functional square-law islimited to segments deeper in the bilayer. Enhanced rates indicate additionalcontributions from slower dynamics, whereas breaking of the square-law sug-gests the emerging surface undulations (2-D director fluctuations). Lipid hydra-tion at the transition of 3-D to 2-D director fluctuations may have importantbiological consequences for biomembrane functioning. The square-lawconfinement suggests restricted water penetration into the hydrophobic bilayerinterior. The slow dynamics at high hydration must be a consequence of

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collective lipid phenomena that explain bulk material properties. Relaxationfrequency dispersions (QCPMG) inform the quantitative Frank elastic con-stants of lipid membranes. Studies of cholesterol in model membranes [3]give added insights into lipid rafts and membrane compositions relevant forbiomembrane functions. [1] T.R. Molugu et al. (2017) Chem.Rev. DOI:10.1021/acs.chemrev.6b00619. [2] J.J. Kinnun et al. (2015) BBA 1848, 246.[3] T.R. Molugu et al. (2016) Chem.Phys.Lipids 199, 39.

1930-PlatPore-Spanning Membranes: Lipid Domains in Confined GeometryClaudia Steinem.IOBC, Georg August Universit€at, Goettingen, Germany.Pore-spanning membranes (PSMs) are well-suited to investigate single lipiddiffusion as well as lipid domain diffusion. Recent findings have highlightedthe dynamic nature of such domains in the plasma membrane and the key roleof the underlying cytoskeleton meshwork in stabilizing them. We used poroussubstrates with different pore radii serving as a static meshwork to modulatethe size of lipid domains in liquid ordered (lo)/liquid disordered (ld) phase-separated continuous PSMs composed of DOPC, sphingomyelin, cholesteroland the globoside Gb3. We analyzed domain formation and domain dynamicsby fluorescence video microscopy. It turned out that the distribution of the lophase of the PSMs on the porous substrate strongly depends on the coolingrate. Analysis of the diffusion of mobile lo domains entrapped in the free-standing parts of the PSMs showed that the domains’ diffusion constantsare slowed down by orders of magnitude due to the confinement ofthe PSM, where the drag force is governed by both the friction in thebilayer and the coupling to the aqueous phase compared to the unrestrictedcase.

1931-PlatMechanical Properties of Membranes under Asymmetric BufferConditionsMarzieh Karimi, Jan Steink€uhler, Debjit Roy, Reinhard Lipowsky,Rumiana Dimova.Theory and BioSystem, Max Planck Institute of colloids and Interfaces,Potsdam, Germany.Biological membranes consist of molecular bilayers which are intrinsicallyasymmetric in nature. This asymmetry can be induced not only by leafletcomposition and specific adsorption but also by differences in the cytosolicand periplasmic solutions containing macromolecules and ions. Membranesare surrounded by aqueous buffers inside and outside the cell exhibiting strongconcentration asymmetry of e.g. sodium, potassium and chlorine ions. Therehas been a long quest to understand the effect of these ions on the physicaland morphological properties of membranes.Ion-lipid interactions and, in particular, the effect of ion trans membraneasymmetry are crucial not only for the membrane phase state [Kubsch et al.Biophys. J. 110:2581-2584, 2016] but also influence the mechanical propertiesof membranes. Here, we set to explore the changes in the mechanical proper-ties of membranes exposed to asymmetric buffer conditions. As a modelmembrane, we employed giant unilamellar vesicles (GUVs) and firstimproved existing protocols for generating GUVs in physiologically relevantsalt concentrations. To assess the membrane mechanical properties, we aspi-rate a GUV into a micropipette and by means of an attached bead manipulatedvia optical tweezers, we pull an outward tube to measure the spontaneous cur-vature and the bending rigidity of the bilayer. With increasing the aspirationpressure, the bead is displaced from the equilibrium position in the opticaltrap, which in return gives us the bending rigidity and spontaneous curvatureof GUVs [Lipowsky, Faraday Discuss. 161:305-331, 2013]. We explore theeffect of asymmetric distribution of salt and sugars across the membrane.This work is part of the MaxSynBio consortium which is jointly funded bythe Federal Ministry of Education and Research of Germany and the MaxPlanck Society.

1932-PlatProtein-Mediated Beads-on-a-String Structure Formation Along Mem-brane Nanotubes in Live CellsHaleh Alimohamadi1, Ben Ovryn2, Padmini Rangamani1.1Mechanical and Aerospace Engineering, University of California San Diego,La Jolla, CA, USA, 2Electrical and Computer Engineering, Colorado StateUniversity, Fort Collins, CO, USA.It is now established that various types of cells form thin tunneling nanotubeswhich connect their plasma membranes and enable intercellular communica-tion. These dynamic structures adopt their configuration and functionalitythrough various constraints including membrane tension, lipid asymmetryand cytoskeletal forces.

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Using a combination of metabolically labeling and bioorthogonal clickchemistry, we implement single-molecule tracking and dynamic super-resolution imaging of tagged glycans on the membrane of live cells. Wehave observed that membrane nanotubes can manifest stable, ‘‘beads-on-a-string’’ structures. However, the physical and biological mechanisms underly-ing these structures are not clear, raising the following fundamental questions:How does the spatial density and type of proteins on the surface relate tothe characteristic shape and formation of these structures? What is theprotein distribution associated with the membrane curvature of the beadsand what are the mechanisms that lead to the often observed stability of thesestructures?To address some of these questions, we model membrane nanotubes using themodified version of the Helfrich energy including surface protein interactionsand diffusion. We find that a localized protein distribution along the membranesurface creates regions of low tension, which are energetically favorable forbead formation. Furthermore, for a fixed membrane area, the beads grow andappear to coalesce as the protein density or areal coverage increases. Diffusiveflux of proteins expands the protein surface coverage, resulting in larger beads.Our results predict that the curvature-gradient at the edges of the beads acts asdiffusion traps, confining the protein and stabilizing the beads-on-a-stringstructures.

Platform: Neuroscience

1933-PlatProbing the Molecular Mechanisms of the Progression of Alzheimer’sDiseaseLee Makowski, Biel Roig Solvas.Northeastern University, Boston, MA, USA.Alzheimer’s disease is characterized by the presence of cerebral deposits ofamyloid fibrils made up of Ab peptides and neurofibrillary tangles containingtau protein. Ab deposition initiates in the neocortex and then expands intoadditional brain regions in a characteristic temporal sequence. The molecularmechanisms that drive this process are poorly understood and insights areneeded to form a foundation for design of therapies to retard or halt disease.The distribution of fibril polymorphs in diseased tissue may provide clues totest models of disease progression, but the in situ study of fibril structure inthese pathological lesions is currently inaccessible to most biophysical ap-proaches. Here we describe studies of these lesions in situ, using x-ray micro-diffraction to generate maps of the structural properties of amyloid fibrils inhistological thin sections of human brain tissue with �5 micron resolution.This work is generating information on the distribution of Ab fibril poly-morphs at multiple length scales, the organization of those fibrils intoplaques, the maturation of plaques during disease and the differences infibril structure in tissues from subjects with distinct clinical histories. Interpre-tation of the observed patterns is providing insights into the molecular pro-cesses causing neurodegeneration, and the role of fibril polymorphism indisease.

1934-PlatModulating Amyloid Formation: Insights from Biophysical StudiesAstrid Graslund1, Ann Tiiman2, Jyri Jarvet1, Vladana Vukojevic2.1Department of Biochemistry and Biophysics, Stockholm University,Stockholm, Sweden, 2Center for Molecular Medicine (CMM), Department ofClinical Neuroscience, Karolinska Institutet, Stockholm, Sweden.The amyloid b (Ab) peptide is composed of 39-43 residues. The peptide isfound in the so called plaques in the brains of patients suffering from Alz-heimer’s disease (AD). The structural conversion of the Ab peptide from anunfolded monomeric state to a b-sheet rich aggregated state (‘‘amyloid’’) isconsidered to be a fundamental event in AD. We study the structure conver-sions and aggregation properties of the Ab peptide using several spectroscopictechniques, including NMR and optical spectroscopy. The kinetic effects onthe aggregation process can be followed by fluorescence using suitableamyloid-binding probes, such as thioflavin T (ThT), which becomes stronglyfluorescent when bound to amyloid material. Fluorescence Correlation Spec-troscopy (FCS) applied to Ab during the amyloid formation process shows inthe presence of ThT the time dependent growing sizes of the formed aggre-gates with amyloid structures (Tiiman et al., Biochemistry 54 (2015) 7203).Metal ions and various small molecules, including peptides, and otherproteins that bind to the Ab peptide, exhibit significant effects on the Abaggregation process, both in terms of kinetics and structural outcome.Cu(II) and Zn(II) bind to amino acid ligands in the N-terminus of the Abpeptide. Zn(II) has been shown to induce increased order in the N-terminus,and at a sub-stochiometric concentration delays the amyloid formation

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(Abelein et al., Proc. Nat. Acad. Sci. US 112 (2015) 5407). The effect of themetal ions can be followed by bulk fluorescence measurements as well as byFCS.

1935-PlatEvidence that the Human Innate Immune Peptide LL-37 May Be a Bind-ing Partner of Abeta and Inhibitor of Fibril AssemblyErsilia De Lorenzi1, Marcella Chiari2, Raffaella Colombo1, Marina Cretich2,Laura Sola2, Renzo Vanna3, Paola Gagni2, Federica Bisceglia1,Carlo Morasso3, Jennifer S. Lin4, Moonhee Lee5, Patrick L. McGeer5,Annelise E. Barron4.1Drug Sciences, University of Pavia, Pavia, Italy, 2Institute of Chemistry ofMolecular Recognition, Milan, Italy, 3Laboratory of Nanomedicine andClinical Biophotonics (LABION), Fondazione Don Carlo Gnocchi ONLUS,Milan, Italy, 4Bioengineering, Stanford University, Stanford, CA, USA,5Kinsman Laboratory of Neurological Research, University of BritishColumbia, Vancouver, BC, Canada.Evidence that the human innate immune peptide LL-37 may be a binding part-ner of ABeta and inhibitor of fibril assemblyBackground: Identifying physiologically relevant binding partners of b-amy-loid (Ab) that modulate in vivo fibril formation may yield new insights into Alz-heimer’s Disease (AD) etiology. Human cathelicidin peptide, LL-37, is aninnate immune effector and modulator, ubiquitous in human tissues and ex-pressed in myriad cell types.Objective:We present in vitro experimental evidence and discuss findings sup-porting a novel hypothesis that LL-37 binds to Ab42 and can modulate Ab fibrilformation.Methods: Specific interactions between LL-37 and Ab (with Ab indifferent aggregation states, assessed by capillary electrophoresis) weredemonstrated by surface plasmon resonance imaging (SPRi). Morphologicaland structural changes were investigated by transmission electron micro-scopy (TEM) and circular dichroism (CD) spectroscopy. Neuroinflammatoryand cytotoxic effects of LL-37 alone, Ab42 alone, and LL-37/Ab complexeswere evaluated in human microglia and neuroblastoma cell lines (SH-SY5Y).Results: SPRi shows binding specificity between LL-37 and Ab, while TEMshows that LL-37 inhibits Ab42 fibril formation, particularly Ab’s ability toform long, straight fibrils characteristic of AD. CD reveals that LL-37 preventsAb42 from adopting its typical b-type secondary structure. Microglia-mediatedtoxicities of LL-37 and Ab42 to neurons are greatly attenuated when the twopeptides are co-incubated prior to addition. We discuss the complementary bio-physical characteristics and AD-related biological activities of these twopeptides.Conclusion: Based on this body of evidence, we propose that LL-37 and Ab42may be natural binding partners, which implies that balanced (or unbalanced)spatiotemporal expression of the two peptides could impact AD initiationand progression.

1936-PlatKinesin-1 and ARL8B-Dependent Targeting of a Prion Mutant intoAxonal Pre-lysosomal Compartments Promotes Prion Aggregation inNeuronsRomain Chassefeyre, Sandra Encalada.Molecular Medicine Department, The Scripps Research Institute, La Jolla,CA, USA.A characteristic of prion pathology is the presence of axonal swellings contain-ing PrP aggregates and accumulations of abnormal amounts of microtubule-associated molecular motor proteins, vesicles, and organelles. Here, we reporta novel mechanism by which aggregation in neurons of a pathogenic mutantPrP is a direct consequence of the direct targeting of these prions to pre-lysosomal compartments by Kinesin-1 and Arl8b. We show that upon expres-sion in neurons, mutant PrP turns on ER stress and in response is constitutivelyexported into the axon in Golgi-derived vesicles by interactions with the smallGTPase Arl8b, which in turn recruits molecular motors to move PrP vesiclestowards the termini. Mutant PrP transiently accesses the cell surface alongthe axon before immediate internalization into late endosomal/pre-lysosomalcompartments. We further show that internalized mutant PrP is poorlydegraded in lysosomes, leading to the progressive accumulation and aggrega-tion of mutant PrP in axons. Our data a direct relationship between intracellulartransport and misfolded protein aggregate formation and demonstrate that for-mation of prion aggregates in axons is a late event strictly dependent onKinesin-1 motor activity and in delivery to- and endocytosis from the plasmamembrane, suggesting that post-Golgi re-routing of abnormal PrP is deleteriousfor neurons.

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1937-PlatSingle Layer Graphene Promotes Neuronal Activity by Regulating Potas-sium Ion Channels in Cultured Neuronal NetworksNiccolo Paolo Pampaloni1, Martin Lottner2, Michele Giugliano3,Alessia Matruglio4, Francesco D’Amico5, Maurizio Prato6, Jose AntonioGarrido2, Laura Ballerini1, Denis Scaini1.1Neurobiology, S.I.S.S.A., Trieste, Italy, 2Walter Schottky Institut andPhysik, Garching, Germany, 3Theoretical Neurobiology &Neuroengineering, University of Antwerp, Antwerp, Belgium, 4CNR-IOM -Istituto Officina dei Materiali, Trieste, Italy, 5ELETTRA Synchrotron LightSource, Trieste, Italy, 6Department of Chemical and PharmaceuticalSciences, University of Trieste, Trieste, Italy.Graphene, with its peculiar bi-dimensional crystal arrangement of pure carbonatoms, is catching the eye of the research community with its extraordinaryphysicochemical properties. In particular, single layer graphene (SLG) poten-tial applications in biology and (nano)medicine have being deeply investi-gated during last years. Although preliminary reports have shown thatgraphene based materials can be safely interfaced with neuronal cells(Fabbro et al., 2015), to date an exhaustive functional study of neuronalnetworks developed interfaced with SLG is missing. Here, for the first time,we show that uncoated SLG is not only fully biocompatible but, surprisingly,induces in cultured neurons an increased network synaptic activity, presum-ably by altering the availability of extracellular Kþ ions. The homeostaticchanges observed in SLG-interfaced cells, as well as the increased networkactivity, were not observed when neurons were grown neither on a many-layers graphene (MLG), nor onto thin gold substrates, suggesting a highlymaterial specificity of this adaptive interaction. In particular, combining ma-terial characterization, electrophysiological patch-clamp recordings andneuronal network simulations, we propose a model in which the peculiarinteraction of SLG with the ionic species present in solution shifts asignificant fraction of phasically firing neurons towards a tonic phenotype,and these changes are reflected as an increased firing activity at the entirenetwork level.

1938-PlatPhotoelectrochemical Modulation of Neuronal Activity with Free-Standing Coaxial Silicon NanowiresRamya Parameswaran1, Joao L. Carvalho-de-Souza2, Yuanwen Jiang3,Michael J. Burke3, John F. Zimmerman4, Kelliann Koehler3,Andrew W. Philips3, Jaeseok Yi3, Erin Adams2, Francisco Bezanilla2,Bozhi Tian3.1MSTP/Biophysical Sciences, University of Chicago, Chicago, IL, USA,2Biochemistry and Molecular Biology, University of Chicago, Chicago, IL,USA, 3Chemistry, University of Chicago, Chicago, IL, USA, 4Engineeringand Applied Sciences, Harvard University, Boston, MA, USA.Extracellular electrical stimulation of excitable cells is the basis for manyimplantable devices that treat diseases such as cardiac arrhythmias, Parkin-son’s disease, depression, epilepsy, and rheumatoid arthritis. While these de-vices have improved the qualities of many lives, they are often limited bytheir bulkiness, requirement for genetic manipulation of target cells, mechan-ical invasiveness, and inability to target single cells. Here, we repurpose thephotovoltaic properties of coaxial p-type/intrinsic/n-type silicon nanowires(PIN-SiNWs), previously designed for solar cell applications, to wirelesslyand photoelectrochemically modulate primary rat dorsal root ganglion(DRG) neuron excitability. We revealed the presence of atomic gold on thenanowire surfaces, likely due to gold diffusion during the material growth.To evaluate how surface gold impacts the photoelectrochemical propertiesof single PIN-SiNWs, we used modified quartz pipettes from a patch clampand recorded sustained cathodic photocurrents from single PIN-SiNWswhen illuminated with a 532 nm laser. We show that using laser pulsesof 532 nm these PIN-SiNWs can elicit action potentials (APs) in primaryrat DRG neurons through a primarily atomic gold-enhanced photoelectro-chemical process. More specifically, we found that the increase in temperatureassociated with AP generation was 0.36 K, a value that is much lowerthan that elicited by photothermally exciting materials (optocapacitance).Additionally, we constructed an excitability curve for these neurons,demonstrating PIN-NW induced generation of APs with laser pulse durationsof up to 5 ms. Remarkably, the amount of laser energy necessary to just elicitAPs in these neurons was essentially constant from pulse durations rangingfrom 0.1 ms to 5 ms, as expected from a classical excitability curve.This work shows that wireless and non-genetic photoelectrochemical neuro-modulation with single nanostructures is possible, and has implicationsfor both basic research and photo-responsive therapies for neurodegenerativediseases.

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1939-PlatQuantifying Molecular Disease Mechanisms in Intact Tissue using Auto-matic and Adaptive Refractive Index Compensation for Light-Sheet Fluo-rescence MicroscopyDouglas Shepherd1, Duncan Ryan2, Elizabeth Gould3, Jasmine Singh4,Taylor Nowlin5, Gregory Seedorf5, Omid Masihzadeh6, Steven Abman5,Sukumar Vijayaraghavan7, Wendy Macklin3, Diego Restrepo3.1Pharmacology, University of Colorado Anschutz Medical Campus, Aurora,CO, USA, 2Physics, Colorado State University, Fort Collins, CO, USA, 3Celland Developmental Biology, University of Colorado Anschutz MedicalCampus, Aurora, CO, USA, 4Bioengineering, University of ColoradoAnschutz Medical Campus, Aurora, CO, USA, 5Pediatric Heart Lung Center,University of Colorado Anschutz Medical Campus, Aurora, CO, USA,6Ophthalmology, University of Colorado Anschutz Medical Campus, Aurora,CO, USA, 7Physiology and Biophysics, University of Colorado AnschutzMedical Campus, Aurora, CO, USA.Optical tissue clearing has revolutionized researchers’ ability to perform fluo-rescent measurements within intact tissue. One common complication to alloptically cleared tissue is a spatially heterogeneous refractive index (RI), lead-ing to light scattering and first-order defocus. We designed C-DSLM (clearedtissue digital scanned light-sheet microscopy) as an adaptive light-sheet fluo-rescence microscopy (LSFM) method intended to automatically generate in-focus images of cleared tissue.1 C-DSLM uses electrotunable lenses andcomputational autofocusing to de-couple volumetric imaging from samplemovement. C-DSLM automatically corrects for first-order defocus and main-tains co-planarity between the exciting light-sheet and focus of the detectionarm. Using C-DSLM and tissue clearing (PACT), we investigated multiple an-imal models difficult to image using standard methods. Here we present our re-sults from two translational animal models of disease. By intentionally under-clearing mouse brain tissue with an endogenous fluorescent reporter of myelin,we created heterogeneous RI tissue that retained both lipid-rich myelin andmyelin progenitor cells. Because C-DSLM can automatically account for thisheterogeneous RI in large samples, we discovered regional differences inbrain-wide myelin progenitor cell distribution linked to changes in circuit func-tion and behavior due to the deletion of proteolipid protein.1 Because C-DSLMimaging is inertia-free, it can accommodate fragile samples such as intact ret-inas. We discovered that the primary plexus vasculature over-compensates fordeficient vertical sprouts and secondary plexus in a rat model of retina devel-opment exposed to severe hyperoxic injury.2 As optical tissue clearing becomesmore widely adopted, simple and affordable high-throughput imaging method-ologies are required. C-DSLM is one such methodology that has already pro-duced multiple advancements in quantifying translational animal models.1) Ryan, D.P. et al. Nature Communications 20172) Singh, J.N. et al. Journal of Biomedical Optics 2017

1940-PlatDe Novo Designed Proteins for Ultrafast Detection of Membrane PotentialChangesMartin J. Iwanicki1, Joshua A. Mancini1, Sohini Mukherjee1,Christopher C. Moser1, Brian Y. Chow2, Bohdana M. Discher1.1Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA,USA, 2Bioengineering, University of Pennsylvania, Philadelphia, PA, USA.Neuronal networks communicate through changes in membrane potentials.Optically recording these changes on the sub-microsecond timescale providesa better understanding of interneuronal communication. While organic probesthat have been developed detect signals with fast temporal resolution, theseprobes lack cell and membrane specificity. Genetically-encoded voltage indica-tors (GEVIs) remedy this problem; however, these protein-based opticalvoltage indicators are dimmer and slower than their organic probe counterparts.Here, we present our progress on the design, development, and characterizationof novel GEVIs based on artificial 4-a-helical bundle proteins, called ma-quettes. We demonstrated that membrane maquettes are able to bind 3 hemesupon assembly in vesicles, each with different redox midpoint potentials. Tosense changes in membrane potential, we are modifying our first prototype ma-quettes to develop two classes of maquette GEVIs. The first class of maquetteGEVIs covalently binds a fluorescent cofactor, and we are using directed evo-lution to improve its fluorescent signal and quantum yield. The second class ofmaquette GEVIs is a fusion construct of a maquette and a fluorescent protein,whose fluorescence can be modulated by a chain of voltage-sensitive hemesembedded within the maquette. Energy transfer has been demonstrated be-tween the fluorescent protein and the membrane maquette. These maquetteGEVI prototypes have been expressed in E. coli and purified for in vitro char-acterization. Parallel maquettes are being developed for expression in hippo-campal neuronsto validate the in vitro biophysical approach in mammaliancells.

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Platform: Replication, Recombination, Repair,Transcription, and Translation

1941-PlatArchitectural Rearrangements during Primer SynthesisMarilyn E. Holt1, Matthew K. Thompson2, Lauren E. Salay1,Walter J. Chazin1.1Center for Structural Biology, Vanderbilt University, Nashville, TN, USA,2Department of Chemistry, University of Alabama, Tuscaloosa, AL, USA.Initiation of DNA replication requires formation of a ‘‘primer’’ on the ssDNAtemplate prior to generation of the new complementary strand by processiveDNA polymerases. In eukaryotes, primers are made by DNA polymerase a-primase (pol a-prim), a heterotetramer containing a primase (p48), a polymer-ase (p180) and two regulatory (p58, p68) subunits. Primase, the only polymer-ase capable of copying de novo from ssDNA, is a DNA-dependent RNApolymerase that generates the initial 8-12 nt RNA primers. These initialRNA primers are released to pol a, which extends the primer by �20 nucle-otides before transferring the hybrid DNA/RNA oligonucleotide to the proc-essive polymerases ε or d. The primase heterodimer contains a DNA-binding domain on the regulatory subunit (p58C) that is connected to the pri-mary scaffold (p48/p58N) by a flexible linker. A combination of small-angleX-ray scattering (SAXS) and biochemical techniques is being used to charac-terize the domain architecture of human DNA primase as primer synthesis isinitiated and elongated. To generate primed template substrates of differentlengths, in vitro RNA translation reactions were performed to generate oligo-mers with the 5’ triphosphate group critical for high-affinity interaction withp58C. Size-exclusion chromatography coupled with multi-angle light scat-tering (SEC-MALS) was used to test experimental conditions and generatehomogenous, monodisperse samples. SAXS data acquired for free primase,under conditions for initiation, and for complexes with primed substrateswill be presented. Transitions in primase structure and dynamics will be dis-cussed in the context of our models for priming, which include primase em-ploying the same active site but different catalytic mechanisms for initiationand elongation functions.

1942-PlatWhen Helicase and Polymerases Collides and Unfolds G4-Quadruplex ontheir TrackVincent Croquette1, Samar Hodeib1, Jean-Baptiste Boul�e2, Shubeena Chib3,Kevin D. Raney3.1CNRS, Paris, France, 2cMus�eum CNRS UMR 7196, Paris, France,3Biochemistry and Molecular Biology, University of Arkansas, Little Rock,AR, USA.Using single molecule technique, we first characterize the kinetics offolding and unfolding as well as the stability of a single G4 inserted in adsDNA: a situation that mimics the G4s in promoters, where the comple-mentary sequence competes with the G-rich structure. We use the factthat a DNA fork rezipping a molecule is arrested by the G4. We find thatthe lifetime of a single telomeric G4 is small (�20s) and thus this G4most likely unfolds before a helicase can reach it. This is not the casefor the very stable c-MYC G4 which remains for typically �2h. For thisG4, we observe in real time how helicases or polymerases behave as theycollide the G4 on their track. We find that the Pif1 helicase resolves thisG4 after marking a pause. On the other hand, this G4 is not resolved bythe RecQ helicase nor by the bacteriophage T4 replicative helicase or theprotein RPA, but it is unwound by the gp43 polymerase and by the T4 re-plisome. We also observe that the G4 may be jumped by those helicaseswhich do not unfold the structure. We shall also discuss the case of otherpolymerases.

1943-PlatNew Insights into Transcriptional Pausing using Ultra-high ResolutionOptical Tweezers and Novel Analysis AlgorithmsRonen Gabizon1, Antony Lee2, Hanif V. Movahed3,4, Richard H. Ebright3,4,Carlos J. Bustamante1,5.1California Institute for Quantitative Biosciences, QB3, University ofCalifornia, Berkeley, Berkeley, CA, USA, 2Physics, University of California,Berkeley, Berkeley, CA, USA, 3Waksman Institute, Rutgers University,Piscataway, NJ, USA, 4Chemistry and Chemical Biology, RutgersUniversity, Piscataway, NJ, USA, 5Molecular and Cell Biology, KavliEnergy Nanoscience Institute, and Howard Hughes Medical Institute,University of California, Berkeley, Berkeley, CA, USA.Transcription by RNA polymerase (RNAP) is interspersed with periods ofpausing, which play critical roles in coordinating transcription with processes

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such as translation, splicing and DNA repair. The process by which RNA po-lymerase enters off-pathway paused states from the main elongation pathwayis not well understood due to the limited time resolution of methods used tostudy these events. By combining high-resolution optical trapping measure-ments of transcription with novel data analysis methods, we characterizedthe dynamics of pausing at unprecedented temporal resolution. We foundthat virtually all RNAP molecules transcribe more slowly when crossing pausesites, even when not entering extended pauses, indicating that pausing mayrequire slow on-pathway transcription dynamics prior to entry into off-pathway paused states. We then used this method to study the dynamics ofbacktracked as well as hairpin-stabilized pauses. We found that backtrackingoccurs stepwise, with non-backtracked or 1 base pair-backtracked statesforming quickly, followed by slow formation of deeper backtracked states.This finding confirms structural studies indicating large conformationalchanges upon transition from 1 base pair backtracks to deeper backtrackedstates. We also found that the nascent RNA structure can either enhance ordiminish pausing in a sequence dependent manner, and functions primarilyby enhancing or attenuating the duration of already accessed sequence-dependent pauses.

1944-PlatCombinatorial Origin of Protein Expression NoiseSangjin Kim, Christine Jacobs-Wagner.Yale University, HHMI, West Haven, CT, USA.Genetically identical cells exhibit diverse phenotypes, even when experi-encing the same environment. This phenomenon, in part, originates fromcell-to-cell variability (noise) in protein expression. While various kineticschemes of stochastic transcription initiation are known to yield transcrip-tional noise, how dynamics of post-transcription initiation events may affectnoise at the protein level remains poorly understood. To address this question,we developed an integrated model of bacterial gene expression. By exploringdifferent scenarios of transcription initiation, transcription elongation dy-namics, mRNA degradation and gene copy number, we found that themRNA lifetime, gene dosage and sequence-dependent transcriptional pausingmodulate the protein expression noise that was initially set by the promoterproperties. Our findings highlight the interplay between transcriptioninitiation, transcription elongation, translation and mRNA degradation inshaping protein number distributions in clonal populations. They alsohave implications for our understanding of genome evolution, and suggestcombinatorial strategies for modulating phenotypic variability by geneticengineering.

1945-PlatThe Interaction between Bacteriophage T7 DNA Polymerase and Gene 2.5Protein at the Single-Molecule LevelJulia Bakx, Jordi Cabanas-Danes, Erwin J.G. Peterman, Gijs J.L. Wuite.Vrije Universiteit Amsterdam, Amsterdam, Netherlands.DNA replication is the process by which two identical DNA copies are pro-duced from a single parental template, in order to guarantee inheritance inall organisms. To this end, a multi-protein complex, the replisome, usesspatiotemporally controlled interactions to perform its function in ahighly-coordinated fashion. While the basic principles of DNA replicationby DNA polymerase (DNAp) on its own is largely understood, the exactinteraction modes and activity mechanisms of the replication proteinswith each other remain difficult to access. A key interaction within bacterio-phage T7 replisome is the one between DNAp and single-stranded DNA-binding protein gene product 2.5 (gp2.5). Both the kinetics of theseproteins on their own and the effect of their interaction on DNA replicationhave been reported before. However, these studies were mainly performedeither using bulk assays which led to averaged results, or when single-molecule assays were performed, they focused on one replicationprotein at a time, while applying high tensions on the DNA. Here we presentresults of single-molecule studies of the bacteriophage T7 replisome usingoptical trapping combined with confocal fluorescence microscopy. First,we investigated how DNA replication is affected by the interaction betweenT7 DNAp and gp2.5. We demonstrate that gp2.5 forms roadblocks topolymerization while biasing DNAp to its proof-reading conformation,exonucleolysis activity. Second, we found two states in which gp2.5 andDNAp can interact. Moreover, we are developing a new analysis method,based on confocal fluorescence kymographs, to study DNA replication atDNA tensions below 10 pN. These results contribute to a better understand-ing of T7 DNAp activity in the presence of gp2.5 and DNA replication ingeneral.

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1946-PlatAnisotropic Fluctuations in the Ribosome Determine aa-tRNA KineticsHuan Yang1, Jeffrey Noel2, Paul Charles Whitford1.1Physics, Northeastern University, Boston, MA, USA, 2Max Delbruck Centerfor Molecular Medicine, Berlin, Germany.The ribosome is a large ribonucleoprotein complex what is responsible for syn-thesizing proteins in all organisms. Accommodation is a key conformationalchange that allows an aminoacyl-tRNA (aa-tRNA) molecule to fully associatewith the ribosome. During tRNA accommodation, there are large-scale fluctu-ations in the L11 stalk and the associated protein. To explore the impact of thisdynamic region on the rate of aa-tRNA accommodation, we used molecular dy-namics simulations with a simplified model to evaluate the free energy as afunction of aa-tRNA position. We find that the free-energy barrier associatedwith the elbow accommodation is dependent on the degree of mobility pre-sented by the L11 stalk. Specifically, when L11 stalk is more rigid, the free-energy barrier is decreased and the A/T ensemble is destabilized. This is dueto the confinement effect introduced by the flexibility of L11 stalk. In addition,when elongation factor Tu (EF-Tu) is present, the A/T ensemble is further de-stabilized. The destabilization effect introduced by EF-Tu and L11 stalk is ad-ditive. These results provide a quantitative foundation to interpret experimentalmeasurements. As well, this work suggests how to design new experiments thatcan precisely control the dynamics of the ribosome.

1947-PlatMechanistic Insight into the Initiation of Repeat-Associated NonaugTranslationRosslyn Grosely, Joseph Puglisi.Structural Biology, Stanford University, Stanford, CA, USA.The predominant genetic cause of amyotrophic lateral sclerosis (ALS) is a hex-anucleotide repeat expansion in the intron of C9orf72. Repeat-associated non-AUG (RAN) translation occurs in all six reading frames of the GGGGCCnucleotide expansion tract resulting in the synthesis of neurotoxic dipeptiderepeat proteins.We have used bulk biochemical techniques and developed a hu-man translation system for single-molecule fluorescence approaches to eluci-date the mechanism of RAN translation initiation.

1948-PlatEvolutionarily-Encoded Translation Kinetics Coordinate Co-Translational SSB Chaperone Binding in YeastNabeel Ahmed1, Kristina Doring2,3, G€unter Kramer2,3, Bernd Bukau2,3,Edward P. O’Brien1,4.1Bioinformatics and Genomics Graduate Program, The Huck Institutes ofLife Sciences, Pennsylvania State University, University Park, PA, USA,2Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany, 3German Cancer Research Center(DKFZ), Heidelberg, Germany, 4Department of Chemistry, PennsylvaniaState University, University Park, PA, USA.Chaperones can bind to the ribosomes and nascent polypeptides co-translationally to assist protein folding. It was not known previously whetherthere is any interplay between chaperone binding and translation kinetics. Tostudy this effect, we utilize the high-throughput transcriptome-wide quantita-tive data for translation kinetics and chaperone binding from ribosome profilingand selective ribosome profiling methods respectively. In vivo selective ribo-some profiling has shown that yeast Hsp70 chaperone Ssb associates broadlywith a major fraction of nascent proteins. Using the ribosome footprint den-sities as a measure of local translation rate, we find that mRNA segments withinthe ribosome are translated faster during periods of Ssb binding to the nascentpolypeptides compared to when Ssb is not bound. The acceleration of transla-tion is maintained even when Ssb is knocked out thus implying an inherent en-coding of faster translation within the mRNA sequence. Testing for mRNAfeatures that can slow translation, we find that mRNA segments translated bySsb-engaged ribosomes are enriched for fast-translated codons (having highercognate tRNA concentrations), are depleted for slowly translated codons, andcontain fewer proline codons. In addition, mRNA segments located 1-15 nucle-otides downstream of Ssb-bound ribosomes have reduced mRNA secondarystructure. Finally, nascent chain segments located in the ribosome tunnel ofSsb-bound ribosomes have average numbers of positively charged residuesbut are enriched in negatively charged residues. Taken together, theseevolutionarily-encoded mRNA and nascent chain features cause faster transla-tion during Ssb binding. This finding is significant as any alteration of transla-tion kinetics due to synonymous mutations can potentially disrupt efficientbinding of Ssb leading to possible misfolding of the protein without any changein its sequence.

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Platform: TRP Channels

1949-PlatMolecular Insights into TRPV1 Polymodal Activation: Is AllostericCoupling between the Toxin and the Vanilloid Binding Sites Mediatedby Annular Lipids?Eleonora Gianti1, Michael Klein1, Tibor Rohacs2, Vincenzo Carnevale1.1Chemistry and the ICMS, Temple University, Philadelphia, PA, USA,2Department of Pharmacology, Physiology & Neuroscience, New JerseyMedical School, Rutgers University, Newark, NJ, USA.The TRPV1 channel is responsible for conducting cations through the cellmembrane in response to a variety of stimuli, amongst which noxious heatand chemical ligands, such as the vanilloid compounds capsaicin and its ultra-potent agonist resiniferatoxin (RTX). Predominantly expressed in sensory neu-rons and nociceptive fibers, TRPV1 is involved in important cellular functions,including heat-sensation and pain. One unresolved question about TRPV1 con-cerns activation by stimuli acting from the extracellular milieu, such as thebinding of the double-knot toxin (Dk/Tx) from spider venom. How is this stim-ulus coupled to the binding of RTX at the vanilloid site? How do Dk/Tx andRTX synergistically act on the gate? Interestingly, both these biomolecular li-gands participate in the formation of the vanilloid-lipid-channel-toxin quadri-partite complex, as shown by recent cryoEM experiments. We performedmolecular dynamics (MD) simulations and confirmed that annular lipids arepermanently bound to and stabilize the open state. While lipid head-groupsinteract with Dk/Tx, the tail-groups are simultaneously in contact with RTXand with a side chain crucial for the movement of the S4-S5 linker. Importantly,mutations at this position were shown to selectively impair capsaicin ability toactivate the channel without appreciably affecting ligand binding. In summary,our results shed light on the mechanism by which vanilloid binding is trans-duced into gating motion and rationalize the experimentally observed allostericcoupling between Dk/Tx and RTX.

1950-PlatThe Role of the Selectivity Filter in TRPV1 Channel GatingAndres Jara-Oseguera, Kenton J. Swartz.Molecular Physiol/Biophys, NINDS, NIH, Bethesda, MD, USA.The TRPV1 channel is a homotetrameric non-selective cation channel thatfunctions in nociceptors as an integrator of external noxious stimuli andendogenous pro-inflammatory molecules. Although the binding sites forsome TRPV1 modulators have been characterized, we still don’t understandhow any of these stimuli influence ion conduction in this receptor. The iden-tification and characterization of the regions that function as activation gatesare therefore central to understanding the mechanisms of gating in the TRPV1and other related channels. The structures of TRPV1 in open and closed statessuggested that in addition to the intracellular gate formed by the pore-liningS6 helices, the selectivity filter could also function as an activation gate.The available structures of other TRP channels also support the existenceof two gates, suggesting that it could be a conserved and unique feature ofthe TRP family. Here we set out to determine whether the selectivity filterof TRPV1 functions as a gate. We have substituted cysteines along the poreusing a cysteineless channel background and assessed their accessibility toexternally applied silver ions in both the open and the closed states in patchclamp recordings. First we found that external silver ions act as high-affinity,voltage-dependent, permeant blockers of the cysteineless TRPV1 channel,with a higher affinity for the closed state relative to the open state, suggestingthat the filter does not function as a gate. We have provided further support forthis hypothesis by showing that accessibility to external silver ions to acysteine more intracellular than the filter is not affected by channel gating.It is possible that the filter adopts a non-conducting conformation under spe-cific conditions yet to be identified, or that it constitutes a highly dynamic re-gion of the receptor.

1951-PlatOxytocin Modulates Nociception as a Direct Agonist of Pain-SensingTRPV1Yelena Nersesyan1, Lusine Demirkhanyan1, Deny Cabezas-Bratesco2,Victoria Oakes3, Ricardo Kusuda4, Tyler Dawson1, Xiaohui Sun1,Chike Cao5, Alejandro Cohen6, Katharina Zimmermann4, Carmen Domene7,Sebastian Brauchi8, Eleonora Zakharian1.1Cancer Biology & Pharmacology, University of Illinois College ofMedicine, Peoria, IL, USA, 2Department of Medicine, Universidad Australde Chile, Faculty of Medicine, Valdivia, Chile, 3Department of Chemistry,King’s College London, London, United Kingdom, 4Department ofAnesthesia, Friedrich-Alexander University Erlangen-N€urnberg, UniversityHospital, Erlangen, Germany, 5Ion Channel Research Unit, Duke University

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Medical Center, Durham, NC, USA, 6Proteomics and Mass SpectrometryCore Facility, Life Sciences Research Institute, Dalhousie University,Halifax, NS, Canada, 7Department of Chemistry, University of Bath, London,United Kingdom, 8Department of Medicine, Universidad Austral de Chile,Valdivia, Chile.Oxytocin is a neuropeptide produced in the hypothalamic paraventricular(PVN) and supraoptic (SON) nuclei that regulates various social behaviorsand reproduction. Oxytocin-containing magnocellular neurons innervate fore-brain regions, and parvocellular neurons project to the brainstem and spinalcord, where oxytocin is known to affect both the acute and chronic nociceptiveresponses. The release of oxytocin from these neurons suppresses nociceptionof inflammatory pain. However, the understanding of the molecular mechanismof anti-nociceptive or anti-inflammatory actions of oxytocin remains inconclu-sive. Here, we report that the noxious stimuli receptor TRPV1 serves a role ofan ionotropic oxytocin receptor. Oxytocin elicits TRPV1 activity both in nativeand heterologous expression systems, irrespective of the presence of the clas-sical oxytocin receptor. In comparison to controls, dorsal root ganglia neuronsof TRPV1 knockout mice exhibit reduced oxytocin sensitivity and in vivo ex-periments display that oxytocin injections significantly attenuate capsaicin-induced nociception. Furthermore, oxytocin potentiates TRPV1 in planar lipidbilayers, which validates its direct agonistic action. The channel demonstratesstrong voltage dependence and rectification, reflected both in the single-channel conductance and the open probability. Molecular modeling and simu-lation experiments provide insight into the oxytocin-TRPV1 interaction, whichresembles that of RTX/DkTx. Together our finding lays the foundation for anexistence of endogenous regulatory pathways that modulate nociception viadirect action of oxytocin on TRPV1, implying its analgesic effect via the chan-nel’s desensitization.

1952-PlatMechanism of TRPV5 Modulation and Gating as Revealed by Cryo-EMTaylor E.T. Hughes1, David Lodowski1, Kevin Huynh2, Aysenur Yazici3,John del Rosario3, Abhijeet Kapoor4, Sandip Basak1, Amrita Samanta1,Sudha Chakrapani1, Z. Hong Zhou2, Marta Filizola4, Tibor Rohacs3,Seungil Han5, Vera Moiseenkova-Bell1.1CWRU, Cleveland, OH, USA, 2UCLA, Los Angeles, CA, USA, 3RutgersUniversity, Newark, NJ, USA, 4Icahn School of Medicine at Mount Sinai,New York, NY, USA, 5Pfizer Research and Development, Groton, CT, USA.The transient receptor potential vanilloid 5 (TRPV5) channel is a distinctmember of the transient receptor potential (TRP) channel family, which ishighly selective for Ca2þ. The transient receptor potential (TRP) superfamily,one of the largest families of polymodal cation channels, is subdivided intosix major branches: TRPV, TRPC, TRPM, TRPA, TRPP, and TRPML.Though the majority of TRP channels are non-selective cation channels,two members of the TRPV subfamily (TRPV5 and TRPV6) are highlyCa2þ selective. TRPV5 is present primarily at the apical membrane of distaltubule epithelial cells in the kidney and plays a key role in systemic Ca2þ ho-meostasis. Though studies have found several residues critical for channelgating, the structural mechanism of ligand induced gating has yet to be eluci-dated. Using cryo-electron microscopy (cryo-EM), we have gained structuralinsights into the mechanism of TRPV5 modulation by exogenous and endog-enous compounds. Differences in the structures solved by our lab have re-vealed key areas of conformational change undergone by TRPV5 tofacilitate gating. Structural comparisons between TRPV5 and other TRPVchannels have revealed potentially conserved modulation mechanisms withinthe TRPV subfamily.

1953-PlatStructures of the Endolysosomal TRPML3 Channel in Distinct StatesReveal Activation and Regulation MechanismsMinghui Li1, Xiaoyuan Zhou2, Deyuan Su1, Qi Jia3, Huan Li4, Xueming Li2,Jian Yang1.1Biological Sciences, Columbia University, New York, NY, USA, 2School ofLife Sciences, Tsinghua University, Beijing, China, 3Department ofOrthopedic Oncology, The Second Military Medical University, Shanghai,China, 4Kunming Institute of Zoology, Kunming, China.The mucolipin transient receptor potential (TRP) channels (TRPML1-3)localize primarily in endosomes and lysosomes. They conduct Ca2þ and Naþ

currents from the lumen to the cytoplasm and play an important role in mem-brane trafficking, autophagy, exocytosis and ion homeostasis. Mutations ofTRPML1 cause mucolipidosis type IV, a severe lysosomal storage disorderwith cognitive, linguistic, visual and motor deficits. Dysfunction of TRPML3causes deafness and pigmentation defects in mice. These severe effects under-score the crucial functional importance of TRPML channels. The activities ofTRPML1 and TRPML3 are differentially regulated by the low endolysosomal

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pH. Thus, in the presence of Ca2þ, TRPML1 currents are greatly enhanced byHþ whereas TRPML3 currents are inhibited by Hþ. To better understand themolecular mechanisms of TRPML channel function and regulation, wedetermined the structures of full length human TRPML3 in the apo, ML-SA1-bound, and low-pH-inhibited states by using cryoelectron microscopy,with resolutions of 4.06, 3.62 and 4.65 A, respectively. The agonist ML-SA1binds between S5 and S6 and opens an S6 gate. The selectivity filter is linedby a combination of carboxylate side-chains and backbone carbonyls anddoes not change significantly between the closed and open states. Apolycystin-mucolipin domain forms a luminal cap. S1 extends into this cap,forming a ‘gating rod’ that connects directly to a luminal pore-loop, which un-dergoes dramatic conformational changes in response to low luminal pH. S2extends intracellularly and interacts with several intracellular regions to forma ‘gating knob’. These unique structural features, combined with electrophys-iological studies, reveal a new mechanism thereby luminal pH and other phys-iological modulators such as PIP2 regulate TRPML3 by changing S1 and S2conformations.

1954-PlatResidues at TRPA1 S4-S5 Linker N-Terminus Are Critical for TranslatingCovalent Modification to Channel ActivationWei Chou Tseng1, Karen Padilla2, Seungil Han3, Aaron Gerlach2.1Pfizer, Cambridge, MA, USA, 2Icagen, Durham, NC, USA, 3Pfizer, Groton,CT, USA.TRPA1, a primary pain target belonging to the transient receptor potentialchannel (TRP) family, is uniquely activated by pungent substances throughcovalent modification of cysteine residues within the N-terminal ankyrin re-peats. Although the biochemistry of reactive cysteines is well defined, themechanism of how covalent modification translates to channel activation re-mains unknown. Here we hypothesize that upon covalent activation residuesfrom the S4-S5 linker couple to those from the TRP-like domain, resulting inthe conformational changes necessary for opening the gate of the channel. Toaddress, we utilized site-directed mutagenesis in the S4-S5 linker to determinethe importance of individual residues in covalent compound activation. Toconfirm which residues are specific for covalent-dependent activation wecounter-screened TRPA1 mutants against the non-covalent opener, PF-04840154, which binds to the S5 pore region and activates independentlyof N-terminal cysteine modification. A total of 23 amino acids spanning theentire S4-S5 linker were mutated with two, R852A and E854R, leading toloss of activation by 300 mM cinnamaldehyde while retaining normal activa-tion by PF-04840154. The highest resolution TRPA1 structure suggests apotential interaction between the N-terminus of the S4-S5 linker and thefirst helix of the TRP-like domain (C.E. Paulsen). Thus we further investi-gated the role of residues at the TRP-like domain for interacting with theN-terminus of the S4-S5 linker including the critical residues R852 andE854. The results demonstrate that residues in the S4-S5 linker are criticalfor TRPA1 activation by covalent modification. Given the importance ofthe S4-S5 linker in the gating of other channel families such as potassiumchannels and HCN channels, this information not only advances our under-standing of TRPA1 gating but also adds more to our understanding of ionchannel biophysics.

1955-PlatA PIP2 Binding Site on a Human TRP Channel: Simulation Studies ofPKD2Qinrui Wang1,2, George Hedger1, Prafulla Aryal1,3, Jiye Shi4,Elizabeth P. Carpenter2, Mark S.P. Sansom1.1Department of Biochemistry, University of Oxford, Oxford, UnitedKingdom, 2Structural Genomics Consortium, University of Oxford, Oxford,United Kingdom, 3Department of Physiology and Biophysics, University ofColorado Anschutz Medical Campus, Aurora, CO, USA, 4Department ofChemistry, UCB Pharma, Slough, United Kingdom.Polycystin-2 (PKD2) is a member of the transient receptor potential (TRP)superfamily of non-selective cation channels. Mutations in this channeland homologous channels lead to a common genetic disease, autosomaldominant polycystic kidney disease (ADPKD). Phosphatidylinositol (4,5)bisphosphate (PIP2) suppresses epidermal growth factor induced PKD2 activa-tion, and hydrolysis of PIP2 can release this inhibition1, suggesting PIP2 maybe a modulator of PKD2 channels. Using multi-scale molecular dynamicssimulations based on a recent electron cryo-microscopy (cryo-EM) structureof PKD2 (PDB ID: 5K47) we revealed a potential PIP2 binding site betweenS3, S4 and S5 transmembrane helices. This binding site is close to thevanilloid/lipid-binding site observed in the TRPV1 channel (PDB ID:5RIZ). Free energy profiles for the interaction of PIP2, phosphatidylserine

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(PS) and of phosphatidylcholine (PC) with PKD2 support the existence of adiscrete binding site, which has a strong selectivity for PIP2 binding. Closeexamination of the protein-lipid interaction at the lowest-energy bindingdistance showed that the head group of PIP2 is coordinated by a cluster ofbasic residues, which are all highly conserved. This suggests that the bindingsite may play a key role in lipid-mediated modulation of PKD2 channelactivity.(1) Ma, R., Li, W., Rundle, D., Kong, J., Akbarali, H. I., and Tsiokas, L. (2005)Mol. Cell. Biol.25, 8285–8298.

1956-PlatMechanism of Regulation of Gi/o-Mediated TRPC4 Activation by Intracel-lular ProtonsQiaochu Wang, Dhananjay P. Thakur, Jinbin Tian, Jaepyo Jeon,Michael X. Zhu.IBP, University of Texas Health and Science Center at Houston, Houston,TX, USA.Transient Receptor Potential Canonical 4 (TRPC4) protein forms non-selective cation channels activated downstream from receptors that signalthrough heterotrimeric G proteins. Although the receptor-operated TRPCchannel activation has mostly been attributed to Gq/11 proteins or receptortyrosine kinases through stimulation of phospholipase C (PLC) isoforms band g, or internal Ca2þ store depletion, our recently work suggests thatTRPC4 channels are particularly coupled to pertussis toxin-sensitive Gi/o

proteins, with a codependence on PLCd1. We showed that the Gi/o-mediatedTRPC4 activation is dually dependent on and bimodally regulated by phos-phatidylinositol 4, 5-bisphosphate (PIP2), the substrate hydrolyzed by PLC,and intracellular Ca2þ, the level of which is increased following PLC stim-ulation due to inositol 1,4,5-trisphosphate induced internal Ca2þ release andCa2þ influx through TRPC4 itself. As a byproduct of PLC-mediated PIP2 hy-drolysis, protons have been shown to play an important role in the activationof Drosophila TRP channels. However, how intracellular pH affectsmammalian TRPC channels was unclear. Using patch-clamp recordings ofHEK293 cells heterologously coexpressing mouse TRPC4b and Gi/o-coupledm opioid receptor, here we investigated the effect of intracellular protons onGi/o-mediated TRPC4 activation. We found that lowering cytosolic pHgreatly accelerated the rate of TRPC4 activation without altering themaximal current density and this effect was dependent on intracellularCa2þ elevation. However, protons did not accelerate channel activation viaa direct action on TRPC4, as shown by the inhibitory effect of low pH onchannel activation by Englerin A, a direct TRPC4 agonist. We proposethat protons exert their effect through sensitization of PLCd1 to Ca2þ, whichin turn promotes TRPC4 opening and further PLCd activities via a positivefeedback mechanism.

Platform: Protein Dynamics and Allostery II

1957-PlatIdentifying Causality in Mutant KRas Residue Pairs from Molecular Dy-namics Data AnalysisSezen Vatansever1, Burak Erman2, Zeynep H. Gumus1.1Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai,New York, NY, USA, 2Chemical and Biological Engineering, KocUniversity, Istanbul, Turkey.K-Ras is the most frequently mutated protein in human cancers. To understandhow an oncogenic mutation impacts K-Ras function, we need to understandhow it alters the regulatory mechanisms of the motions of the protein, whichhave so far remained elusive. Here, we present the structural, conformationaland dynamical changes in K-Ras in response to the most recurrent oncogenicmutation, G12D, using a new integrated Molecular Dynamics (MD) simulationdata analysis approach. Our results show that the G12D mutation leads Switch-II (SII) region motions to negatively correlate with and drive those of theP-loop, Switch-I (SI) and a3 regions. Ours is arguably the first study to revealcausal correlations between the motions of G12D mutant K-Ras residues and tocorrelate them to decay times, which, taken together, can inform new therapeu-tic possibilities.

1958-PlatProtein Hydraulics: Water Mediated Cooperativity of Substrate Bindingin PKAPiotr Setny.University of Warsaw, Warsaw, Poland.Catalytic subunits of protein kinases share a common fold centered aroundseveral precisely arranged key motifs. Multiple amino acids mutations within

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such conserved regions have been identified to either abrogate kinase catalyticfunction or switch it to permanently active state, in any case leading to severedisorders. The analysis of crystallographic structures representing diverse ki-nases reveals the presence of at least several water molecules whose positionswithin the protein core are equally well preserved as amino acid types in manyfunctionally important locations. It remains unknown whether those water mol-ecules play any important role, and whether their removal - disturbing localinteraction patterns to no smaller degree than amino acid mutations - can affectkinase stability and function.We present the results of long computer simulations of PKA catalyticsubunit targeted at the analysis of water structure, kinetics and affinity toburied hydration sites. In addition to already available structural informationour simulations give insights into hydration pockets filled by disordered,highly mobile water molecules, not fully resolved in X-ray structures. Weshow that such regions are vital for the existence of flexible or partially disor-dered protein segments such as the activation and peptide binding loops inkinases.We further demonstrate that communication between the ATP and proteinbinding sites involves an isolated water molecule embedded between theDFG and YRD regions. Modification of its hydrogen bonding patterninduced by ATP binding affects the configuration of the DFGþ1 residue.This in turn shifts the conformational equilibrium of the Pþ1 peptidepositioning loop, promoting more frequent occurrence of peptide bindingconformation. The above findings complement well NMR-based data onchanges in local kinase mobility upon substrate binding, and provide mecha-nistic explanation for experimentally observed binding cooperativity of thetwo substrates.

1959-PlatAn Allosteric Region of Src Tyrosine Kinase Allows for Stabilization of ItsActive-Like ConformationLalima G. Ahuja1, Yilin Meng2, Alexandr P. Kornev3, Benoit Roux2,Susan Taylor4.1Pharmacology, University of California San Diego, San Deigo, CA, USA,2Biochemistry and Molecular Biology, University of Chicago, Chicago, IL,USA, 3Pharmacology, University of California San Diego, San Diego, CA,USA, 4Pharmacology, Chemistry Biochemistry, University of California SanDiego, San Diego, CA, USA.Protein kinases play an essential role as signaling enzymes and their sensi-tive action in the needs of the cell requires their tight regulation. Theiractive conformations are exquisitely regulated and their populationscontrolled by various post-translational modifications. The inactive confor-mation of Src tyrosine kinase is characterized by its non-phosphorylatedactivation loop and the rolled-out aC helix in the N-lobe. The inactiveconformation has the regulatory kinase spine disassembled as the RS3residue from the aC helix is displaced. Our umbrella sampling methods tomap the energy landscape of these conformations has allowed for definingfour states; the inactive, active conformations and two intermediate states.In the present study we extend our umbrella sampling technique to under-stand the activation allostery in Src tyrosine kinase in three different mu-tants. The first mutant is the L297F that occupies an allosteric region inthe N-terminal of the aC helix and allows for activation of Src tyrosine ki-nase analogous to RAFs. The second mutant is the V281F mutation in thecatalytic spine that allows for the closing of the two kinase lobes in theabsence of nucleotide. A double mutant of Src tyrosine kinase V281F/L297F allows for stabilization of a unique catalytically dead kinaseconformation in the active-like state. This unique conformation allows forunderstanding of kinase switching function independent of its catalyticrole. A third M314V mutation in the regulatory spine allows us to explorethe maneuvering of hydrophobicity in the Src tyrosine kinase core. Thesestudies provide a fresh perspective to understanding conformationalswitching in Src family of tyrosine kinases and the dynamic allostery thatunderlies this molecular communication. Our community map analysis ofresidue networks provides an additional layer of understanding to theseobservations.

1960-PlatDirect Observation of GDP Unbinding Reveals Multiple Allosteric Path-ways Underlie G-Protein ActivationSukrit Singh1, Xianqiang Sun1, Kendall J. Blumer2, Gregory R. Bowman1.1Biochemistry and Mol. Biophysics, Washington Univ. in St. Louis, St.Louis, MO, USA, 2Cell Biology and Physiology, Washington Univ. in St.Louis, St. Louis, MO, USA.

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Heterotrimeric G-proteins are molecular switches that play crucial roles insignaling processes throughout biology. Binding of an activated G-Protein-Coupled-Receptor (GPCR) to the G-protein heterotrimer induces GuanosineDiphosphate (GDP) release from the Ga subunit, allowing for activation ofGa via binding of Guanosine Triphosphate (GTP). However, the GPCR-Gabinding interface is �30A away from the GDP binding pocket, and the allo-steric mechanism coupling these two sites remains unclear. Structural andbiochemical studies have elucidated major details and intermediates of therelease pathway, but are insufficient to obtain a full understanding of thismulti-state process. Using long-timescale Molecular Dynamics simulations,we observe the complete mechanism of GDP release, as well as the key struc-tural and entropic changes driving this process. We first detect allosteric path-ways connecting the GPCR- and GDP-binding sites by applying our CARDSmethodology to measure structural and dynamical correlations between resi-dues. Then by combining two powerful simulation methods, Metadynamicsand Markov State Models, we directly observe GDP release from Ga andextract the rate-limiting step of dissociation - a previously unattainable featdue to the long timescale of this process. Using these two perspectives weconstruct an allosteric network of GPCR-mediated activation of Ga proteins.This integrated approach could also identify allosteric networks in other biolog-ical systems or aid drug design efforts by maximizing sampling of boundcomplexes.

1961-PlatConformational Dynamics of Histone Methyltransferase SET8 Probed byMillisecond-Timescale Molecular Dynamics, Markov State Modeling andBiochemical ExperimentsRafal P. Wiewiora1,2, Shi Chen2,3, Minkui Luo3, John D. Chodera1.1Computational and Systems Biology Program, Memorial Sloan KetteringCancer Center, New York, NY, USA, 2Tri-Institutional PhD Program inChemical Biology, Weill Cornell Medicine, New York, NY, USA, 3ChemicalBiology Program, Memorial Sloan Kettering Cancer Center, New York, NY,USA.Histone lysine methyltransferases (HKMTs) are a family of epigeneticenzymes responsible for catalyzing the methylation of lysine residues in his-tones and other proteins. During the past decade, the knowledge of HKMTshas been largely extended from structural and biochemical perspectives,laying foundations for potential therapeutics against multiple diseases, inparticular cancer. However, while evidence is available that HKMTs are con-formationally dynamic in the context of histone methylation and inhibitorbinding, limited knowledge is available about their dynamic conformationalensembles.In this research, multiple approaches were combined to explore the conforma-tional dynamics of SET8, which is responsible for the mono-methylation ofH4K20 and essential for key biological functions, including cell cycle regula-tion. A series of new X-ray structures of SET8 in previously unseen confor-mations were solved with various ligands. Molecular dynamics simulationswere conducted to generate aggregate tens of milliseconds of data andanalyzed by Markov State Models (MSMs) to reveal the free energy land-scapes, hidden conformations, and kinetic pathways between different confor-mations. Steady-state and pre-steady-state kinetics of SET8-mediatedmethylation were characterized to dissect the stepwise details of the bindingevents. A series of crucial residues were identified and validated by mutationsto characterize the conformational change pathways between differentconformations. Taken together, a model describing the conformational dy-namics of SET8 in the process of assembling all catalytic cycle complexeswas derived. Additionally, the structure-function relationships of SET8 cancermutations were predicted and validated based on the proposed model. Weenvision that this work will not only deeper the understanding of the molec-ular mechanism of SET8’s function, but also shed light on addressing similarquestions about other HKMTs and accelerating the rational design of novelHKMT inhibitors through capturing unique conformations of individualenzymes.

1962-PlatHow Collagen Fibrils Dynamically Distribute and Measure StressesAgnieszka Obarska-Kosinska1,2, Christopher Zapp1, Frauke Gr€ater1,3.1Heidelberg Institute for Theoretical Studies, Heidelberg, Germany,2Heidelberg University, Cell Networks, Heidelberg, Germany,3Interdisciplinary Center for Scientific Computing (IWR), HeidelbergUniversity, Heidelberg, Germany.Collagen is the most abundant protein in mammals and a major componentof load-bearing tissues such as tendon, bone or cartilage. Most of the

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collagen in these tissues corresponds to type I collagen, which forms heavilycrosslinked protein fibers. Much is known about collagen’s mechanicalproperties from experiments and simulations. However, the preferred modesof covalent bond scission events and subsequent biochemical processes remainelusive.To investigate how physiological load on collagen relates to the forces in in-dividual molecular bonds of collagen and which bonds are subjected to thehighest forces we employed Force Probe Molecular Dynamics simulations.First, we built a full-atom model of collagen fibril using an integrativestructural biology approach. Then, we reconstructed covalent crosslinksat conserved lysine residues to represent physiological collagen as realis-tically as possible. This resulted in the first large-scale (1.8 Mio atomsincluding water) and yet atomistically resolved structural model of crosslinkedcollagen.Force Probe Molecular Dynamics simulations and internal stress distributionin collagen determined by Force Distribution Analysis suggested lysine-based crosslinks as the most susceptible sites to bond rupture. Quantumcalculations and electron paramagnetic resonance experiments showed thatthe bond rupture leads to formation of radicals, which are known to actas signaling molecules. This suggests collagen to play a hithertounknown role as an intrinsic force gauge by successive and specific ruptureof covalent bonds. Our results have interesting implications for the roleof the different extent and nature of crosslinks in tissues of different kindand age.

1963-PlatInvestigating Chemokine Receptor CCR2 Dynamics and Druggability byEnsemble Based ApproachesBryn C. Taylor1, Irina Kufareva2, Tracy Handel2, Rommie E. Amaro3.1Biomedical Sciences, UC San Diego, La Jolla, CA, USA, 2Skaggs School ofPharmacy and Pharmaceutical Sciences, UC San Diego, La Jolla, CA, USA,3Chemistry and Biochemistry, UC San Diego, La Jolla, CA, USA.Chemokine receptor CCR2 is implicated in a wide range of diseases such asinflammation, autoimmunity, and cancer, and thus is an important drug targetin the pharmaceutical industry. However, CCR2 antagonists have been largelyunsuccessful to date. Recently CCR2 was crystallized for the first time by ourcollaborator Prof. Handel, opening up new opportunities for rational drugdesign. While crystal structures provide valuable snapshots of proteins andprotein complexes, they lack the ability to reveal chemokine receptor dy-namics at a level that is required to develop new drug (lead) molecules. There-fore, key challenges are: (i) understanding how small molecule antagonistsmodulate the protein dynamics, and (ii) developing compounds that bind tonovel (allosteric) binding sites on the receptor. Here, we present our novelapproach to understanding molecular recognition and the inactivation mecha-nism of CCR2, using long-timescale molecular dynamics (MD) simulationsand differential dynamics Markov state modeling (ddMSMs). By comparingthe difference in conformational ensembles (differential dynamics), we aredeveloping new insights into molecular recognition for CCR2, which willidentify unique druggable pockets and ultimately facilitate the design ofnew potential therapeutic compounds for inflammatory and autoimmunediseases.

1964-PlatMHC Class II Complexes Sample Intermediate States along the AntigenicPeptide Exchange PathwaySebastian Stolzenberg1, Marek Wieczorek1, Jana Sticht1,Sebastian G€unther1,2, Christoph Wehmeyer1, Zeina El Habre1,Miguel Alvaro-Benito1, Frank No�e1, Christian Freund1.1Freie Universit€at Berlin, Berlin, Germany, 2University of Maryland Schoolof Medicine, Baltimore, MD, USA.The presentation of antigenic peptide-MHCII complexes (pMHCIIs) for sur-veillance by T cells is a well-known immunological concept in vertebrates,yet the conformational dynamics of antigen exchange remain elusive. Bycombining NMR-detected H/D exchange with Markov modelling analysis ofan aggregate of milliseconds of adaptive molecular dynamics simulations,we reveal that a stable pMHCII spontaneously samples intermediate conforma-tions that are both relevant for peptide exchange and consistent with X-raycrystallography. More specifically, these conformations represent two majorpeptide exchange pathways: the kinetic stability of a pMHCII’s ground statedefines its propensity for intrinsic peptide exchange, while the population ofa rare, intermediate conformation correlates with the propensity of the HLA-DM-catalysed pathway. Helix-destabilizing mutants designed based on ourmodel shift the exchange behavior towards the HLA-DM-catalysed pathway

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and further allow us to conceptualize how allelic variation can shape an indi-vidual’s MHC restricted immune response. This research used resources ofthe Oak Ridge Leadership Computing Facility at the Oak Ridge National Lab-oratory (project IDs: BIP103 and BIP149), which is supported by the Office ofScience of the U.S. Department of Energy under Contract No. DE-AC05-00OR22725.

Workshop: Probing Atomic Single Sites in Cellsand Bio-Assemblies: Advances in In-Cell NMR

1965-WkshpIn Cell NMR: Its Contribution for Understanding Functional ProcessesLucia Banci.Department of Chemistry ‘Ugo Schiff’, University of Florence, SestoFiorentino, Italy.In-cell NMR, i.e. the collection of high resolution NMR spectra of biomole-cules in intact, living cells, represents one of the highest impact applicationsof magnetic resonance.These experiments allow us to obtain information on the conformational andfunctional properties of biomolecules at atomic resolution in conditions ascloser as possible to the physiological ones.Methodological aspects and innovations will be discussed and a few examplesof the striking power of this approach presented. Particular focus will be on theobserved meaningful differences of properties of biological molecules in livingcells with respect to those in vitro.Barbieri L, Luchinat E and Banci L . In-cell NMR spectroscopy in HEK293Tcells: a protocol to characterize proteins in their physiological environment.Nature Protocols 11: 1101, 2016Luchinat E, Barbieri L, Rubino JT, Kozyreva T, Cantini F and Banci L. In-cellNMR reveals potential precursor of toxic species from SOD1 fALS mutants.Nat Commun 5: 5502, 2014Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y and Aricescu AR.Atomic-resolution monitoring of protein maturation in live human cells byNMR. Nat Chem Biol 9: 297-299, 2013.

1966-WkshpStudying Proteins Inside Eukaryotic Cells in NMRIchio Shimada.Division of Physical Chemistry, University of Tokyo, Tokyo, Japan.In-cell NMR spectroscopy is a method used to observe isotopically labeledmolecules within living cells. The first in-cell NMR experiment was performedwith an E. coli overexpressing a 15N-labeled protein. For the first application ofthe in-cell NMR method with eukaryotic cells, isotopically labeled target pro-teins were introduced, by microinjection, into Xenopus laevis oocytes, and acell-penetrating tag was also utilized. Our group reported an in-cell NMRmethod for mammalian cells; we used a pore-forming toxin, streptolysin O(SLO), to introduce target proteins by diffusion. By using these methods,protein-drug interactions and intracellular post-translational modifications,such as phosphorylation and acetylation, were successfully detected in vivo.However, the major limitation of the in-cell NMR experiments is the occur-rence of cell death during the NMR measurement. As the suspension containsa high density of cells, nutrient depletion occurs rapidly in the anaerobic envi-ronment within the NMR tube, thus causing the deterioration of conditions andresulting in cell death during NMR measurements. Therefore, in-cell NMR ex-periments for eukaryotic cells currently have limited applications, such as forobtaining a single NMR spectrum measured within a very short time. Althoughsparse sampling methods have been utilized to shorten the time required to ac-quire multidimensional NMR spectra, many existing in vitro NMR experimentsthat are used to provide information regarding dynamics and protein interac-tions take several hours to perform. In this study, we will show a bioreactor sys-tem for in-cell NMR, which we developed, to suppress the cell death duringNMR measurements, and its recent application in our lab.

1967-WkshpCellular Solid-State NMR Applied to Bacterial and Human cellsMarc Baldus.NMR Group. Bijvoet Center for Biomolecular Research, Utrecht University,Utrecht, Netherlands.Solid-state NMR (ssNMR) is a method that can be applied to gain atomic-levelinsight into heterogeneous molecular systems. Over the last 15 years, ssNMRhas seen strong methodological and instrumental developments that have al-lowed for the characterization of complex molecules including membrane

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proteins, amyloid fibrils or protein biopolymers with remarkable structural ac-curacy and comprehensiveness. More recently, ssNMR has profited from rev-olutionary enhancements in sensitivity, mainly due to the advent of DynamicNuclear Polarization (DNP) and it has seen significant progress in the fieldof ssNMR 1H detection. In our contribution we show how to make combineduse of such methods to probe biomolecules in bacterial and human cells. Ap-plications include protein translocation and insertion machines in bacteriaand extend to membrane associated as well as soluble protein complexes in hu-man cells. We also describe how to combine such ssNMR-based studies withmodalities such as cryo-electron tomography (CET) and Fluorescence micro-scopy to obtain structural insight into cell organization from the atomic tosub-micrometer scale.

1968-WkshpIn-cell NMR Spectroscopy for the Investigation of the Conformation ofMacromoleculesVolker Dotsch.Goethe University Frankfurt, Frankfurt, Germany.The non-invasive character of NMR spectroscopy allows researchers to inves-tigate the conformation and dynamics of biological macromolecules in theirnatural environment, for example the cytoplasm of cells. In the past we haveused in-cell NMR investigations to study several proteins as well as thebehavior of telomeric DNA in different cellular systems. We will show dataon two different systems: The telomeric G-overhang is the 3’ single strandedprotrusion of double stranded telomeres and consists of repeatingd(TTAGGG)n elements. These elements form G-quadruplexes, which howeverunder different in vitro conditions can adopt several different conformations. Inorder to investigate which of these conformations is the biologically relevantconformation we have injected quadruplexes of different length into Xenopusoocytes or investigated them in oocytes extracts. These investigations revealedthat G4 units coexist in two conformations, the hybrid-2 and the 2-tetrad anti-parallel basket. In addition, we have used in-cell NMR to investigate thebehavior of Pin-1, a peptidyl-prolyl isomerase and show that the protein usesits WW domain to nonspecifically investigate other proteins as potential sub-strates. This non-specific interaction can be blocked by phosphorylation inthe WW domain.

1969-WkshpDissecting Bacteria and Mammalian Cells by Whole-Cell NMR: CellWalls, Ribosomes, Nuclei, Oh My!Joseph A.H. Romaniuk, Sabrina Werby, Michelle Park, Lynette Cegelski.Chemistry, Stanford University, Stanford, CA, USA.Our research program is inspired by the challenge and importance of eluci-dating chemical structure and function in complex biological systems and intro-ducing new strategies to prevent and treat human disease. We integrate solid-state NMR spectroscopy with biochemical and microbiological methods todetermine atomic- and molecular-level detail in macromolecular assemblies,intact cells, and bacterial biofilms. In this presentation, I will present our effortsto quantify and identify differences in bacterial cell wall composition by whole-cell NMR and to characterize the modes of action of antibiotics in intact bac-terial cell samples. Traditional biochemical methods have delivered most of theinformation to date regarding antibiotic modes of action, but can be accompa-nied by molecular ambiguities. A new mode of action has even been recentlyascribed to beta-lactams such as penicillin. Solid-state NMR methods enablea comprehensive analysis of cell-wall and cellular pools of carbon and nitrogenand can be used to measure distances between labeled small molecules andlabeled sites in the bacterial cell. We have identified differential spectral signa-tures to quantify cell-wall parameters and to quantify the balance of peptido-glycan and teichoic acid in the bacterial cell wall, even in the absence ofspecific isotopic labeling, and have examined inhibitors with alternate modesof action, such as ribosome-targeting antibiotics. Towards a view of what car-bon pools make up a cell, I will also compare and contrast compositional poolsamong bacteria and mammalian cells.

Workshop: Atoms to Cells: Modeling BiologicalComplexity

1970-WkshpBiomolecular Simulation for AllRon O. Dror.Stanford University, Stanford, CA, USA.

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Molecular dynamics simulations are increasingly recognized as a powerfulcomplement to experimental structural biology, but their use has historicallybeen limited to a relatively small group of specialists. I will discuss progresstoward making atomic-level simulation a broadly accessible tool for molecularbiology, together with key remaining challenges.

1971-WkshpCrowded and Complex: Molecular Simulations of Biological MembranesMark S.P. Sansom1, Anna L. Duncan1, Matthieu Chavent1,2.1Biochemistry, University of Oxford, Oxford, United Kingdom, 2Institut dePharmacologie et de Biologie Structurale IPBS, Universit�e de Toulouse,CNRS, Toulouse, France.Biological membranes are complex in terms of their lipid composition andcrowded with proteins, which occupy between 25 to 40 % protein of the mem-brane area. Furthermore many membrane proteins form strong and specific in-teractions with selected lipids. Coarse-grained molecular dynamics (CG-MD)simulations can be used to explore the consequences of this in terms of theemergent dynamic organization of realistic models of cell membranes. Thesesimulations highlight the role of lipid-mediated interactions between mem-brane proteins. Recent applications include models of bacterial [1], mammalian[2], and viral membranes. The results of very large scale CG-MD simulationsmay be used to parameterize ‘meso’ scale models which allow a more directlink to be made between simulations and experimental data from e.g. singleparticle tracking studies [3].[1] Rassam, P., Copeland, N.A., Birkholz, O., Toth, C., Chavent, M., Duncan,A.L., Cross, S.J., Housden, N.G., Seger, U., Quinn, D.M., Garrod, T.J., San-som, M.S.P. Piehler, J., Baumann, C.G., & Kleanthous, C. (2015) Supramolec-ular assemblies underpin turnover of outer membrane proteins in bacteria.Nature 523:333-336.[2] Koldsø, H., & Sansom, M.S.P. (2015) Organization and dynamics of recep-tor proteins in a plasma membrane. J. Amer. Chem. Soc. 137: 14694-14704.[3] Chavent, M., Duncan, A.L., & Sansom, M.S.P. (2016) Molecular dynamicssimulations of membrane proteins and their interactions: from nanoscale tomesoscale. Curr. Opin. Struct. Biol. 40: 8-16.

1972-WkshpRas Signaling: Allostery, Conformation, and FunctionRuth Nussinov, Hyunbum Jang.Cancer and Inflammation Program, NCI-Frederick, Leidos BiomedicalResearch, Inc., FNL, Frederick, MD, USA.Ras is a small GTPase, controlling signal transduction pathways andpromoting cell proliferation and survival. KRAS is frequently mutated incancer. Ras consists of highly homologous catalytic domains and flexibleC-terminal hypervariable regions (HVRs) that differ significantly acrossRas isoforms. Ras activation is regulated by guanine nucleotide exchangefactors that catalyze the exchange of GDP by GTP, and inactivation is termi-nated by GTPase-activating proteins that accelerate the intrinsic GTP hydro-lysis rate by orders of magnitude. Ras has multiple partners, signals throughseveral key pathways and fulfills critical functions in the cell life. Mutationsin Ras are common in a variety of cancers; yet it is still undruggable.Elucidation of Ras conformational ensembles including the ligand-boundconformations, the activated (or inactivated) allosteric modulated states,post-translationally modified states, mutational states, transition states, andnonfunctional states are essential for deciphering Ras functions from itsconformational landscapes. Our recent works highlight how these mayhelp in elucidating vital mechanistic questions in Ras biology and hopefullycontribute to therapeutic strategies. Funded by Frederick National Labora-tory for Cancer Research, National Institutes of Health, under contractHHSN261200800001E.

1973-WkshpAllostery and Conformational Dynamics in Protein EvolutionS. Banu Ozkan.Physics, Arizona State University, Tempe, AZ, USA.The critical role of protein dynamics has become well recognized in variousbiological functions, including allosteric signaling and protein ligand recog-nition electron transfer. Likewise, in protein evolution, the classical view ofthe one sequence-one structure-one function paradigm is now beingextended to a new view: an ensemble of conformations in equilibriumthat can evolve new functions. Therefore, understanding inherent structuraldynamics are crucial to obtain a more complete picture of protein evolution.A small local structural change due to a single mutation can lead to a

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large difference in conformational dynamics, even at quite distant residuesdue to allostery. We have recently analyzed evolution of different proteinfamilies including GFP proteins, beta-lactamase inhibitors, and nuclear re-ceptors and observed that alteration of conformational dynamics throughallosteric regulations leads to functional changes. Moreover, our site-specific dynamics-based metric reveals that enzymatic function is regulatedby dynamically-coupled residues, which forms an allosteric communicationnetwork with the active sites. Disease causing mutations trigger a globalloss in dynamic coupling, which disrupts the communication network ulti-mately inhibiting function. Analysis of over 200 missense mutations alsoshows that Gaucher disease (GD) mutations are abundant at dynamic allo-steric residue coupling sites which we call DARC spots. Further tests using75 human enzymes revealed that diseases emerging from DARC spot muta-tions are not isolated to GD; indeed, this phenomenon is observed across theproteome.

1974-WkshpCell Biophysics with Virtual CellLeslie Loew.Center for Cell Analysis and Modeling, U. Conn. School of Medicine,Farmington, CT, USA.Virtual Cell (VCell.org) is a problem solving environment for cell biologicalmodeling built on a central database and disseminated as a client-server appli-cation. The philosophy behind VCell is to enable cell biologists andbiophysics to perform experiment driven analysis and modeling. It does thisthrough a powerful and user-friendly user interface. VCell supports multiplebiophysical mechanisms: reaction kinetics, diffusion, flow, membrane trans-port, lateral membrane diffusion, electrophysiology and rule-based modelsof multi-state/multimolecular interactions. Reaction-diffusion simulationscan be based on 3D analytical geometries or segmented experimental imagesof cells and tissues. VCell includes solvers for ODEs, PDEs, stochastic reac-tion kinetics, network-free simulations, spatial particle-based simulations andspatial hybrid stochastic/deterministic simulations. (supported by NIH grantP41 GM103313).

Workshop: From Molecules to Mammals:Imaging, Sensing, and Light Control

1975-WkshpSmall and Bright: Tailoring Luminescent Nanoparticles for BiologyGang Han.University of Massachusetts Medical School, Worcester, MA, USA.Functional luminescent nanoparticles are promising materials for in vitro andin vivo optical imaging and therapy due to their unique optical and chemicalproperties. In this talk, I will present three new types of biocompatible lumi-nescence nanoparticles. The first type of materials is upconversion nanopar-ticles (UCNPs). I will present new developments regarding engineeringUCNPs towards optogenetic applications in neuroscience and immunotherapy.The second type of nanoparticles is persistent luminescence nanoparticles(PLNPs). They are bioluminescence-like and possess unprecedented in vivodeep tissue energy rechargeability, outstanding signal-to-noise-ratio with noneed for an excitation resource (light) during imaging, and they can bedirectly detected with existing imaging systems. These nanoparticles continueto emit light for minutes or hours and, in some cases, days, after turning offthe excitation source. These long-lasting, light-emitting nanocrystals can pro-vide noninvasive imaging technology for evaluating structural and functionalbiological processes in living animals and patients. The third is a type oforganic Biodpy nanoparticles that were tailored with outstanding NIRabsorbing ability. Rather than the conventional laser light needed in PDT, Iwill present their ultralow power lamp operable PDT applications in deep tis-sue tumor treatment.

1976-WkshpOptogenetic FMRI and the Investigation of Global Brain CircuitMechanismsJin Hyung Lee.Stanford University, Stanford, CA, USA.Understanding the functional communication across brain has been a longsought-after goal of neuroscientists. However, due to the widespreadand highly interconnected nature of brain circuits, the dynamic relationshipbetween neuronal network elements remains elusive. With the development

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of optogenetic functional magnetic resonance imaging (ofMRI), it isnow possible to observe whole-brain level network activity that resultsfrom modulating with millisecond-timescale resolution the activity ofgenetically, spatially, and topologically defined cell populations. ofMRIuniquely enables mapping global patterns of brain activity that result fromthe selective and precise control of neuronal populations. Advances in themolecular toolbox of optogenetics, as well as improvements in imagingtechnology, will bring ofMRI closer to its full potential. In particular, theintegration of ultra-fast data acquisition, high SNR, and combinatorial opto-genetics will enable powerful systems that can modulate and visualize brainactivity in real-time. ofMRI is anticipated to play an important role in thedissection and control of network-level brain circuit function and dysfunc-tion. In this talk, the ofMRI technology will be introduced with advancedapproaches to bring it to its full potential, ending with examples of dissect-ing whole brain circuits associated with neurological diseases utilizingofMRI.

1977-WkshpBuilding Proteins to Peek and Poke at GTPase Circuits In VivoKlaus M. Hahn.Pharmacology, University of North Carolina, Chapel Hill, NC, USA.This presentation will focus on new approaches to visualize and manipulatesignaling networks in living cells, including what we believe are broadly appli-cable methods to control proteins with light, and less perturbing approaches tovisualize signaling. We are probing the role of Rho family GTPase circuits inimmune cell function, using engineered allosteric switches to photoinhibit orphotoactivate guanine exchange factors, kinases, and GTPases as we visualizeeffects on downstream signals in real time. Success with three different proteinfamilies leaves us optimistic that there is a simple way to identify and controlallosteric networks within proteins.

1978-WkshpIlluminating the Biochemical Activity Architecture of the CellJin Zhang.University of California, San Diego, La Jolla, CA, USA.The complexity and specificity of many forms of signal transduction are widelysuspected to require spatial microcompartmentation and dynamic modulationof the activities of signaling molecules, such as protein kinases, phosphatasesand second messengers. We have developed a series of fluorescent biosensorsto probe the compartmentalized signaling activities. In this talk, I will firstintroduce a series of fluorescent biosensors, focusing both on their designsand general applications, and then present studies where we combined geneti-cally encoded fluorescent biosensors, superresolution imaging, and targetedbiochemical perturbations to probe the biochemical activity architecture ofthe cell.

1979-WkshpEngineering of Bacterial Phytochromes for Near-Infrared Imaging,Sensing and Light-Control in MammalsDaria M. Shcherbakova1, Andrii A. Kaberniuk1, Taras A. Redchuk2,Vladislav V. Verkhusha1,2.1Albert Einstein College of Medicine, Bronx, NY, USA, 2University ofHelsinki, Helsinki, Finland.Near-infrared light is favorable for imaging in mammalian tissues due tolow absorbance of hemoglobin, melanin and water, and low light-scattering.Therefore, fluorescent proteins, biosensors and optogenetic constructs foroptimal imaging, optical readout and light manipulation in mammals shouldhave fluorescence and action spectra within the near-infrared window. Inter-estingly, natural bacterial phytochrome photoreceptors (BphPs) utilize thelow molecular weight biliverdin, found in most mammalian tissues, as aphotoreactive chromophore. Due to their near-infrared absorbance BphPsare preferred templates for designing optical molecular tools for applicationsin mammals. Based on the analysis of the photochemistry and structure ofBphPs we developed several BphP-based fluorescent proteins, biosensors,and optogenetic tools. Near-infrared fluorescent proteins and biosensorsextend the methods developed for conventional microscopy into a deep-tissue in vivo macroscopy including multicolor cell and tissue labeling, fluo-rescence resonance energy transfer, cell photoactivation and tracking, anddetection of enzymatic activities and metabolites in tissues. Near-infrared op-togenetic tools allow noninvasive light-control of biochemistry and physi-ology of an animal directly through the skin. Moreover, all BphP-basedreagents spectrally complement existing genetically encoded probes in thevisible range.

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Workshop: Biomembrane Models and Tools

1980-WkshpGiant Vesicles as Handy Tools for Assessing Membrane Mechanics, Wet-ting and ReshapingRumiana Dimova.Theory & Bio-Systems, Max Planck Institute of Colloids and Interfaces,Potsdam, Germany.Giant vesicles are a fascinating model membrane system, which has beeninitially established and used as a workbench for studying basic propertiesof simple lipid bilayers. Nowadays, they are increasingly employed by bio-physicists to unravel the mechanisms driving various biological processesoccurring at the level of the cell membrane. The success of this enterpriseis evidenced by the widening acceptance of giant vesicles as a mimetic sys-tem among biologists. Methodologies for assessing the membrane materialproperties and effects of membrane remodeling factors as deduced frommeasurements on giant vesicles become increasingly important. In thistalk, we will introduce such methods and showcase approaches formeasuring mechanical properties such as bending rigidity and spontaneouscurvature. The latter is readily generated by asymmetries across the mem-brane. Two examples will be considered: spontaneous curvature generatedby asymmetric distribution of GM1 in the bilayer leaflets, and asymmetricadsorption of poly(ethylene glycol), both of which result in the spontaneousformation of cylindrical or necklace-like lipid nanotubes (see e.g. ACSNano 10:463, 2016). In addition, the process of wetting of the membraneby crowded aqueous phases will be discussed as a membrane reshaping fac-tor leading to a variety of vesicle shape transformations (Soft Matter 8:6409,2012; Adv. Mater. Interfaces 4:1600451, 2017). The presented exampleswill demonstrate that even in the absence of proteins and active processes,the membrane is easily remodeled by simple physicochemical effects. Thiswork is part of the MaxSynBio consortium, which is jointly funded by theFederal Ministry of Education and Research of Germany and the MaxPlanck Society.

1981-WkshpConstructing and using Phase Diagrams of Multi-component LipidMixturesGerald W. Feigenson.Field of Biophysics, Cornell University, Ithaca, NY, USA.For most studies of lipid mixture models of biomembranes, researchersmust understand which phases are present and in what fractions in order tointerpret almost any kind of signal information from their samples. This work-shop talk describes both the conceptual value of phase diagrams for under-standing the lipid bilayer as a fascinating state of matter, and also details ofuseful methods that we have found to yield reliable phase boundaries andphase identification. In addition, highlighting some problematic experimentalmethods might be of value to those who want to solve their own phasediagrams.

1982-WkshpThe Styrene-Maleic Acid Copolymer: A Versatile Tool in MembraneResearchJ. Antoinette Killian.Membrane Biochemistry & Biophysics, Utrecht University, Utrecht,Netherlands.

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A promising tool in membrane research is the solubilization of membranes bystyrene-maleic acid (SMA) copolymers. These amphipathic molecules areable to solubilize membranes in the form of nanodiscs, allowing solubilizationof membrane proteins in their native lipid environment and characterizationby a range of techniques. Properties of the polymers, environmental condi-tions and membrane composition all appear to play an important role in deter-mining the efficiency of membrane solubilization and the properties of theresulting nanodiscs. Here I will discuss recent insights into these mattersbased on experiments in model membrane systems and in biologicalmembranes.

1983-WkshpPlasma Membrane ModelsKalina Hristova.Johns Hopkins University, Baltimore, MD, USA.The biophysical characterization of proteins in plasma membranes often neces-sitates the development of simplified membrane model systems that capture thecomplexity of the native cellular environment and enable quantitative measure-ments. I will describe two such model systems: plasma membrane vesicles andcells under reversible osmotic stress. I will discuss the production and charac-terization of these model membrane systems, and I will focus on their utilityand their limitations.

1984-WkshpNanopore-Confined Bilayers: A Model of Biomembranes with DefinedCurvature and a Tool for Oriented Sample Magnetic ResonanceAlex I. Smirnov.Chemistry, North Carolina State University, Raleigh, NC, USA.It is well established that biological function of cellular membranes is deter-mined by both membrane proteins and the membrane biochemical composi-tion. Moreover, the membrane shape/local curvature may also play a role inmodulating membrane properties and the proteins’ function. While specificbiomembrane compositions are more readily replicated in biophysicalbench experiments, formation of bilayers of specific shapes and curvaturerepresents a more challenging task. Macroscopic alignment of such mem-branes – an important prerequisite of high resolution magnetic resonanceand other studies – is even more difficult especially over a broad range ofexperimental conditions such pH, ionic strength, and temperature. Here wedescribe methods for forming self-assembled lipid nanotubular bilayers in-side cylindrical nanopores composed of anodic aluminum oxide (AAO).Such hybrid nanostructures, named lipid nanotube arrays, represent a newtype of substrate-supported and macroscopically-aligned lipid bilayers ofdefined curvature that have many attractive features for both membranebiophysics and structure-function protein studies by spectroscopic tech-niques. Optical properties of AAO allow for assessing the integrity of mem-brane protein complexes by UV-vis while high density of the depositedlipids and proteins enable examination by other biophysical methods,including DSC, QCM, and magnetic resonance. The latter studies haveshown that the individual lipids in such nanopore-confined structures main-tain fast uniaxial diffusion and a high degree of macroscopic alignment. Themacroscopic alignment enables detailed studies of effects of lipid composi-tion on structure of integral membrane proteins by solid state oriented sam-ple NMR, EPR and DEER. Accessibility of either both or mainly innerleaflet of the nanotubular bilayers to water-soluble species provides forstudies of protein, peptides, and drug binding. Supported by DE-FG02-02ER15354 to AIS.

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Posters

Posters: Protein Structure and Conformation II

1985-Pos Board B1Mechanism of Phospholipase iPLA2b Activity and Regulation Revealed bythe Novel Crystal StructureSergey V. Korolev.Biochemistry, Saint Louis University, St. Louis, MO, USA.Calcium-independent phospholipase iPLA2b (also known as PLA2G6A orPNPLA9) is a signaling enzyme which hydrolyzes phospholipids to generatepotent lipid second messengers in response to stress or injury. The enzyme isa product of the PARK14 gene with strong genetic link to a spectrum of neuro-degenerative disorders including Parkinson’s disease (PD). It is also linked toidiopathic PD and represents one of the major phospholipase activities in thebrain. Alterations in iPLA2b function have demonstrated its role in other humanpathologies including cardiovascular disease, cancer and diabetes. Mechanismsof its activation and tissue-specific functions remain poorly understood. This isin contrast to known enzymatic activity and several well-characterized down-stream signaling cascades implicated in agonist-induced arachidonic acidrelease, insulin secretion, vascular constriction/relaxation, store-operated cal-cium-entry, cellular proliferation, migration and autophagy.We have solved a crystal structure of the full length mammalian iPLA2b andinvestigated mechanisms of the protein activity and interaction with calmod-ulin. The crystal structure of significantly revises existing mechanistic models.It demonstrated unexpected oligomeric structure and the conformation of cat-alytic and auxiliary protein-interaction domains. The structure suggests themechanisms of inhibition by calmodulin, activation through the autoacylationreaction and the potential role of ATP in stabilizing ankyrin repeats. The novelcrystal structure together with biochemical studies has immediate implicationsfor the mechanisms of the phospholipase activity, of the inhibition and activa-tion as well as of the potential mechanism of tissue specific cellular localiza-tion. It provides a well-defined framework to investigate the role ofneurodegenerative mutations and the function of iPLA2bin the brain as wellas its role in other diseases.

1986-Pos Board B2Exploring a Novel Oligomerization Mechanism of Thermostable DirectHemolysin, a Pore-Forming ProteinNidhi Kundu, Kausik Chattopadhyay.Biological Sciences, Indian Institute of Science Education and ResearchMohali, Mohali, India.Thermostable direct hemolysin (TDH) is a pore-forming toxin and a majorvirulence factor of pathogen Vibrio parahaemolyticus . It permeabilizes targethost cells by generating pores on the cell membranes. Unlike other pore-forming toxins, TDH follows a distinct mode of action by forming oligomersin solution, without any prior membrane binding event. However, the reasonbehind this unique in-solution oligomerization propensity of TDH still remainsunknown. In the present study, we show the role of the C-terminal region ofTDH in solution oligomerization by dissecting its interaction at theprotomer-protomer interface. Our study also highlights the mechanism bywhich a single conserved disulphide bond within the C-terminal region regu-lates the formation of the in-solution oligomers by TDH. Our study for the firsttime attempts to establish the possible mechanistic basis behind the unique ten-dency of TDH to form active oligomers without prior binding to the host cellmembranes. Present work also opens new avenues to address the questionslike why in-solution oligomerization is absent in the structurally-related eu-karyotic pore-forming toxins.

1987-Pos Board B3Can b-Cyclodextrin Encapsulated Polyphenols Combat Oxidative Stress?A Case Study with Ribonuclease a ProteinPritam Roy, Swagata Dasgupta.Chemistry, Indian Institute of Technology Kharagpur, Kharagpur, India.Aging in living cells is a direct consequence of oxidative stress in proteins,caused due to the generation of reactive free radical species in the biologicalsystem. Protein oxidation leads to modification of amino acid residues whichcan serve as a marker for estimating oxidative stress. Out of several techniquespromoted for studying protein oxidation, one of the widely used markers inoxidative stress is the formation of dityrosine (DT) cross-linkages in proteinssince it gives a strong fluorescence emission. Oxidative stress not only causesa major structural change in proteins but also affects regular function. One ofthe well-known proteins, Ribonuclease A (RNase A) which has 6 Tyr residues,shows a characteristic DT fluorescence peak upon oxidation using potassium

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persulfate and cobalt (II). RNase A is generally associated with cleaving ofRNA during the transcription and translation processes thus proper functioningof RNase A is essential for protein synthesis. The RNase A dimer formed dur-ing oxidation shows significant changes in its secondary structure compared tothe native protein. Upon incubation with polyphenols like Gallic Acid (GA),Epicatechin gallate (ECG) and Epigallocatechin gallate (EGCG) there is signif-icant decrease in DT formation. Although polyphenols like ECG and EGCGexhibit strong antioxidant effect, it promotes protein oligomerization due toprotein-polyphenol cross-linking. On the other hand encapsulation of polyphe-nols in b-cyclodextrin (b-CD) prevents protein oligomerization as well as DTformation. This may be attributed to the fact that the quinone forming rings ofECG and EGCG are encapsulated in the cavity of b-CD and hence no longeravailable for protein cross-linking.

1988-Pos Board B4The Conformation of Human Phospholipid Scramblase 1, as Studied byInfrared Spectroscopy. Effects of Calcium and DetergentNagore Andraka, Lissete Sanchez-Magraner, Marcos Garcia-Pacios,Felix M. Goni, Jose L. Arrondo.Instituto Biofisika (CSIC-UPV/EHU), Leioa, Spain.Human phospholipid scramblase 1 (SCR) is a membrane protein that catalyzesthe transmembrane (flip-flop) motion of phospholipids. It can also exist in a nonmembrane-bound form in the nucleus, where it modulates several aspects ofgene expression. Catalysis of phospholipid flip-flop requires the presence ofmillimolar Ca2þ, and occurs in the absence of ATP. Membrane-bound SCRcontains a C-terminal a-helical domain embedded in the membrane bilayer.The latter domain can be removed giving rise to a stable truncated mutantSCRD that is devoid of scramblase activity. In order to improve our understand-ing of SCR structure infrared spectra have been recorded of both the native andtruncated forms, and the effects of adding Ca2þ, or removing detergent, or ther-mally denaturing the protein have been observed. Under all conditions the mainstructural component of SCR/SCRD is a b-sheet. Removing the C-terminal 28aa residues, which anchor SCR to the membrane, leads to a change in tertiarystructure and an increased structural flexibility. The main effect of Ca2þ is anincrease in the a/b ratio of secondary structure components, with a concomitantincrease in the proportion of non-periodic structures. At least in SCRD, deter-gent (Zwittergent 3-12) decreases the structural flexibility, an effect somewhatopposite to that of increasing temperature. Thermal denaturation is affected byCa2þ, detergent, and by the presence or absence of the C-terminal domain, eachof them influencing in different ways the denaturation pattern.

1989-Pos Board B5Structural and Functional Characterization of Vitronectin and Ail forHost Cell Invasion by Y. pestisLuz M. Meneghini, L. Miya Fujimoto, Yong Yao, Francesca M. Marassi.Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.Ail (attachment invasion locus) is a membrane protein that is highly expressedon the surface of the bacterium Yersinia pestis, which is the causative agent ofplague. Ail interacts with various human host factors to promote pathogenesisand bacterial survival including: (1) resistance to lysis by the human comple-ment system, a major effector of innate immunity; (2) adhesion to human cells;(3) suppression of blood clotting; and (4) enhanced fibrinolysis. Recently, wehave demonstrated that Ail also recruits Vitronectin (Vn) from human serumto the Y. pestis cell surface and promotes its degradation by the Y. pestis outermembrane protease Plasminogen activator (Pla). Vn is a 459-residue multido-main, multifunctional protein that regulates the complement, coagulation andfibrinolysis pathways. It contains binding sites for a wide range of molecules,including Heparin, Integrins and pathogenic proteins. Despite its importance,little is known about the structure of Vn, or the molecular interaction betweenAil and Vn to promote Y. pestis pathogenesis. Here, we demonstrate that Vnfragments can be expressed and purified from E. coli for in vitro binding assaysand NMR structural studies. The data provide molecular information about theinteraction of Ail with this central human protein and help explain the func-tional role of Ail in Y. pestis pathogenesis.

1990-Pos Board B6Probing the Elongated Structure of a Streptococcal Surface Protein usingStructural and Single-Molecule Biophysical ApproachesJames A.H. Gilburt, Christoph G. Baumann, Jennifer R. Potts,Fiona Whelan.Biology, University of York, York, United Kingdom.The protein Sgo0707 from Streptococcus gordonii is expressed on the bacte-rial cell surface and is proposed to be involved in binding to oral keratino-cytes. Similar to several other surface proteins of Gram-positive bacteria,Sgo0707 contains a repetitive C-terminal region; in this case, repeats contain84-88 amino acids. The aim of this work is to determine if the repetitive

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region forms a rod-like structure that projects the N-terminal functionaldomain of the protein from the bacterial cell surface. We have usedSHRImP-TIRFM, a super-resolution fluorescence microscopy technique, tomeasure the end-to-end distance of a 7 repeat protein construct, and SEC-MALS-QELS to measure its hydrodynamic radius. Combined with crystalstructures of both single and double repeat structures, we will assess the elon-gation of the repetitive region.

1991-Pos Board B7Solution Structures of Wildtype and Deglycosylated Neuropilin 1Raphael Reuten1, Natalie Krahn2, Matthew McDougall2,Denise Nikodemus1, Makrus Meier2, Manuel Koch3, Joerg Stetefeld2,Trushar R. Patel4.1Biotech Research and Innovation Centre, University of Copenhagen,Copenhagen, Denmark, 2Chemistry, University of Manitoba, Winnipeg, MB,Canada, 3Institute for Dental Research and Oral Musculoskeletal Biology,University of Cologne, Cologne, Germany, 4Alberta RNA Research andTraining Institute, University of Lethbridge, Lethbridge, AB, Canada.Neuropilin-1 (NRP1) is the cellular growth factor that interacts with sema-phorin 3A, placenta growth factor-2 and vascular endothelial growth factor(VEGF165). The interactions of NRP1 with these proteins initiate signalingpathways that have implications on fundamental cellular processes. NRP1have been designated as a potential target for the treatment of various typesof cancers. It is composed of two CUB domains (a1 and a2) that are con-nected with F5/8 type C1 (b1) and C2 (b2) domains, followed by a flexiblelinker, a MAM domain, membrane-anchored region and a cytoplasmic tail.The high-resolution structures of various domains of human NRP1 (a2, b1and b2 and MAM domains) are available. However, the information ofhow the a1 domain is connected to the a2, b1 and b2 domains and howthe overall assembly behaves is unavailable. Recently, a crystal structureof mouse a1, a2, b1 and b2 (PDB:4Z9) domains suggested that the a1domain is linked with the rest of the domains via a flexible linker. We char-acterized the wild-type NRP1 composed of a1, a2, b1 and b2 domains and amutant version that lacks glycosylation using the dynamic light scattering,analytical ultracentrifugation, and small angle X-ray scattering techniques.The results from all three techniques suggest that the glycosylation is crucialfor the stability and homogeneity of NRP1. Furthermore, all though no majordifference was found between these two versions of NRP1 in terms of theirlow-resolution structures obtained using small angle X-ray scattering, theirsolution conformation differs significantly compared to the crystal structureof the deglycosylated version of mouse a1, a2, b1 and b2 structure. Based onour preliminary data, we hypothesize that the flexible linker between the a1and a2 domains allow efficient interaction with ligands to initiate signalingpathways.

1992-Pos Board B8Monomerization of XIAP by Executioner CaspasesJamshid Davoodi, Hossein Hozhabri, Hossein Hozhabri.University of Tehran, Tehran, Islamic Republic of Iran.A class of cysteine proteases known as caspases are responsible for a physio-logical process called Apoptosis. These enzymes are classified as initiatorand executioner caspases based on the stage they enter the apoptosis process.The initiator caspases of�8 and�10 become activated by extracellular signalsin the extrinsic apoptosis pathway and initiator caspase-9 is activated by intra-cellular signals during the intrinsic apoptosis pathway. These pathways inter-sect at the executioner caspase levels which consists of �3 and �7. Caspasesinvolved in the intrinsic apoptotic pathway possess a tetrapeptide motif calledIAP binding Motif, IBM, which plays crucial role in antagonizing and regu-lating their activities by Inhibitor of Apoptosis Proteins, IAPs. This evolu-tionary conserved motif is found at the N-termini of the small subunits ofcaspase-9, �7, and �3. We also identified an IBM motif at the large subunitof caspase-7 which showed higher affinity toward XIAP as compared to itssmall subunit. We therefore hypothesized that as far as XIAP interaction is con-cerned, caspase-3 and caspase-7 should behave differently. To decipher XIAP-caspase interactions size exclusion high pressured liquid chromatography wasperformed. The retention time analysis revealed that procaspase-3 is a dimericprotein as reported previously. However active caspase-3 as well as caspase-7appeared as heterodimer of the large and the small subunits as opposed to a ho-modimer of a heterodimer. XIAP protein consisting of the BIR1-2-3 domainson the other hand appeared as dimer. Interestingly, interaction of eithercaspase-3 or caspase-7 caused XIAP dissociation into monomers while cas-pases maintaining their monomeric states. Given that dimerization is requiredfor caspases activation it is intriguing to propose that one mechanism for cas-pase inhibition by XIAP is through locking the executioner caspases in themonomeric state.

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1993-Pos Board B9The Stability, Reduction Potential and Ligand State of Two Conformationsof a C-Type Cytochrome from the Diatom Thalassiosira PseudonanaSaveeta May Rampur, Evelyn Bordeaux, Emily Tabaie, Katherine Frato.Chemistry, Seattle University, Seattle, WA, USA.In this study we investigate the structure and function of a c-type cytochromefrom the diatom Thalassiosira pseudonana, Tp34211. The axial iron ligand of ac-type cytochrome is often either a methionine or histidine residue. The axialligand plays an important role in determining the midpoint potential of theheme cofactor, which in turn determines the possible physiological electron do-nors and acceptors. Initial experiments suggested that recombinantly expressedTp34211 forms two alternative conformations that can be differentiated bySoret band changes upon imidazole binding or quenching of the tryptophanfluorescence emission signal. Based on sequence alignments of Tp34211 andknown cytochrome c6 proteins, we predict that Tp34211 has an axial histidineligand (H78), but a neighboring lysine residue (K79) could serve as an alternateligand. To test the possible role of ligand switching in the two alternative con-formations observed for Tp34211, we recombinantly expressed and purifiedboth wild-type Tp34211 and a K79A variant that should force bis-His ligation.Following purification by ion-exchange and size-exclusion chromatogrpahy,guanidine induced unfolding assays were performed on the various proteinsamples, with extent of unfolding measured via intrinsic tryptophan fluores-cence. The DG�

unfolding of the unquenched conformation of WT Tp34211and the K79A mutant were determined to be the same within error, suggestingthat histidine coordination is favored under normal solution conditions.Midpoint potentials of the K79A variant and the two conformations of thewild-type protein will also be measured to further identify the role of histidineversus lysine ligation in Tp34211.

1994-Pos Board B10Environmental Calcium Controls Alternate Physical States of the Caulo-bacter Surface LayerJonathan Herrmann1, John Smit2, Lucy Shapiro3, Soichi Wakatsuki1.1Structural Biology, Stanford University, Stanford, CA, USA, 2University ofBritish Columbia, Vancouver, CA, Canada, 3Developmental Biology,Stanford University, Stanford, CA, USA.Surface layers (S-layers) are paracrystalline, proteinaceous structures found inmost archaea and many bacteria. Often the outermost cell envelope component,S-layers serve diverse functions including aiding pathogenicity and protectingagainst predators. We report that the S-layer of Caulobacter crescentus exhibitscalcium-mediated structural plasticity, switching irreversibly between an amor-phous aggregate state and the crystalline state. This finding invalidates the com-mon assumption that S-layers serve only as static wall-like structures. In vitro,the Caulobacter S-layer protein, RsaA, enters the aggregate state at physiolog-ical temperatures and low divalent calcium ion concentrations. At higher con-centrations, calcium ions stabilize monomeric RsaA, which can then transitionto the two-dimensional crystalline state. Caulobacter requires micromolar con-centrations of calcium for normal growth and development. Without an S-layer,Caulobacter is even more sensitive to changes in environmental calcium con-centration. Therefore, this structurally dynamic S-layer responds to environ-mental conditions as an ion sensor and protects Caulobacter from calciumdeficiency stress, a unique mechanism of bacterial adaptation. These findingsprovide a biochemical and physiological basis for RsaA’s calcium-bindingbehavior, which extends far beyond calcium’s commonly accepted role in aid-ing S-layer biogenesis or oligomerization and demonstrates a connection tocellular fitness.

1995-Pos Board B11Species Differences in Visual Arrestin Multimerization Revealed byAnalytical UltracentrifugationCassandra Barnes1, Kevin Namitz2, Michael Cosgrove2, Peter Calvert1.1Dept. Ophthalmology, SUNY Upstate Medical University, Syracuse, NY,USA, 2Dept. Biochemistry, SUNY Upstate Medical University, Syracuse,NY, USA.Arrestins play important roles in GPCR signaling and desensitization. Thephotoreceptor specific visual arrestin (Arr1) is a soluble protein with noknown post-translational modifications, that is differently distributedthroughout light and dark-adapted photoreceptor. Second only to its targetGPCR, rhodopsin in concentration, mechanisms that underlie the transloca-tion of such an abundant protein are not understood. Arr1 is predicted toself-associate up to tetramers across multiple mammalian species, varyingmostly in the KD of the dimer and tetramer forms. One hypothesis on thelocalization of Arr1 in the cell body of dark-adapted photoreceptors is thatthe Arr1 tetramer is partitioned there due to differences in densities of subcel-lular structures in the two compartments and the impact of steric volume

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exclusion on the accessible volumes. To investigate whether this self-associative behavior applies across a broader range of species and other modelorganisms, we examined Xenopus laevis Arr1 (xArr1) which bears �68%sequence identity to mammalian Arr1s. Homology modeling based on thebovine crystal structure suggests that it is likely to have a very similar tertiarystructure. Residues of mammalian Arr1 hypothesized to be at dimer andtetramer interfaces by their ability to affect the extent of oligomerizationare mostly conserved in X. laevis as well, suggesting xArr1 would likelyfollow the same self-association pattern. We performed analytical ultracentri-fugation experiments on xArr1 as well as bovine Arr1 (bArr1) for comparison.Our data for bArr1 agrees well with KDs from published studies, and the KD

dimer for xArr1 fits well into the range published for human, mouse, and bovineArr1. Surprisingly, at concentrations up to 210 micromolar, our results showxArr1 as monomer and dimer only, with no evidence of higher order oligo-mers. The mechanism underlying this difference is under investigation.

1996-Pos Board B12Non-symbiotic Hemoglobin Conformational Space Dependence on theHEME Coordination using NESI-TIMS-TOF MSDavid Butcher1, Sophie Bernad2, Valerie Derrien2, Pierre Sebban2,Jaroslava Miksovska1, Francisco Fernandez-Lima1.1Chemistry and Biochemistry, Florida International University, Miami, FL,USA, 2Laboratoire de Chimie Physique, Universit�e Paris-Sud 11, Orsay,France.In this study, for the first time, the conformational space of the rice non-symbiotic hemoglobin type 1 (rHb1) was studied as a function of the startingsolution pH using trapped ion mobility spectrometry coupled to mass spectrom-etry (TIMS-MS) and molecular dynamics. Comparison of the charge state dis-tribution, apo to holo form ratio, and the collision cross section (CCS) profilesas a function of the solution pH showed higher stability of the rHb1 wild-type(WT) when compared with the H73L mutant at mildly acidic conditions. Com-parison of the CCS profiles of the rHb1 WT and H73L holo and apo formshowed that only the initial unfolding pathways involved the heme cavity,with and without a heme loss, followed by unfolding pathways not necessarilyinvolving the environment of the heme prosthetic group. Representative candi-date structures for the nine transitions observed in the CCS profiles were pro-posed using molecular dynamic simulations. Inspection of the representativestructures of the CCS ensemble suggests that the rHb1 unfolding is driven byinitial distancing of the A, B, and H helices, while the heme cavity and hemegroup remains intact, followed by the distancing of the E, F, and G helicesand subsequent loss of the a-helical structure leading to a final random coilconformation.

1997-Pos Board B13Studies of the Behavior of Individual (and Combined) Domains of HumanE- and N-CadherinPrince Tiwari.Biological Scicnces, Indian Institute of Science Education and ResearchMohali, Mohali, India.Cadherins are calcium-dependent cell surface proteins which play a key rolein cell-cell interactions. We have cloned and expressed over 20 different con-structs of domains derived from human E- and N-cadherins, both singly and incombinations (i.e., as fused domains), with the intention of obtaining a betterunderstanding of the structural contents and structural-biochemical behav-iour(s) of domains within the context of the whole protein, and upon excisionand production as truncated forms. Using a variety of biophysical andbiochemical methods, we have examined domain structural contents and ef-fects of Ca-binding, and domain-domain interactions (involved in cell-cellinteraction), upon these domains. Insights obtained will be presented. Mainly,they support the notion that the extracellular domains numbered 1, and 2, ofboth E- and N-cadherins, are involved in the most important intermolecularand inter-cellular interactions, with domains 3, 4 and 5 performing supportiveroles.

1998-Pos Board B14Interaction of the ASAP1 PH Domain with the N Terminus of ARF1 isControlled by Conformational SwitchingNeeladri S. Roy1, Peng Zhai1, Xiaoying Jian1, Lisa Jenkins2, Ruibai Luo1,Marielle E. Yohe3, Paul A. Randazzo1.1Lab of Cellular & Molecular Biology, NCI/NIH, Bethesda, MD, USA, 2Labof Cell Biology, NCI/NIH, Bethesda, MD, USA, 3Pediatric OncologyBranch, NCI/NIH, Bethesda, MD, USA.Arfs belong to the Ras superfamily of small GTP binding proteins that playcrucial roles in membrane trafficking and actin cytoskeleton remodeling.Arfs are active in GTP bound form and switching off Arfs involve hydrolysisof its bound GTP, a process catalyzed by Arf GTPase- activating proteins (Arf

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GAPs). Humans have 31 genes for Arf GAPs. 21 of these encode proteinscontaining a pleckstrin homology (PH) domain that is essential for GAP ac-tivity and is positioned immediately N-terminal to the catalytic Arf GAPdomain. In the Arf GAP ASAP1, activity is increased by more than 10,000-fold by binding of the membrane phospholipid phosphatidylinositol 4,5-bi-sphosphate (PIP2) to the PH domain. The effect of PIP2 depends on the PHdomain and on an N-terminal extension of 16 amino acids in Arf1. These ob-servations led us to hypothesize that the PH domain binds the N-terminus ofArf dependent on PIP2. Here we found that the N terminus of Arf1 specif-ically binds ASAP1 PH domain in presence of PIP2 using fluorescence anisot-ropy, Forster Resonance Energy Transfer (FRET) and chemical crosslinkingexperiments. Chemical crosslinking coupled with mass spectrometry furtherenabled us to identify a possible binding site which was further examinedby mutational analysis. As far as we are aware, this is the first example ofligand-regulated protein binding by a PH domain and explains, at least inpart, the mechanism by which PIP2 binding to the PH domain leads toincreased Arf GAP activity. The switch-like behavior of this PH domainmay extend to other PH domains found in different regulatory proteins forRas superfamily proteins.

1999-Pos Board B15A Histidine-Lysine Axial Ligand Switch in a HemoglobinDillon Nye, Matthew Preimesberger, Ananya Majumdar, Juliette Lecomte.Johns Hopkins University, Baltimore, MD, USA.The cyanobacterium Synechococcus sp. PCC 7002 uses a monomeric hemo-globin, GlbN, to defend itself against reactive oxygen and nitrogen species.Bis-histidine coordination of the heme iron contributes to the resistance ofthe protein to oxidative damage. To characterize the stability of the iron liga-tion, we pursued pH titrations of GlbN and found by NMR spectroscopy that alysine is able to displace the distal histidine from the ferrous iron, but not theferric iron, at pH above 10.5. With mutagenesis, this lysine was identified asLys42, a residue that interacts with one of the heme propionates in the bis-histidine complex. Indeed, when the heme is replaced with its dimethylesterderivative, lysine ligation occurs at lower pH, which facilitated the study ofthe His-Fe-Lys complex. Absorbance spectroscopy was used to determinethe apparent pKa of the conformational transition (9.2 5 0.1), and NMR spec-troscopy quantified the partitioning between the His-Fe-Lys and His-Fe-Hisstates as well as the rate of interconversion. Slow exchange on the chemicalshift time scale allowed for the description of key structural features. Ligandswitching in GlbN is reminiscent of the alkaline transition of cytochrome c, inwhich one or more lysine residues displace the axial methionine in the ferric,but not ferrous, oxidation state. Selection of endogenous heme ligands hasprofound effects on the redox potential, electron transfer kinetics, and ligandbinding reactions of a heme protein. Our results implicate the lysine-hemepropionate interaction as an important determinant of the ligation state inGlbN.

2000-Pos Board B16STIM1-Induced Conformational Transition of ORAI-1 Leads to ChannelActivationZainab Haydari, Hengameh Shams, Mohammad Mofrad.University of California Berkeley, Berkeley, CA, USA.Calcium release-activated calcium (CRAC) channels in the plasma membraneare integral membrane proteins that play a central role in cellular signaling bygenerating the calcium influx. The calcium influx results in a decrease in theconcentration of Ca2þ within the endoplasmic reticulum (ER) that triggersthe immune system response.The stromal interaction molecule (STIM1) detects the decline of ER Ca2þ con-centration and activates the channel. The molecular details of STIM1 interac-tion with Orai1 (a pore subunit of the channel) that causes the channel openingare not yet clear. In order to understand the molecular details of this signaltransduction pathway, we developed all-atomic molecular models of theSTIM1/Orai-1 complex and studied the binding of STIM1 to C-terminus ofOrai-1 leading to conformational changes in Orai-1 protein complex followedby the activation of the CRAC channels. In this study, we examined the effectof different point mutations of the C-terminus of Orai-1 and ER tension on theSTIM-induced conformational transition of Orai-1 that was further comparedwith previous experimental studies.Our results showed that the M1 helices of Orai1 are slightly bent in the regionthat spans the junction between the hydrophobic and basic portions of the pore.This raises the possibility that the conformation of the basic region couldchange, perhaps as part of the gating process, while the hydrophobic sectionand glutamate ring remain fairly fixed. Furthermore, we observed that the dis-tance between M1 helices on the cytoplasmic site is increased resulting in theexpected widening of the cytoplasmic region of the pore.

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2001-Pos Board B17Bioactive 3D Structure of Phenylalanine Ammonia-Lyase Reveal Key In-sights into Ligand Binding DynamicsZsofia Bata1,2, Erzsebet Madaras1, Ibolya Leveles2,3,Friedrich Hammerschmidt4, Csaba Paizs5, Laszlo Poppe1,Beata G. V�ertessy2,3.1Department of Organic Chemistry and Technology, Budapest University ofTechnology and Economics, Budapest, Hungary, 2Research Centre forNatural Sciences, Hungarian Academy of Sciences, Budapest, Hungary,3Department of Applied Biotechnology and Food Science, BudapestUniversity of Technology and Economics, Budapest, Hungary, 4Institute ofOrganic Chemistry, University of Vienna, Vienna, Austria, 5Faculty ofChemistry and Chemical Engineering, Babesx-Bolyai University, Cluj-Napoca, Romania.Aromatic amino acid ammonia-lyases and 2,3-aminomutases contain thepost-translationally formed prosthetic 3,5-dihydro-4-methylidene-5H-imida-zol-5-one (MIO) group. The so-called MIO-enzymes may be used for thestereoselective synthesis of enantiopure a- or b-amino acids, making thesechemical processes more environmentally friendly and more affordable.Even though, a number of structures are available in the PDB for MIO en-zymes, the only structure for a eukaryotic phenylalanine ammonia-lyase(PAL) (PDB:1W27) is in catalytically inactive conformation, due to the‘‘loop-out’’ conformation of catalytically important Tyr-loop. Lack of pro-tein structure in a catalytically competent conformation impeded under-standing the PAL mechanism and the key reasons for the difference inlyase and mutase activity. Our recent study characterized novel amino phos-phonic acid inhibitors of MIO enzymes. Enzyme kinetic measurements andisothermal titration calorimetry demonstrated that addition of a methylidenesubstituent to the b-carbon atom of the phosphonic acid analogue of the nat-ural substrate, l-Phe, enhanced binding (Kd = 40nM) and reversed the enan-tiopreference of the enzyme. Here we provide an in-depth structural andmechanistic analysis of Petroselinum crispum PAL, based on our presentlydetermined crystal structure complexed with the methylidene substitutedphosphonic acid inhibitor at 1.5A resolution. Our strategy in using thevery strong binding, mirror image enantiomer was essential to growingwell diffracting crystals. Unlike the currently available crystal structure,in this new structure the catalytically essential Tyr-loop is in a catalyticallycompetent ‘‘loop-in’’ conformation. The high resolution enabled straightfor-ward molecular dynamics studies of the entire tetrameric protein. RandomAccelerated Molecular Dynamics investigated ligand binding pathways,and loop dynamics related to the binding process.

2002-Pos Board B18Resolving theMechanism of AdhesionMediated by a Non-Clustered Delta-1 ProtocadherinDebadrita Modak.Chemistry, The Ohio State University, Columbus, OH, USA.Cadherins form a large family of calcium-dependent cell adhesion proteinsinvolved in cell differentiation, tissue morphogenesis and neuronal connec-tivity. Non-clustered delta-protocadherins are a subgroup of cadherins with 6or 7 extracellular cadherin (EC) repeats and cytoplasmic domains distinctfrom classical cadherins. These proteins mediate homophilic adhesion andhave been implicated in various diseases, including asthma, autism, epilepsyand cancer. However, how delta-1 protocadherins use their extracellulardomain to mediate adhesion is unknown. Here we present the first X-raycrystal structures of a delta1-protocadherin member (Protocadherin-1)involved in asthma. Protocadherin-1 (PCDH1) is a delta-1 protocadherin ex-pressed mainly in the airway epithelium, skin keratinocytes, and lungs. Thestructure revealed binding modes that involve antiparallel overlap of multi-ple EC repeats. Mutagenesis, binding assays and other biochemical experi-ments validated the modes of adhesion as predicted from these structures.Overall, our studies reveal the molecular mechanism of adhesion mediatedby PCDH1 and shed light on its role in maintaining epithelial integrity,the loss of which causes asthma.

2003-Pos Board B19Zinc Availability-Dependent Unfolding of Loz1 Zinc FingerVibhuti Wadhwa1, Amanda J. Bird2, Mark P. Foster1.1Department of Chemistry and Biochemistry, The Ohio State University,Columbus, OH, USA, 2Department of Human Nutrition, The Ohio StateUniversity, Columbus, OH, USA.The transcription factor Loz1 (Loss Of Zinc-sensing 1), in Schizosaccharomy-ces pombe represses gene expression under conditions of zinc excess, presum-ably through zinc regulated DNA binding, and plays a central role in

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maintaining zinc homeostasis. A 96-residue construct (Loz1(426-522), consist-ing of a pair of C2H2-type zinc fingers at the extreme C-terminus and an adja-cent 40 amino acid ‘‘accessory domain’’ are sufficient for zinc responsiverepression by Loz1. The accessory domain does not contain identifiable struc-tural motifs; however, zinc responsive activity is lost in constructs lacking apart of this domain.The 1H-15N HSQC spectrum of Loz1(426-522) prepared in Zn-containingbuffers has fewer dispersed signals than expected from its primarysequence. Backbone resonance assignments show that these dispersed sig-nals arise from the zinc fingers, indicating an unstructured accessorydomain under these conditions. In addition, broad signals and doublingare observed for a few residues, suggesting conformational exchange atdifferent timescales in the free protein. Signal doubling is lost when proteinis bound to DNA.Dialysis of the protein into zinc free buffers results disappearance of resonancesarising from the first zinc finger, concomitant with increased spectral crowdingat the center of the HSQC spectrum; these observations are consistent with lossof zinc and unfolding of the first zinc finger. 15N relaxation dispersion (CPMG)experiments revealed ms-ms timescale exchange dynamics in the first zincfinger. These observations suggest that dynamics and weak binding to zincby the first zinc finger may be responsible for the zinc-sensing function ofthe protein. On-going experiments aim to elucidate the function of the zincfinger domain as a zinc sensor and its role in regulating DNA binding and tran-scription repression.

2004-Pos Board B20Structural Insights to Toxic Amylin Oligomers from 2D IR SpectroscopyKacie Rich, Megan Petti, Martin Zanni.UW-Madison, Madison, WI, USA.Type II Diabetes is classified as an amyloid disease due to the characteristicaggregation of amylin into fiber-like structures. Amylin is a 37 residue pep-tide that is co-secreted with insulin and is responsible for helping controlblood glucose levels. Amylin aggregation is linked to beta-cell death, as itis found aggregated postmortem in patients, but exact nature of its toxicityis unknown. The FGAIL region of the peptide was predicted to be themost amyloidogenic portion of the sequence, but it was not found to be inthe beta-sheet region of the final fiber structure as predicted by decades ofprevious work. Using 2D IR spectroscopy, our group has identified an olig-omeric species in the aggregation pathway that contains a beta-sheet in theFGAIL region. By isotope labeling amino acids, we monitor the structure ofindividual residue throughout the aggregation. The effect of temperature andsalt have been investigated on the oligomeric assembly mechanism and sta-bility of the intermediate, as has peptide concentration, which alters the freeenergy. Toxicity on INS-1 cells is consistent with this intermediate being thecytotoxic species. Our observations tie together disparate observations madeover many years and provide a unifying description for the aggregationmechanism of amylin.

2005-Pos Board B21Structural Insights into Mitochondrial Endog in Response to OxidativeStressHanna S. Yuan1, Jason L.J. Lin1, Woei-Chyn Chu2.1Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan,2Department of Biomedical Engineering, National Yang-Ming University,Taipei, Taiwan.Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial proteinthat degrades chromosomal DNA during apoptosis. In mitochondria, EndoGalso processes DNA and participates in mitochondrial DNA metabolism.EndoG-deficient mice have high levels of mitochondria reactive oxygen spe-cies (ROS) and develop heart and mental diseases, showing that the EndoGdefect is linked to mitochondrial dysfunction and oxidative stress-linked dis-eases. However, it remained unclear how EndoG degrades DNA as a homo-dimer and why EndoG’s function is linked to ROS levels in mitochondria.We reported the crystal structure of the C. elegans EndoG homolog, CPS-6,in complex with single-stranded DNA at a resolution of 2.3 A. Two separateDNA strands are bound at the active site of each protomer in the homodimerwith their nucleobases pointing away from the enzyme, explaining why CPS-6degrades DNA without sequence specificity. We also showed that underoxidative stress, homodimeric EndoG becomes oxidized and converts tomonomers with a diminished endonuclease activity. High ROS levels arethus linked to mitochondrial dysfunction by repressing EndoG’s activity.Modulation of EndoG dimer conformation and endonuclease activity couldpresent an avenue for prevention and treatment of the diseases resultedfrom oxidative stresses.

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2006-Pos Board B22Structure, Function, and Dynamics of Xanthomonas albilineans Cas2 inType I-C CRISPR-Cas SystemEuiyoung Bae1, Nayoung Suh2.1Agricultural Biotechnology, Seoul National University, Seoul, Republic ofSouth Korea, 2Pharmaceutical Engineering, Soon Chun Hyang University,Asan, Republic of South Korea.CRISPRs and CRISPR-associated (Cas) proteins provide a microbial adaptiveimmunity against phage infection. Cas2 is a universal Cas protein found in alltypes of CRISPR-Cas systems, and its role is implicated in the new spaceracquisition into CRISPR loci. In type I-C CRISPR-Cas systems, Cas2 proteinsare metal-dependent double-stranded DNA (dsDNA) nucleases, and a pH-dependent conformational transition has been proposed as prerequisite forthe nuclease function. Here, we report the crystal structure of Xanthomonas al-bilineans Cas2 (XaCas2), and provide experimental evidence of a pH-dependent conformational change during functional activation. XaCas2 crys-tallized at an acidic pH represented a catalytically inactive conformationalstate, and exhibited dsDNA nuclease activity only under neutral and basic con-ditions. In the crystal structure, significant conformational heterogeneity wasevident in the putative hinge regions, suggesting that XaCas2 engages inhinge-bending conformational switching. The presence of a Trp residue inthe hinge region enabled investigation of hinge dynamics by fluorescence spec-troscopy. The pH dependence of the fluorescence intensity overlapped pre-cisely with that of nuclease activity. Mutational analysis further confirmedthat the conformational activation of the nuclease function was in play.Together, our results reveal strong correlations among the conformationalstates, catalytic activity, and hinge dynamics of XaCas2, and suggest thatCas2 may play a dual role as a dsDNA nuclease or as a central adaptor proteinwithin the CRISPR-adaptation complex.

2007-Pos Board B23Flexibility of the Myelin Scaffolding Protein PeriaxinArne Raasakka1, Huijong Han2,3, Matti Myllykoski2, Petri Kursula1,2.1Department of Biomedicine, University of Bergen, Bergen, Norway,2Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu,Finland, 3German Electron Synchrotron (DESY), Hamburg, Germany.Rapid nerve impulse conduction is necessary for a correctly functioning verte-brate nervous system. This is enabled by myelin, an insulating lipid-rich struc-ture that encloses selected axonal segments. The insulation arises from compactmyelin, which is composed of tightly stacked lipid bilayers devoid of cytosoliccontent. Non-compact myelin is richer in cytosol, has a role in myelin mainte-nance, and resides mostly under the inner- and outermost myelin membranes.In the peripheral nervous system, myelin is produced by Schwann cells, whichin addition to their outermost abaxonal membrane, are surrounded by acarbohydrate-rich basal lamina that is connected to the abaxonal membranevia dystroglycans. These penetrate the membrane and connect to a networkof intramyelin proteins, including periaxin (PRX) - a scaffolding moleculefor other proteins within this network that links the abaxonal membrane tothe cytoskeleton. Both PRX isoforms, S- and L-PRX, contain a folded 100-amino acid PDZ-like domain. Additionally, the longer isoform contains a�1300-residue tail, which - based on sequence analysis - can be divided intoseveral distinct regions that are predicted to be disordered. This tail is a proteinscaffolding unit, which is crucial for the correct morphology and stability ofperipheral myelin, as point mutations in the tail region result in Charcot-Marie-Tooth disease (CMT) - an incurable demyelinating neuropathy. Wecharacterized L-PRX using different biophysical methods, including X-raycrystallography, NMR spectroscopy, synchrotron-radiation circular dichroism(SRCD) spectroscopy, and small-angle X-ray scattering (SAXS). The crystalstructure is an intertwined, domain-swapped PDZ-like homodimer with poten-tial membrane interactions, as suggested by detergent titrations. Different partsof the tail are very flexible and functional in vitro. Quantitative studiesregarding the affinities and structural implications of these interactions are un-derway. Our study will contribute to understanding the structure-function rela-tionships of myelin proteins in health and disease.

2008-Pos Board B24Effects of Hydroxylation at Proline 567 in HIF-1a on the Binding to pVHLHongsheng Qian1, Junhang Hu2, Qingwen Zhang1.1Shanghai University of Sport, Shanghai, China, 2Henan Quality Polytechnic,Pingdingshan, China.Hypoxia is a physiological condition commonly present in tissue tumorgrowth and hypoxia training. Hypoxia inducible factor-1a (HIF-1a) playsan essential role in the regulation of hypoxia in animal cells. This mechanismis mediated by the von Hippel–Lindau tumor suppressor protein (pVHL)through the hydroxylation of HIF-1a proline 564. However, previous study

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has shown proline 567 was hydroxylated by prolyl hydroxylase-3 (PHD-3)in vitro. In addition, recent computational work has illustrated that a newhydrogen bond network localized around HYP567 was rearranged. In thiswork, we performed all-atom molecular dynamics (MD) simulations to inves-tigate the structural effect and the interaction between pVHL and HIF-1a instatus of non-hydroxylation proline (noHYP), hydroxylation proline564 (HYP564), hydroxylation proline 567 (HYP567), and hydroxylation pro-lines 564 and 567(2HYP). Our cumulative 5 ms MD simulations revealthat HYP564 shows a lower RMSD value than the rest of variations, while2HYP increases the structural flexibility of loop L7 (V137-P146) and a-domain (V155-P192) in pVHL comparing with HYP564. Hydrogen bond pro-file analysis illustrates that both HYP564 and 2HYP variations have two morehydrogen bonds (HYP564-HIS115 and SER111-HYP564) presented in anextremely steady condition, while 2HYP slightly reduces the intensity of H-bonds than HYP564. After the binding free energy calculation, HIF-1a showsa strong binding affinity to pVHL in HYP564 system, while the 2HYP obvi-ously decreases the binding free energy between HIF-1a peptide and pVHL.In addition, our study highlights the conformational dynamics process be-tween pVHL and variations of HIF-1a, suggesting that the hydroxylation ofproline 567 may disturb the capture of HIF-1a by pVHL. Considering otherevidences, we suggest that proline 567 in HIF-1a may not be hydroxylatedin humans, at least in normal condition.

2009-Pos Board B25Non-Enzymatic Self-Association of Fibrinogen in Solution Studied with 1HNMR SpectrometryRustem I. Litvinov1,2, Yuriy F. Zuev3, Bulat Z. Idiyatullin3,Dilyafruz R. Bakirova3, Alexander E. Sitnitsky3, Artem Zhmurov4,Valeri Barsegov4,5, John W. Weisel1.1University of Pennsylvania School of Medicine, Philadelphia, PA, USA,2Kazan Federal University, Kazan, Russian Federation, 3Institute ofBiochemistry and Biophysics, Kazan, Russian Federation, 4Moscow Instituteof Physics & Technology, Moscow Region, Russian Federation, 5Universityof Massachusetts, Lowell, MA, USA.Fibrinogen is a blood plasma protein that polymerizes to form the fibrin clot oncleavage of its fibrinopeptides. This work provides quantitative characteristicsof the molecular hydrodynamics of fibrinogen in a broad 0.3-60 mg/ml range ofconcentration and 5-42�C temperature obtained using pulsed-field gradient 1HNMR. Arrhenius plots revealed the activation energy for fibrinogen diffusionEd = 21.3 kJ/mol at 1.4 mg/ml and 28.4 kJ/mol at 38 mg/ml. The diffusive mo-tion of fibrinogen underwent a remarkable slowdown with concentrationsbeginning at 1.7-3.4 mg/ml, which deviated from the standard hard-particlebehavior, suggesting concentration-dependent intermolecular entanglement.By contrast, diffusivity of fibrinogen variant I-9 with truncated C-terminal por-tions of the Aa chains was much less concentration-dependent, indicating theimportance of intermolecular linkages formed by the aC regions. The remark-able concentration dependence was observed regardless of the absence or pres-ence of the GPRP peptide (inhibitor of fibrin polymerization) and in samplesfree of fibrin oligomers, confirming that the observed steep decrease in fibrin-ogen diffusivity was not due to potential contamination by oligomeric fibrin-fibrinogen complexes. Theoretical models combined with all-atom MolecularDynamics simulations revealed that fibrinogen in solution has a bendableconformation that interpolates between a flexible chain and a rigid rod observedin the crystal. The results obtained illuminate the important role of the aC re-gions in modulating the fibrinogen molecular shape through formation of weakintermolecular linkages that control the bulk properties of fibrinogen solutions.The results emphasize the importance of fibrinogen self-assembly in modu-lating the hydrodynamic properties of semi-diluted and concentrated fibrinogenpreparations, and point to the potential role of fibrinogen self-assembly in bio-logical and clinical applications.

2010-Pos Board B26Botulinum Toxins a and E Inflict Dynamic Destabilization on T-SNARE toImpair Snare Assembly and Membrane FusionRyan Khounlo.BBMB, Iowa State University, Ames, IA, USA.Botulinum toxins (BoNT) A and E block neurotransmitter release by specif-ically cleaving the C- terminal ends of SNAP-25, a plasma membrane SNAREprotein. We find that SNAP-25A and E, the cleavage products of BoNTA and Erespectively, terminate membrane fusion via completely different mechanisms.Combined studies of single molecule FRET and single vesicle fusion assaysreveal that SNAP-25E is incapable of supporting SNARE pairing and thus,vesicle docking. In contrast, SNAP-25A facilitates robust SNARE pairingand vesicle docking with somewhat reduced SNARE zippering, which leadsto severe impairment of fusion pore opening. The EPR results show that the

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discrepancy between SNAP-25A and E might stem from the extent of the dy-namic destabilization of the t-SNARE core at the N-terminal half which plays apivotal role in nucleating SNARE complex formation. Thus, the results provideinsights into the structure/dynamics-based mechanism by which BoNT A and Eimpair membrane fusion.

2011-Pos Board B27Modulation of the Peroxidase Activity of Human Cytochrome C by ULoops C and DHaotian Lei, Shiloh Nold, Bruce Bowler.Chemistry, University of Montana, Missoula, MT, USA.Cytochrome c (Cytc) is a globular protein within the inner mitochondrial mem-brane that shuttles electrons from Complex III to Complex IV in the electrontransport chain. Upon release from the mitochondria, Cytc participates in initi-ation of the caspase cascade leading to apoptosis. Cytc binds to cardiolipin inthe inner mitochondrial membrane leading to loss of Met80-heme ligation,enabling Cytc to function as a peroxidase capable of oxidizing cardiolipin.The affinity of Cytc for oxidized cardiolipin is decreased and therefore Cytccan dissociate from the membrane facilitating release from mitochondria andpropagation of apoptosis. In order for the heme coordination site to open forperoxidase activity, the heme crevice loop must undergo structural rearrange-ment similar to the alkaline transition. Unlike most residues in the heme creviceloop, positions 81 and 83 evolve to more sterically demanding residues inhigher eukaryotes. Previous results suggest that although residues 81 and 83are key residues for regulating the peroxidase activity of Cytc, there is evidencethat substitutions in U-loop C can affect the stability of the heme crevice loop.We hypothesize that U loops C and D have evolved interactively to optimizethe peroxidase activity of Cytc. To perform this test, we prepared a series ofhuman-to-yeast substitutions in U-loop C of human Cytc. Using circular di-chroism and UV-visible spectroscopies, we have investigated global stabilityof the protein and local stability of the heme crevice loop. Using stopped-flow methods, we investigated the peroxidase activity for both I57V andN54K human Cytc. We observe that the N54K substitution strongly enhancesthe peroxidase activity of human Cytc, whereas the I57V substitution has littleeffect.

2012-Pos Board B28Probing Local Solvation Environments in H-NOX Proteins using Unnatu-ral Amino AcidsCaroline Kearney, Trexler D. Hirn, Gwendolyn D. Fowler,Lukasz T. Olenginski, Daniyal Tariq, Scott H. Brewer,Christine M. Phillips-Piro.Chemistry Department, Franklin and Marshall College, Lancaster, PA, USA.Heme Nitric Oxide and/or Oxygen (H-NOX) binding proteins, which can befound in both prokaryotic and eukaryotic cells, are gas-sensing heme proteinsthat act as effector domains initiating a cascade of signals for cells to function.H-NOX proteins are a diverse group of hemoproteins that bind diatomic gaseslike nitric oxide (NO), carbon monoxide (CO), and/or oxygen (O2). Here, twoH-NOX proteins, O2-binding Caldanaerobacter subterraneus H-NOX (Cs H-NOX), and non-O2-binding Shewanella oneidensis (So H-NOX), are underinvestigation. Specifically local protein environments in Cs H-NOX wereprobed using the vibrational reporter unnatural amino acid (UAA) 4-cyano-L-phenylalanine (pCNF). This UAA was genetically site-specifically incorpo-rated into CsH-NOX at multiple positions individually including near the hemepocket using amber codon suppression technology. UV-visible spectroscopicanalysis was used to confirm heme incorporation and the ligation state ofeach construct. Infrared analysis of each construct probing the position of thenitrile symmetric stretch of pCNF revealed a myriad of local environmentsin the protein. These results will be presented in addition to preliminarywork on So H-NOX.

2013-Pos Board B29Experimental and Computational Studies of Obscurin’s FlexibilityJake Whitley, Daniel Marzolf, Oleksandr Kokhan, Nathan Wright.Chemistry and Biochemistry, James Madison University, Harrisonburg, VA,USA.Obscurin is a giant modular muscle protein that functions to connect the sarco-plasmic reticulum to the contractile apparatus. Obscurin is made up of multipleIg domains in a chain connected by short linkers. Previous structural papersshow that short linkers are less flexible, yet MD papers show these linkers tobe very flexible. Our research reconciles these divergent data. Here we testthe flexibility of 5 dual obscurin domain systems. Using NMR and SAXS,we show that these domains all adopt an extended architecture. However,MD and SMD data demonstrate obscurin to be significantly flexible. Therefore,we believe obscurin to be an extended, flexible protein, despite its short linkers.

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Posters: Protein Structure, Prediction, andDesign I

2014-Pos Board B30Analysis of Relative Binding Affinity Predictions for Protein-ProteinComplexesXavier Bonner1, Brian Kuhlman2, Hayretin Yumerefendi2.1Morehouse College, Atlanta, GA, USA, 2University of North Carolina,Chapel Hill, Chapel Hill, NC, USA.Proteins are responsible for a multitude of chemical reactions within livingcells across all species. Proteins mediate most of their functions by interact-ing with themselves or other proteins, thus forming complexes. These com-plexes are widely studied because mutations affecting protein-proteininteractions often lead to various diseases. Moreover, understanding the bio-physical principles of protein interactions will allow researchers to engineerbetter therapeutics. Here we take a computational approach using Rosetta tomodel how different mutations affect the overall structure of proteins andtheir relative binding affinity. My work focuses on expanding and improvingthe current prediction algorithm by applying it to a very diverse and largedataset of mutational data. One avenue that we explore to achieve more ac-curate predictions is by introducing distance constraints on neighboring res-idues and complexes.

2015-Pos Board B31Molecular Design of Artificial Ring Fingers for Detecting UbiquitinationActivitiesKazuhide Miyamoto, Ayumi Yamashita, Kazuki Saito.Himeji Dokkyo University, Himeji, Japan.Protein ubiquitination is an enzymatic cascade reaction consisting ofubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. E2 enzymes are associated with various cancerssuch as leukemia, breast cancer, lung cancer, and colorectal cancer. Todate, the monitoring of E2 activity for cancer diagnosis is challenging dueto its intricate cascade reaction. To overcome this hurdle, we recentlyhave developed the novel strategy for monitoring E2 activities. Here, we de-signed the concise machinery of ubiquitination with artificial RING fingerproteins (ARFs) functioning as E3 enzymes. The machinery enabled thesimplified detection of E2 activities without a substrate for the ubiquitintransfer. Furthermore, the system of combining a signal accumulation ISFETbiosensor with ARFs enabled the high-sensitivity quantitative detection ofE2 activities over a response range of femtomolar to micromolar concentra-tions. The real-time monitoring of pathological conditions was demonstratedusing human acute promyelocytic leukemia cells following treatment withthe anticancer drug bortezomib. The strategy for E2 activities is extremelysimple and convenient.

2016-Pos Board B32Rational Engineering and Rosetta Design of a Genetically Encoded Fluo-rescent Reporter of Protein Conformational ChangeJan Maly1, Yann Thillier2, Grace Or2, Kit Lam2, Jon T. Sack3, Lin Tian2,Vladimir Yarov-Yarovoy3.1Biophysics, UC Davis, Davis, CA, USA, 2Biochemistry and MolecularMedicine, UC Davis, Davis, CA, USA, 3Physiology and Membrane Biology,UC Davis, Davis, CA, USA.The goal of this project is to rationally design a genetically encoded fluorescentreporter of conformational change using molecular rotor dyes, which exhibitvery high fluorescence increase while bound specifically to an appropriatemacromolecule. We focus on probing movements of voltage-gated ion chan-nels that undergo large electrostatic and conformational changes. As part ofthis longer-term investigation, we use RosettaDesign, homology modeling,and ligand docking methods to develop a variant of the E. coli ethidium bro-mide multi-drug binding protein (EbrR) for use as a fluorescent reporter ofvoltage-gated ion channel activation. Initial fluorescence binding assaysshow strong fluorescence signal increase from malachite green (MG) bindingto EbrR. Furthermore, RosettaLigand models indicate that our novel nonmembrane-permeable analog of MG, where a b-alanine residue is linked tothe dye backbone via a peptide bond (MG-bA), has a much more convergentbinding pose than MG, and mimics the hydrogen-bonding orientation of watermolecules and amino acids in the binding pocket of the EbrR crystal structure.These preliminary results allow us to move forward with Rosetta-based bindingoptimization of MG-bA and EbrR. We aim to incorporate the redesigned EbrRsequence into the voltage sensors of ion channels to transform MG-bA bindinginto a spatial and temporal fluorescent reporter of voltage-gated ion channel ac-tivity.

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2017-Pos Board B33Successful Rational Affinity Maturation of an Alpha-Synuclein AntibodySai Pooja Mahajan1, Bunyarit Meksiriporn2, Dujduan Waraho-Zhmayev2,Fernando Escobedo3, Matthew P. Delisa2.1Intel Corporation, Portland, OR, USA, 2Cornell University, Ithaca, NY,USA, 3Chemical Engineering, Cornell University, Ithaca, NY, USA.The ability to modulate protein-protein interactions is of great interest to bothfundamental biological science and applied research such as drug design.Whilemany studies have demonstrated the ability to use structure-based informationto rationally improve protein-protein interactions, the rational design of target-specific binders is still an unsolved problem. In this context, single domain an-tibodies (VHHs) are promising designable molecules, given their small size,stability, and ability to bind surfaces of any shape. Alpha-Synuclein (AS) isa natively disordered protein, implicated in the pathogenesis of Parkinson’s dis-ease (PD) and related neurological disorders. The Non-Amyloid Component(NAC) region of AS has been previously targeted for inhibiting the aggregationand/or reducing toxicity of AS. Various methods such as yeast surface display,phage display and, more recently, rational design approaches have been usedfor generating antibody fragments for this purpose. In this work, we have pur-sued a dual modeling-experimental approach for rationally designing an AS-specific VHH.Starting from an immunized Camelid library against the NAC region of A53Tmutant of AS (A53T), a single-round of a bacteria-based selection techniquewas used to obtain a NAC-specific VHH. Atomistic models of the VHH andthe VHH-NAC binding were constructed using a combination of MolecularDynamics techniques (Replica Exchange Molecular Dynamics, Umbrella Sam-pling) and numerical calculations (Finite-Difference Poisson-Boltzmann Equa-tion). Using this dual approach of prediction and experimental verification, wecould enhance the affinity of the VHH by an order of magnitude. Hence, ourwork, demonstrates the ability to develop a predictive in-silico model of bind-ing for the VHH-NAC system. This approach can be especially powerful fortargeting natively disordered and weakly immunogenic antigens such as ASfor which crystal structures are not readily available and experimentally vali-dated computational models can be leveraged for rational design and affinityenhancement.

2018-Pos Board B34The Dynamic Self-regulation of Modular Cullin-Ring LigasesRyan Lumpkin, Elizabeth Komives.Chemistry and Biochemistry, UC San Diego, La Jolla, CA, USA.E3 Ubiquitin (Ub) Ligases (UBLs) trigger cellular signaling through the cova-lent attachment of Ub (ubiquitylation) to lysine residues on bound proteins. Themulti-subunit Cullin-RING ligase (CRL) represents the largest family of E3 Li-gases and is responsible for protein degradation through the proteasome. CRLscontain a Cullin protein (CUL 1-7) with a RING-box (RBX1 or 2) proteinbound at its C-terminus. Adapter proteins link a substrate receptor to the N-ter-minus of CUL and a ubiquitin-conjugating protein (E2) binds to RBX. Anactive CRL facilitates the transfer of ubiquitin from E2 across a gap of morethan 50 A to a substrate at the other end of the ligase. The Ankyrin Repeatand SOCS-box (ASB) substrate recognition proteins associate with CUL5and RBX2 through the Elongin B and C (EBC) adapter proteins. Modificationof CUL by Nedd8 (neddylation) regulates substrate-receptor exchange and theE2 specificity of RBX.We have expressed and purified all of the proteins in the ASB9-containng CRL,which ubiquitylates creatine kinase (CK). We show that the purified complexbecomes neddylated at K724 of CUL5, and creatine kinase is ubiquitylatedat several lysines on the protein surface. We are pursuing cryoEM andhydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments toelucidate the structure and dynamics of the complex. Initial cryo-electron mi-croscopy reveals the ASB9 protein is highly structured, consistent with its lowincorporation of deuterium in HDXMS. HDXMS also revealed that binding ofEBC alters exchange within CK bound on the other end of ASB9.

2019-Pos Board B35Identification of a Schiff Base Adduct In Cyp3A4Parker Flanders, Matthew Schwartz, Justice Spriggs, Tom Larson,Larry R. Masterson.Department of Chemistry, Hamline University, Mahtomedi, MN, USA.Cyp3A4 is a liver enzyme responsible for metabolizing almost 50% of alldrugs. Previous research in our lab has shown that acetaldehyde, a major prod-uct of ethanol metabolism, to be a strong inhibitor of this enzyme in its meta-bolism of sedatives, such as a variety of benzodiazepines. Modifications ofproteins by acetaldehyde is a known mechanism of alcoholic liver disease, aswell as cancer of the liver. To further understand the details of acetaldehydeinhibition of Cyp3A4, liquid-chromatography coupled to mass spectroscopy

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was utilized to map digests of Cyp3A4 in order to identify the residues thatmay be modified. Analysis of acetaldehyde adducts was done by reducingthe acetaldehyde modified lysine sidechain with sodium borohydride(NaBH4), resulting in a stable secondary amine. Further data on the differencesin acetaldehyde modified and unmodified Cyp3A4 will be presented.

2020-Pos Board B36Designing Selective Protein Binding SitesFrancesca Nerattini1, Luca Tubiana2, Chiara Cardelli1, Valentino Bianco2,Christoph Dellago2, Ivan Coluzza3.1Faculty of Physics, University of Vienna &VDSP, Vienna, Austria, 2Facultyof Physics, University of Vienna, Vienna, Austria, 3ComputationalBiophysics, CIC biomaGUNE, San Sebastian, Spain.Molecular recognition is a critical process for many biological functions andconsists in non-covalent binding of different molecules, such as protein-protein, antigen-antibody and many others. The host-guest molecules involvedoften show a shape complementarity, and one of the main specification for mo-lecular recognition is that the interaction must be selective, i.e. the host shouldbind strongly to one selected guest and poorly, if at all, to all other biomole-cules. Our work focuses on the role played by the chemical heterogeneityand the steric compatibility on the selectivity power of the binding site betweentwo proteins. We developed a computational design procedure, based on thecaterpillar coarse-grained model, for a reference system where we fashionedthe protein binding site as a protein-like surface which perfectly shapes aportion of the partner protein. The investigated range of surface area for theartificial binding sites falls in typical natural sizes (750 �1500 A2). On theprotein-like surface, we decorated anchoring points whose chemical function-alization is chosen so to optimise the binding with the protein. Using highly-efficient Monte Carlo simulations methods we explore the binding and foldingproperties of our artificial proteins. A significant result is that, despite the factthat protein and surface chemical sequences are interdependent and simulta-neously generated to stabilise the bound folded structure, the protein is stablein the folded conformation even in the absence of the protein-like partner forall investigated systems. Moreover, we observe that an increase of the surfacearea results in a decrease of specificity for the binding, therefore imposing anupper limit for molecular recognition binding sites at the typical physiologicaltemperature range.

2021-Pos Board B37Are Proteins Such Unique Polymers? - The Role of Directional Interac-tions in the Designability of Generalized HeteropolymersChiara Cardelli1, Valentino Bianco2, Lorenzo Rovigatti3,Francesca Nerattini1, Luca Tubiana2, Christoph Dellago2, Ivan Coluzza4.1Computational Physics Group, Department of Physics, University of Vienna& VDSP, Vienna, Austria, 2Computational Physics Group, Department ofPhysics, University of Vienna, Vienna, Austria, 3Universita�la Sapienza,Roma, Italy, 4CIC biomaGUNE Parque Cientıfico y Tecnologico deGipuzkoa, Donostia / San Sebastian, Spain.Proteins are an example of designable heteropolymers able to fold in uniquetarget structures. Instead, self-assembly of artificially designed heteropoly-mers cannot still reach, to the best of our knowledge, the control over thesingle chain structure and the high variability of structures that natural pro-teins can achieve. Here we try to answer the following questions: ‘‘Whatartificial heteropolymers lack compared to bio-heteropolymers that grantthe latter such precision and variability? Is the geometry of the proteinskeleton the only choice to be designable?’’ We propose that directional in-teractions are critical to the answer as they can reduce the number of ener-getically accessible configurations. With the added directionality, artificialheteropolymers gain control over the structure comparable to the one achiev-able in proteins. We demonstrate our prediction with our computationalapproach by exploring different directional interactions and different aminoacids alphabets. We calculate the configurational entropy, that is connectedto the number of accessible configurations, for various directional interac-tions. We verify their designability for many alphabet sizes, and we comparethe results with the predictions of the current mean field theories describingheteropolymer freezing. Moreover, we introduce a simple qualitative crite-rion to discriminate a priori designable from not-designable polymer back-bones. We identify as designability criterion the appearance of a particularpeak in the radial distribution function that dominates over the random pack-ing peak. We show that the presence of such a peak indicates that the systemis designable and it is a universal feature of all designable heteropolymers,as it is also dominating the radial distribution function of proteins. We pro-pose that our novel criterion can guide the engineering of new types of self-assembling modular polymers that will open new applications for polymer-based materials science.

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2022-Pos Board B38Rational Design of PDZ Domain SpecificityYoung Joo Sun1, Titus Hou1, Lokesh Gakhar2, Sahezeel Awadia3,Rafael Garcia-Mata3, Ernesto Fuentes1.1Biochemistry, University of Iowa, Iowa City, IA, USA, 2Biochemistry,Crystallography Facility, University of Iowa, Iowa City, IA, USA,3Biological Sciences, University of Toledo, Toledo, OH, USA.PDZ (PSD-95/Dlg/Zo-1) domains are structurally conserved protein-proteininteraction modules found in signal scaffolding proteins. There are thousandsof PDZ domains and they have low sequence homology. PDZ domains bindto the C-terminus or internal amino acid motifs of target proteins (PBMs,PDZ-binding-motif). Moreover, PDZ domains generally interact with a set ofPBMs within a structurally conserved pocket. However, PDZ-PBM interac-tions are context-dependent, such that each PDZ-PBM combination has aunique PDZ structural motif and PBM binding mode. Due to context-dependence and low PDZ sequence homology, predicting and engineeringPDZ domain specificity remains challenging. We used structure-based align-ment in combination with automated structural analysis of PDZ-PBM struc-tures to define the ‘‘binding pocket characteristics’’ of the PDZ domainfamily. We tested this approach using two model PDZ domains: CASK PDZthat binds C-terminal PBMs and Scribble PDZ1 that binds internal PBM se-quences. First, we identified six subpocket positions in the CASK PDZ domainpredicted to switch specificity to that of the Tiam1 PDZ. Individual or doublesubstitutions had variable effects on CASK PDZ specificity. In contrast,combining sets of substitutions from the three subpockets successfully switchedthe PBM specificity such that the difference in DGbinding of CASK PDZsextuple mutant to that of Tiam1 PDZ was within 0.25 kcal/mol for a set ofPBMs. Second, we identified two positions in the Scribble PDZ1 domain thatpotentially dictate internal ligand specificity. Single substitutions at each posi-tion were marginally successful at modulating specificity of internal and C-ter-minal PBMs. However, the double substitution showed synergy betweenspecificity subpockets resulting in a modified Scribble PDZ domain that doesnot bind the internal ligand while preserving the ability to bind C-terminalPBMs. Together, our results indicate that the ‘‘binding pocket characteristics’’analysis is a powerful general approach for engineering PDZ-PBM interactions.

2023-Pos Board B39Glass Chip for Nanopore Based Low Noise Resistive Pulse SensingLennart J. de Vreede, Cuifeng Ying, Michael Mayer.BioPhysics Research Group, Adolphe Merkle Institute, University ofFribourg, Fribourg, Switzerland.Resistive-pulse sensing provides structural insights on individual proteins whentranslocated through nanopores. The substrate capacitance, however, inducesnoise in the ionic current signal, which increases strongly with increasing band-width of the recording. For this reason, minimizing substrate capacitance is crit-ical for characterizing single molecules through resistive-pulse sensing.Inspired by recent reports on low-noise nanopore substrates, we engineered aglass chip for low noise nanopore resistive-pulse measurements using micro-fabrication. Replacing silicon substrate material with fused silica gives anapproximately 5-fold reduction of noise at a bandwidth of �57 kHz. For fabri-cation, we deposited a 30 nm thick silicon nitride (SiNx) layer on glass by low-pressure chemical vapor deposition. After etching the glass a SiNx membranewith a diameter of �20 mm remained and prepared a �20 nm diameter nano-pore in the membrane by controlled dielectric breakdown. For a glass chip, thestandard deviation of the ionic current during resistive-pulse measurements was8 pA at 15 kHz recording bandwidth and 19 pA at 57 kHz and for a silicon chip,the standard deviation of the ionic current was 29 pA at 15 kHz and 95 pA at 57kHz. We demonstrate improved sensitivity for resistive-pulse sensing by usingIgG protein as test particle translocating through the glass-based nanopore. Thelow noise from the glass chip at high bandwidth improves the shape character-ization of the protein and potentially allows the detection of subtle changes inprotein dynamics within the nanopore. Additionally, we have shown low laser-induced noise from glass chips during ionic current measurements, in contrastto silicon-based chips, which respond to laser by significantly increased noise.The absence of light-induced noise from electrically insulating and opticallytransparent chips for nanopore recordings makes them compelling for synchro-nized electrical and optical measurements.

2024-Pos Board B40De Novo Design of Cross-a Amyloid-Like Fibrils with Cellular ActivityShao-Qing Zhang1, Lijun Liu2, Junjiao Yang1, Marco Lolicato1,Huong Kratochvil1, Xiaokun Shu1, William F. DeGrado1.1UCSF, San Francisco, CA, USA, 2DLX Scientific, Lawrence, KS, USA.Deposition of tight and orderly insoluble b-amyloids is a hallmark of manyneurodegenerative diseases, including Alzheimer, Huntington’s, and prion dis-

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eases. Whether a-helix-based amyloid-like fibrils induce similar pathologicalsymptoms is unknown. To investigate this problem, as the first step we havede novo designed cross-a amyloid-like self-assembled suprastructures. Electronmicroscopy has shown fibril structures for our designed sequences. We havealso determined the crystal structures of these fibrils. The structures show thatour designed amyloid-like fibrils have a twist along the helical fibril axis be-tween the successive rungs, resembling the recently solved cryo-EM structuresof tau and amyloid-b filaments. In our design, the building blocks in our designare coiled-coil-like helix bundles, which offer great benefits of modularity forstructure design. By tuning the size of the residues at the interfaces betweenbuilding blocks, designed fibrils possess distinct inter-rung rises and twists,which match with our design models. The thioflavin-T assay shows that theinterfacial residues between the building blocks also affect the fibrillization ki-netics, which resembles the effects of pathological mutations in the b-amyloidstructures. Therefore, our newly designed cross-a amyloid-like fibrils can be anew testing model for neurodegenerative diseases on the molecular level.

2025-Pos Board B41Rational Protein Design via Structure-Energetics-Function Relationshipsin the Photoactive Yellow Protein (PYP) Model SystemJohan H. Both1, Robert M. Parrish1,2, Todd J. Martınez1,2, Steven G. Boxer1.1Chemistry, Stanford University, Stanford, CA, USA, 2PULSE Institute,Stanford University, Stanford, CA, USA.Rational protein design uses biophysical principles to inform the generation ofnew, purpose-built proteins. However, this methodology is hampered by ourlimited understanding of these biophysical principles relating a protein’s struc-ture to its function. In an initial attempt at using structure-energetics-functionrelationships to rationally design protein function, we recently developed amodel that guides qualitative suggestions for engineering light-activated disso-ciation of split GFPs [1]. We analyzed the potential energy surface of the photo-dissociation reaction coordinate to identify critical energetics features likebarriers and population branching ratios, which can be tuned to increase thephotoswitching quantum yield and decrease the fluorescence quantum yield.To develop this structure-energetics-function design methodology further, weare using QM/MM non-adiabatic dynamics studies to predict the effects ofmutating key residues in the PYP chromophore environment on observablephotoactivated processes. These predictions will then be evaluated by compar-isons to spectroscopic measurements.References1. Lin CY, Both J, Do K, Boxer SG (2017) Mechanism and bottlenecks instrand photodissociation of split green fluorescent proteins (GFPs). Proc NatAcad Sci USA E2146-E2155.

2026-Pos Board B42Structural Design of Novel Protein AcetyltransferasesLogan Kaler, Yadilette Rivera-Colon.Science, Bay Path University, Longmeadow, MA, USA.Acetylation is a common but poorly understood co-translational and post-translational protein modification in which an acetyl group is attached to pro-tein residues. Acetyltransferases are responsible for this process in cells andrequire the presence of the co-factor Acetyl Coenzyme-A. The acetyltransfer-ase enzyme family has specific members that can catalyze the transfer of anacetyl group to a lysine side chain (KAT) or the N-terminus (NAT) of a protein.In this study, we identified amino acids on an acetyltransferase where we hy-pothesized that changes in specific structural features might contribute to sub-strate specificity and mutated specific amino acids in NATs and KATs. We willtest whether these changes alter their substrate specificity by utilizing the re-sulting novel proteins in a fluorescence-based enzymatic assay. The resultsof these assays will be compared to those of the wild-type acetyltransferasesto assess substrate specificity in comparison to native enzymes. The goal isto confirm the key elements that have been hypothesized to confer specificityof the enzymes and also determine how these elements differ between NATsand KATs. Furthermore, any mutants with engineered specificity will be crys-tallized and their structures will be determined, intended for understanding themechanistic basis of specificity at atomic resolution.

2027-Pos Board B43Engineering a Promiscuous AcetyltransferaseJenna Morris, Yadilette Rivera-Colon.Science, Bay Path University, Longmeadow, MA, USA.Acetyltransferases have been well-studied mechanistically and in relation tomany diseases that potentially result from malfunctioning acetyltransferases,such as cancer. However, there has yet to be an acetyltransferase discoveredthat is able to act on multiple amino acid substrates; a ‘‘promiscuous’’ acetyl-transferase like this could help us to understand the active site requirements fordistinct acetyltransferase activities and potentially aid in the treatment of

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acetyltransferase-related diseases. Therefore, I aim to engineer a lysine acetyl-transferase (KAT) to have N-terminal acetyltransferase activity which willwiden the activity of acetyltransferases. To accomplish this, I will compareand contrast the active sites of an N-terminal acetyltransferase (NAT) and aLysine acetyltransferase (KAT) to generate a list of the differing amino acids,and then conduct mutagenesis on the KAT. Our goal is to ultimately convert aKAT into NAT, which will reveal information concerning which amino acidscontribute to both the specificity and binding capabilities of the twoacetyltransferases.Following this, we will compare and contrast our new functioning versions ofthe NAT to an archaeal N-terminal acetyltransferase with the goal of alteringthe archaeal NAT to be more like our new NAT versions generated before,which contain properties of the original KAT and NAT. The archeal NAThas a wide variety of substrates compared to other NATs. Using the samemethods as above, we want to engineer a new version of the archaeal NATthat successfully acetylates both the N-terminal and lysine side chains, therebyproducing the first acetyltransferase that is promiscuous enough to acetylateboth substrates. By creating this ‘‘promiscuous’’ acetyltransferase, we willhave created an acetyltransferase that could potentially become a molecularprobe that could aid in the development of treatments of human diseases andcertain types of cancer that are linked to acetyltransferases.

2028-Pos Board B44Engendering Methane Monooxygenase and Hydrogen Peroxide OxidaseActivity into a Designed Dimetal Protein by Increasing Protein DynamicsRonald L. Koder1, Jonathan M. Preston1, Bernard H. Everson1,Emma Bjerkefeldt1, Florika C. Macazo2, Fabien Giroud2, Shelly D. Minteer2,David J. Vinyard3, Gary W. Brudvig3.1Physics, The City College of New York, New York, NY, USA, 2Chemistry,The University of Utah, Salt Lake City, UT, USA, 3Chemistry, YaleUniversity, New Haven, CT, USA.It has become relatively easy to design proteins which are extremely rigid butlack the degree of dynamic motion required for catalytic activity. Here wereport the rational redesign of DF2, an extremely rigid, computationally de-signed homodimeric diiron protein, into two functional enzymes - a methanemonooxygenase when bound to two iron ions and a hydrogen peroxide oxidasewhen bound to two manganese ions. This was achieved by simply reversing ofthe amino acid sequence of two of the helices, greatly reducing the packingcomplementarity in the protein core - both decreasing the protein stabilityand greatly increasing the dynamic motion of the enzyme. The resulting diironenzyme catalyzes the hydroxylation of methane to methanol when suppliedeither with molecular oxygen and reducing equivalents or hydrogen peroxide.The dimanganese enzyme oxidizes hydrogen peroxide by two electrons form-ing molecular oxygen at a rate of at least 400 s�1 while only catalyzing thereduction of hydrogen peroxide to water at a rate of 0.08 s�1. Most natural en-zymes with peroxide as a substrate are catalases - hydrogen peroxide dismu-tases which catalyze both reactions at high rates in order to lower thecellular concentration of hydrogen peroxide. Because it favors the oxidizinghalf-reaction over the other by a factor of 5000, dimanganese CDM3 promisesto serve as a unique alternate version of an oxygen evolving cluster in thedesign of new photoautotrophic organisms which perform carbon fixation uti-lizing light and low energy electrons originating from what is typically consid-ered to be a toxic intercellular waste product.

2029-Pos Board B45A Multi-scale Approach to the Study of Protein Design, Folding and Ag-gregationFrancesca Nerattini1, Valentino Bianco1, Chiara Cardelli1, Luca Tubiana1,Ivan Coluzza2.1Faculty of Physics, University of Vienna, Vienna, Austria, 2CICbiomaGUNE, Donostia, Spain.In nature, proteins are dissolved in water, which has a profound impact on pro-tein folding and aggregation properties. In order to study such effect at a funda-mental level, we employ a multiscale coarse-grained approach. First, wedemonstrate with a multiscale approach that a simple design procedure can pro-duce proteins that fold into their target structure both at a simple representationlevel (lattice model1-3) as well as at a realistic representation level (caterpillarmodel4,5). In fact, we show that, for a large set of real protein structures, thecaterpillar protein model produces designed sequences with similar physicalproperties to the corresponding naturally occurring sequences. For an indepen-dent set of proteins, previously used as a benchmark, the correct folded struc-ture of both the designed and the natural sequences is also demonstrated.Within the lattice model, we introduce an explicit solvent and we show thatfundamental features of natural proteins can be reproduced on large popula-tions of artificial sequences designed taking into account the properties of water

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at different temperatures and pressures. Finally, we push our model to solutionsof proteins studying their aggregation properties.References1.Coluzza, I. & Frenkel, D. Monte Carlo study of substrate-inducedfolding and refolding of lattice proteins. Biophys. J. 92, 1150-6(2007).2.Shakhnovich, E. I. & Gutin, a M. Engineering of stable and fast-folding sequences of model proteins. Proc. Natl. Acad. Sci. U. S. A. 90,7195-7199 (1993).3.Abeln, S., Vendruscolo, M., Dobson, C. M. & Frenkel,D. A simple lattice model that captures protein folding, aggregation and amy-loid formation. PLoS One 9, (2014).4.Coluzza, I. A Coarse-Grained approachto protein design: Learning from design to understand folding. PLoS One 6,e20853 (2011).5.Coluzza, I. Transferable coarse-grained potential for denovo protein folding and design. PLoS One 9, e112852 (2014).

2030-Pos Board B46Monitor and Control Upstream Bioprocessing using a Point-of-Need MassSpectrometerRichard W. Moseley, Alexander I. McIntosh.Microsaic Systems, Woking, United Kingdom.The applications of mass spectrometry in the pharmaceutical industry havelong been established. Mass spectrometers are highly versatile and commonlyused to identify reagents, products, contaminants, and impurities from samplesin drug research and development, scale-up, and high volume manufacturinglines. However, the instruments are normally large, power demanding, andexpensive pieces of equipment operated in centralized laboratories by MS spe-cialists. The new demands of bioprocessing, used to make biologics, means thatthere is a need for analytical instruments, such as mass spectrometers, to pro-vide real-time information (on-line and at-line) at the point-of-need. Theinformation-rich data provided by MS also helps to address the increased com-plexities of bioprocessing. We show how our deployable, and compact massspectrometer sampling the media from a bioreactor environment in real-timecould provide important process monitoring, and critical quality attributes ofthe product. This information can then be used to maximize yields of biologicsby optimising the cell media, feeding, and harvesting strategies of the targetbiologic being produced. In addition, MS point-of-need analysis providestimely safety assurance as any harmful PTMs of the product, and otherdangerous host cell proteins can be controlled and minimized upstream.

2031-Pos Board B47PEBank: A Comprehensive Database for Protein Engineering and DesignConnie Wang1, Paul Chang1, Marie Ary1, Stephen Mayo2, Barry Olafson1.1Protabit, Pasadena, CA, USA, 2California Institute of Technology,Pasadena, CA, USA.Recent advances in gene synthesis, microfluidics, deep sequencing, and micro-array techniques have made it possible to construct and assay large libraries ofvariant protein sequences. This rapid generation of large sets of mutational datahas significantly enhanced researchers’ ability to study how proteins functionand to engineer proteins with new and improved properties. Although manygroups around the world are currently generating large amounts of protein en-gineering data, there is no standardized format to report this data and no simplemechanism for groups to share the data that they generate.We have developed PEBank (Protein Engineering data Bank), a comprehensivedatabase for protein engineering data where users can store their data as well asquery and analyze data submitted by themselves and others. PEBank stores thedata in a relational database using a standardized schema that requires full pro-tein sequence information and detailed assay descriptions. These features allowfor accurate comparison of measurements made across different proteins and bydifferent groups. PEBank is comprehensive in that it accepts data for severaldifferent protein properties, including those related to stability, folding, activ-ity, and binding. PEBank thus provides a central repository for data that is oftenscattered across many different specialized databases. PEBank features a webinterface and REST API that streamlines data deposition and allows for batchinput and queries. A suite of analysis tools are provided to allow for discoveryand analysis of relationships between mutated sequences. We demonstrate theimportance of a standardized format for reporting protein engineering data thatallows for accurate comparisons between different data sets and enables futuredata mining and machine learning approaches to be applied.

2032-Pos Board B48A Knob-Socket Based Rule Set for Designing Peptide Binding to PDZDomainsShivarni Patel, Hyun Joo, Jerry Tsai.Chemistry, University of the Pacific, Stockton, CA, USA.PDZ domains are regulatory proteins binding the C-termini of proteins. Knowl-edge of the binding mechanism of PDZ domains provides new avenues for can-cer treatment. Application of the knob-socket packing model in mapping thequaternary packing surface topology (PST) allows a direct analysis of the

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residues important for peptide specificity and affinity to the PDZ domain. PST’sof all eight solved crystal structures of T-cell lymphoma invasion and metas-tasis 1 (Tiam1) PDZ domains are mapped using knob-socket analyses. Thecomparison among the PSTmaps of all Tiam1 PDZ-ligand structures reveal de-terminants of peptide affinity and specificity. The PST maps of PDZ complexesallow categorization of the important interactions between ligand and the PDZdomain binding pockets. Interestingly, all the ligands bind as a 6th sheet to thePDZ’s 5-strand b-sheet. The extension of the b-sheet provides additional knobsand sockets to pack with PDZ’s second a-helix H2. In agreement with previousexperimental analyses, the peptide P0 and P-2 positions directly interact with thePDZ domain, but the binding is more complex. Comparison between differentPDZ domains and their respective peptides show that the P0 position packs pri-marily against the first coil, and the P-2 position packs into H2. Therefore, thepeptide interaction with a PDZ domain can be separated into 3 distinct interac-tions: 1) extend the b-sheet, 2) interactions of this extended b-sheet with the a-helix H2 (specifically P-2), and 3) P0 packing into the first coil. This set of rulesprovides a simple yet discrete guide to designing better binding peptides to aPDZ domain. Moreover, this result demonstrates the utility of the Knob-Socket construct in the analysis and design of peptide ligand binding with a pro-tein domain.

Posters: Protein Stability, Folding, andChaperones II

2033-Pos Board B49Quantitative Prediction of Bacterial Fitness from a Protein’s EnergyLandscapeCatherine R. Knoverek1, Kathryn M. Hart1,2, Gregory R. Bowman1.1Biochemistry and Molecular Biophysics, Washington University in St.Louis, St. Louis, MO, USA, 2Chemistry, Williams College, Williamstown,MA, USA.The biophysical properties of proteins provide a link to how molecular muta-tions might exert their effects on cellular fitness. A good candidate for exam-ining this relationship between genotype and phenotype is TEM beta-lactamase, a bacterial enzyme that confers antibiotic resistance by hydrolyz-ing penicillin-like antibiotics. It is known that four missense mutations inTEM result in increased activity against third-generation cephalosporins,such as cefotaxime. These mutations affect both the catalytic efficiency andthe stability of TEM, but neither of these properties alone are enough to pre-dict bacterial fitness. Circular dichroism (CD) data suggest that TEM foldingincludes at least one intermediate, and western blots provide evidence thatchanges in the stability of this intermediate ensemble relative to the nativeensemble correlates with changes in cellular protein abundance. This isfurther supported by pulse proteolysis experiments indicating that the inter-mediate ensemble is susceptible to degradation. We hypothesize that interme-diate stability can be quantitatively related to protein abundance, thusallowing us to create a mathematical model to predict bacterial fitness, asmeasured by minimal inhibitory concentration (MIC), from the activity andstability of a TEM mutant.

2034-Pos Board B50Prediction of New Stabilizing Mutations Based on Mechanistic Insightsfrom Markov State ModelsMaxwell I. Zimmerman1, Kathryn M. Hart2, Carrie A. Sibbald1,Thomas E. Frederick1, John R. Jimah3, Catherine R. Knoverek1,Niraj H. Tolia3, Gregory R. Bowman1.1Biochemistry and Molecular Biophysics, Washington University in St.Louis, St. Louis, MO, USA, 2Department of Chemistry, Williams College, St.Louis, MO, USA, 3Molecular Microbiology, Washington University in St.Louis, St. Louis, MO, USA.Protein stabilization is fundamental to enzyme function and evolution, yet un-derstanding the determinants of a protein’s stability remains a challenge. This islargely due to a shortage of atomically-detailed models for the ensemble ofrelevant protein conformations and their relative populations. For example,the M182T substitution in TEM b-lactamase, an enzyme that confers antibioticresistance to bacteria, is stabilizing but the precise mechanism remains unclear.Here, we employ Markov state models (MSMs) to uncover how M182T shiftsthe distribution of different structures that TEM adopts. We find that M182Tstabilizes a helix that is a key component of a domain interface. We then predictthe effects of other mutations, including a novel stabilizing mutation, andexperimentally test our predictions using a combination of stability measure-ments, crystallography, NMR, and in vivo measurements of bacterial fitness.We expect our insights and methodology to provide a valuable foundationfor protein design.

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2035-Pos Board B51Probing Protein Folding Landscape by Using Combined Force Spectros-copy and Molecular Dynamics SimulationsHa H. Truong, Susan Marqusee.Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA,USA.Most proteins, even those with complex folding trajectories and rough energylandscapes, successfully navigate the funneled folding landscape to achieve thecorrect native conformations. However, some proteins misfold, sometimes re-sulting in some neurodegenerative diseases and cancers. Such misfolding isthought to occur via partially folded high-energy structures accessible fromthe native state or during the folding process. Structural and energetic charac-terization of such high-energy species is important but experimentally chal-lenging due to their rare and transient nature under native conditions. Single-molecule force spectroscopy allows investigation of the force dependent move-ment of the transition state ensemble and detection of high-energy intermedi-ates, which may be populated at high force. Molecular dynamics simulationsallow atomistic characterization of structures and multiple folding pathwayson the energy landscape. Here, we probe important protein sub-states andfolding pathways of the second PDZ domain of PTP-BL by using a combina-tion of single molecule optical tweezers experiments and molecular dynamicssimulations.

2036-Pos Board B52The Structural Basis of Thermostability in an Engineered Variant of theEngrailed HomeodomainJennifer T. Young, Catrina Nguyen, Michelle E. McCully.Biology, Santa Clara University, Santa Clara, CA, USA.The Engrailed Homeodomain (EnHD) is a three-helix bundle transcriptionfactor with an ultra-fast folding rate, making it an attractive choice forcomputational and experimental thermostability studies. The Mayo groupdesigned an engineered variant of EnHD called UVF, which both folded suc-cessfully and had a melting temperature >99�C, much higher than themelting temperature of 52�C for EnHD. We constructed hybrid proteinmodels to test several hypotheses. First, we hypothesize that the buried res-idues of UVF contribute more to its thermostability than its surface residues.To test this hypothesis, we constructed two hybrid models combining thesets of buried and surface residues of EnHD and UVF. We also hypothesizethat the lack of hydrogen bonds between buried and surface residues of UVFenable these regions of the protein to move independently and potentially in-crease thermostability. To test this, we created models of EnHD withoutthese hydrogen bonds and compared its stability with UVF. Finally, engi-neering proteins for stability often comes at the expense of function,and the stability-focused design of UVF removed EnHD’s DNA-bindingresidues. We constructed various models of UVF reintroducing theDNA-binding residues of EnHD to find a suitable model. Here, wereport all-atom, explicit-solvent molecular dynamics simulations of EnHD,UVF, and our hybrid models at 25�C and 100�C and assess the proteins’stabilities.

2037-Pos Board B53Chemical Chaperone Activity of NADD in Protein FoldingChen Chen, Pei-Fen Liu, Chiwook Park.Purdue University, West Lafayette, IN, USA.Folding of most proteins occurs in the complex and crowded cellular environ-ment, and the efficient folding in this environment is critical for the fitness ofthe organism. Still, our understanding of how the complex chemical environ-ment affects protein folding in cells is largely limited. Recently, we discov-ered that NADþ has a significant effect on the folding of E. coliglyceraldehyde-3-phosphate dehydrogenase (GAPDH). Originally, we foundfrom our proteomics screen that GAPDH is apparently destabilized in thepresence of ATP. A follow-up biophysical characterization demonstratedthat the apparent destabilization results from selective stabilization of adimeric intermediate of GAPDH, not the native tetramer, by ATP. Moreover,GAPDH folds significantly faster in the presence of a physiological concen-tration of not only ATP but also NADþ, which is a cofactor for GAPDH.Investigation of the effects of the structural fragments of NADþ on the foldingof GAPDH revealed that NADþ facilitates the folding of GAPDH through theinteraction between its adenosine moiety and a partially folded intermediateof GAPDH. A mutagenesis study of the binding pocket elucidated that theadenine-binding subsite of the NADþ-binding pocket is responsible for the ef-fect of NADþ on GAPDH folding. Based on the results, we propose a novelmechanism by which NADþ functions as a chemical chaperone in protein fol-ding.

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2038-Pos Board B54The Influence of the Apical Domain of Groel Chaperone on the Kineticsand Thermodynamics of Zebrafish Dihydrofolate Reductase Under Ther-mal StressCharu Thapliyal1, Pratima Chaudhuri2, Tapan K. Chaudhuri1.1School of Biological Sciences, Indian Institute of Technology Delhi, NewDelhi, India, 2Amity Institute of Biotechnology, Amity University Noida,Uttar Pradesh, India.In the present study, we have attempted to characterize the chaperoning abilityof the apical domain (residue 191–345) of bacterial chaperone GroEL (mini-GroEL) in isolation. In native form, miniGroEL constitutes a hydrophobicpocket where partially folded substrate protein binds. In the present studyvarious aspects of the thermal aggregation prevention of dihydrofolate reduc-tase from zebrafish (zDHFR) were studied in the presence of miniGroEL.The thermal denaturation of zDHFR is irreversible, thus it is a very good modelsystem for chaperone mediated aggregation prevention studies. The thermalaggregation of zDHFR was considerably prevented (42%) in the presence ofminiGroEL. This miniature form of GroEL was able to delay the rate of thermalinactivation of zDHFR as monitored through Arrhenius plots. Furthermore, thereactivation yield of thermally inactivated zDHFR was significantly enhanced(35%) in the presence of miniGroEL. Through these findings we were able toascertain the chaperoning ability of the miniature form of GroEL which ex-hibited all attributes of aggregation prevention, which could in turn be usedas a very good in vitro and in vivo chaperone system akin to small heat shockproteins. It further delayed the thermal aggregation of zDHFR which couldprove useful during in vivo over-expression of miniGroEL where the partiallyaggregating protein could be further acted upon by ATP-dependent chaper-oning machineries. Since, it is non-homologous to known sHSPs, studyingits chaperoning mechanism can provide novel mechanistic insights.

2039-Pos Board B55Nanomechanics of Protein Unfolding Outside Protease NanoporesBinquan Luan.IBM Research, Yorktown Heights, NY, USA.Protein folding and unfolding have been the subject of active research for de-cades. Most of previous studies in protein unfolding were focused on temper-ature, chemical and/or force (such as in AFM) induced denaturations. Recentstudies on the functional roles of proteasomes (such as ClpXP) revealed a novelunfolding process in cell, during which a target protein is mechanicallyunfolded and pulled into a confined, pore-like geometry for degradation. Whilethe proteasome nanomachine has been extensively studied, the mechanism forunfolding proteins with the proteasome pore is still poorly understood. Here,we investigate the mechanical unfolding process of ubiquitin with (or reallyoutside) an idealized proteasome pore, and compare such process with thatin the AFM pulling experiment. Unexpectedly, the required force by a proteo-some can be much smaller than that by the AFM. Simulation results also un-veiled different nanomechanics, tearing fracture vs. shearing friction, in thesetwo distinct types of mechanical unfoldings.

2040-Pos Board B56Identifying Novel Interacting Partners for the UNC-45 Chaperone inDrosophila melanogasterDaniel Smith.San Diego State University, San Diego, CA, USA.The UNC-45 protein is a member of the UNC-45/CRO1/She4p (UCS) familythat is required for myosin accumulation and thick filament assembly duringmuscle development in several model organisms. UNC-45 interacts with thechaperone Hsp90 and appears to be responsible for folding the myosinmotor domain. However, additional factors that interact with UNC-45during this process are poorly characterized. To approach this problem, weare using the Drosophila melanogaster model system together with co-immunoprecipitation and mass spectrometry to identify novel UNC-45 inter-acting proteins. We have generated a transgenic fly that expresses an UNC-45:GFP fusion protein under the control of a Drosophila UNC-45 promoterfragment. Similar to wild-type UNC-45, the recombinant fusion protein ex-hibits in vitro chaperone activity for the non-native substrate citrate synthasein thermal aggregation assays. The transgene is expressed at roughly 45% ofendogenous levels and is capable of rescuing UNC-45 knockout flies in vivo.These data indicate that the tethered GFP moiety does not significantly alteror inhibit UNC-45 activity. The UNC-45:GFP fusion protein can be efficientlyimmunoprecipitated using anti-GFP Nano-Traps� (ChromoTek) and we areexploring conditions for the efficient pull-down of UNC-45 interacting partnersusing this ex vivo system. These putative binding partners will then be identifiedusing mass spectrometry and interrogated in vivo using standard genetic tech-niques available to the Drosophila system. Identifying novel UNC-45 clients

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could further our understanding of the role of this highly conserved chaperone,while identification of co-chaperone partners could provide us with novelmechanistic insights. This work has been funded through R01 AR055958 toSIB and F32 AR067654 to DAS.

2041-Pos Board B57Mechanistic Basis for Client Recognition and Amyloid Inhibition ofNMNATShengnan Zhang, Xiaojuan Ma, Dan Li, Cong Liu.Interdisciplinary Research Center on Biology and Chemistry, ShanghaiInstitute of Organic Chemistry, Chinese Academic of Science, Shanghai,China.Imbalance of proteostasis leads to abnormal protein aggregations as a commonpathological hallmark in a variety of devastating human neurodegenerative dis-eases such as AD and PD. Chaperones play essential roles in maintaining pro-teostasis and preventing protein from abnormal aggregation. NMNAT, a NADsynthase firstly identified as a key protector in Wallerian axon degeneration,was recently found to possess chaperone activity and exhibits protective effectsin cells and animal models of neurodegeneration. However, it remains enig-matic how an enzyme evolves chaperone activity and prevents pathologicalamyloid aggregation.Here, we found that NMNAT recognizes a full spectrum of client amyloid pro-teins (e.g. a-syn, Tau, Ab and IAPP), and prevents their aggregation and cyto-toxicity. By using multiple biophysical approaches, we identified the clientbinding pocket of NMNAT which is partially overlapped with its catalyticpocket, providing the structural basis of how NMNAT exhibits both chaperoneand enzyme activity. We found that both electrostatic and hydrophobic interac-tions are crucial for the client-chaperone recognition. In addition to bindingvarious amyloid client proteins, NMNAT shows high binding affinity andselectively to phosphorylated Tau, which is closely related to diseases. More-over, NMNAT can inhibit both liquid-liquid phase separation and amyloid ag-gregation of phosphorylated Tau. Our work helps to explain how a chaperonehalt its client proteins from aggregation and highlight the importance ofNMNAT as a maintenance factor in neurodegenerative diseases.Reference:

2042-Pos Board B58Mapping Interactions between the Chaperone Domain of UNC-45B andMyosinMichael Villarreal, Eleno Garza, Andres Oberhauser.Neuroscience and Cell Biology, University of Texas Medical Branch,Galveston, TX, USA.Force generation in striated muscle comes from the myosin motor domain, an110kDa globular protein that allows conversion of the chemical potential en-ergy in ATP into mechanical work. This complex protein is incapable ofself-folding and assembly. Instead, molecular chaperones work in a precisenetwork to allow a nascent myosin polypeptide to be protected from aggrega-tion and folded into its native and functional conformation. The UNC-45Bchaperone is associated with the proper folding and function of the sarcomericmyosin. Hence, deciphering the precise mechanisms by which the chaperoneUNC-45B helps to fold myosin heads is a challenge at the core of muscle devel-opment and function. Which are the key regions in UNC-45B mediate itsmyosin-chaperonin function? Guided by the available crystal structures, bio-physical, biochemical data we have mapped putative key amino acid regionsin UNC-45B that may mediate its myosin-binding and chaperoning functions.We are using a combination of complementary assays that probe effect of mu-tations on UNC-45B: i) structure (by circular dichroism and Bis-ANS fluores-cence); ii) binding to myosin (by using a myosin-chaperone fluorescence assay)and iii) chaperone function (by using a light scattering assay for myosin aggre-gation). Together, these biophysical studies have started to shed light upon thecritical centers for UNC-45B function, which may serve as target sites forfuture drug development.

2043-Pos Board B59Cryo-EM Analysis of the AAAD Quality Control Protease ClpXPMia Shin.Department of Integrative Structural and Computational Biology, TheScripps Research Institute, La Jolla, CA, USA.To prevent off-pathway folding trajectories, mitochondria require a diversenetwork of chaperones and proteases to maintain protein homeostasis, or pro-teostasis. ClpXP, along with Lon, is the primary quality control protease withinthe mitochondrion. Though the requirement for these proteases is not clear,ClpXP-mediated degradation is involved in several different signaling path-ways, including the mitochondrial unfolded protein response. Glynn and col-leagues solved the crystal structure of E. coli ClpXDN in 20091. This 3.25Aasymmetric reconstruction revealed two different conformations of the large

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and small subdomains of each ATPase subunit with respect to each other -either nucleotide loadable or unloadable. This observation, paired withbiochemical data, suggested that the ClpX ring exists in a L-L-U-L-L-U confor-mation. In our previous work2, we present the first atomic model of a substrate-bound inner mitochondrial membrane AAAþ quality control protease, YME1.Our structure reveals how the ATPases form a closed spiral staircase encirclingan unfolded substrate, directing it toward the flat, symmetric protease ring. Oneof the structure-guided inquiries upon solving the YME1 structure and deter-mining the mechanism of substrate translocation was whether this mechanismis conserved for other AAAþ proteases. By analyzing the structure of ClpXPby cryo-EM, we hope to determine its mechanism of substrate translocationand whether this mechanism is conserved.

2044-Pos Board B60Teasing Apart the Role of the Ribosome and Molecular Chaperones inCellular Protein FoldingRayna M. Addabbo1, Matthew D. Dalphin1, Yue Liu2, Miranda F. Mecha1,Silvia Cavagnero1,2.1Biophysics Graduate Program, University of Wisconsin-Madison, Madison,WI, USA, 2Department of Chemistry, University of Wisconsin-Madison,Madison, WI, USA.All cells rely on protein folding to support and sustain life, yet protein foldingpathways in vivo are not well understood. Inside E. Coli cells, proteins are pro-duced by ribosomes in the presence of the trigger factor and DnaK molecularchaperones. Prior studies established that both the ribosome and molecularchaperones facilitate in vivo protein folding. However, it has not yet beenpossible to determine their independent roles since cells cannot survive at tem-peratures over 30�C when the genes for trigger factor and DnaK have beenconcurrently knocked out. In this investigation, we attain the latter importantyet elusive experimental condition by biosynthesizing a model protein in anE. Coli cell-free system derived from a trigger factor- deleted cell strain inthe presence of an in-house-designed peptide inhibitor of DnaK. We showthat the process of being translated through the ribosome is sufficient to grantsolubility to the de novo-synthesized protein at submicromolar protein concen-trations. The cotranslationally acquired structure favors proper folding but it isnot a sufficient condition to ensure it. We employed time-resolved fluorescenceanisotropy in the frequency-domain in combination with multidimensionalNMR spectroscopy to study the quality of the de novo-produced protein. Wefound that even soluble ribosome-released proteins may be improperly folded,and minimal concentrations of molecular chaperones are required to ensureproper folding.

2045-Pos Board B61Structures and Dynamics of Protein Folding on the Ribosome by NMRSpectroscopyAnais M. Cassaignau, Christopher Waudby, Tomasz Wlodarski,Lisa Cabrita, John Christodoulou.Structural and Molecular Biology, University College London, London,United Kingdom.We have developed a strategy to enable the study of co-translational foldingusing solution-state NMR spectroscopy, the only technique able to characterizethis dynamic process at atomic resolution. Using isotopically-labelled, ribo-some-nascent chain complexes (RNCs), we have created snapshots that mimicthe emergence of an immunoglobulin-like domain, FLN5, within a multido-main protein. The ribosome appears to modulate the folding process, as thecomplete sequence of FLN5 is required to emerge well beyond the tunnelbefore acquiring native structure, whereas FLN5 in isolation folds spontane-ously, even when truncated. To understand the behaviour of this nascent chainwhile bound to the ribosome, we use a combination of protein engineering andNMR dynamics to describe residue-specific sites of nascent-chain-ribosome in-teractions of different magnitude of strength, and biochemical assays as well aschemical shift-restrained MD simulations of RNCs to underpin our spectral ob-servations. The multi-pronged studies presented here are providing some of thefirst high-resolution insights of these fundamental processes, and begin todissect the energy landscape sampled by a nascent chain on the cusp of initia-tion of folding on the ribosome.1. Cassaignau, Cabrita et al., NSMB 2016 2. Cassaignau et al., Nat Prot 2016

2046-Pos Board B62Thermodynamic Stability of Polar and Non-polar FibrilsFarbod Mahmoudinobar, Zhaoqian Su, Cristiano L. Dias.Physics, New Jersey Inst of Tech, Newark, NJ, USA.Protein aggregation into fibril-like structures is the hallmark of amyloid dis-eases that included Alzheimer’s, Parkinson’s and type 2 diabetes. An under-standing of the molecular forces driving fibril formation may provide insights

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into strategies to prevent these diseases. Thermodynamics has played animportant role in unraveling the molecular mechanisms of different confor-mational changes in proteins, e.g., protein folding. However, equilibriumthermodynamic quantities of amyloid fibrils are not easily accessible experi-mentally and they remain largely unknown. In this work, we discuss resultsfrom all-atom molecular dynamics simulations in which we measured equilib-rium thermodynamic quantities related to addition/dissociation of a peptideto/from a fibril. In particular, we will highlight differences in the thermody-namic properties of polar (IAPP28-33) and non-polar (Ab16-21) fibrils. Wealso study the effect of a small molecule inhibitor on stability of these fibrils.Simulations were performed using an umbrella sampling protocol combinedwith replica exchange molecular dynamics to compute potential of mean force(PMF) of peptide addition as a function of temperature. The PMF correspondsto the free-energy to ad a peptide to a fibril. The temperature dependence ofthe PMF is used to compute changes in entropy (DS), enthalpy (DH) and heatcapacity (DC) of peptide addition/dissociation. We find that the non-polarfibril becomes more stable with increasing temperature and its stability isdominated by entropy. In contrast, the polar fibril becomes less stable withincreasing temperature while it is stabilized by enthalpy. These behaviorsare consistent with the nature of the interactions in their dry core and theyhighlight the importance of side chains accounting for stability of amyloidfibrils.

2047-Pos Board B63Effect of Gene Polymorphisms on the Structural Dynamics of Prion Pro-teins: A Comparative StudyNoah Yoshida, India Claflin, Oscar Coello, Patricia Soto.Creighton University, Omaha, NE, USA.Transmissible spongiform encephalopathies (TSEs) are a group of fatal andrare neurodegenerative diseases that affect mammals, including humans. Theinfectious agent in TSEs is a proteinaceous entity, termed prion, which isdevoid of nucleic acids and capable of replication, infectivity, and toxicity.The main component of the prion agent is the prion protein PrPSc, which resultsfrom the autocatalytic misfolding of the host-encoded cellular form of the prionprotein PrPC. The molecular factors that determine PrPC to PrPSc conversionare not well understood. The secondary structure of the globular-shaped C-ter-minal of PrPC is well conserved across mammal species in spite of the presenceof gene polymorphisms. Our structural bioinformatics study examines the ef-fect of the polymorphisms on the dynamic network of prion protein residue in-teractions and on prion protein - model cell membrane interactions for anumber of mammal species. We will discuss the outcomes of our study in rela-tion to the initial steps of PrPC to PrPSc conversion.

2048-Pos Board B64Membrane Binding of Parkinson’s Protein Alpha-Synuclein: Effect ofPhosphorylation at Positions 87 and 129 by the S to D Mutation ApproachPravin Kumar1, Nathalie Schilderink2, Mireille M.A.E. Claessens2,Vinod Subramaniam2,3, Martina Huber1.1Huygens-Kamerlingh Onnes Lab, Department of Physics, LeidenUniversity, Leiden, Netherlands, 2University of Twente, Nanobiophysics,MESAþ Institute for Nanotechnology & MIRA Institute for BiomedicalTechnology and Technical Medicine, Enschede, Netherlands, 3FreeUniversity of Amsterdam, Amsterdam, Netherlands.Human alpha-synuclein (aS), a protein relevant in the brain with so-far un-known function, plays an important role in Parkinson’s disease. The phos-phorylation state of aS was related to the disease, prompting interest in thisprocess. The presumed physiological function and the disease action of aSinvolves membrane interaction. Here, we study the effect of phosphoryla-tion at positions 87 and 129, mimicked by the mutations S87A, S129A(non-phosphorylated) and S87D, S129D (phosphorylated) on membranebinding [1]. Local binding is detected by spin-label continuous-wave elec-tron paramagnetic resonance, by monitoring the mobility of the spin label.Low mobility shows membrane binding, high mobility local unbinding. ForS87A/D, six positions (27, 56, 63, 69, 76, and 90) were spin labelled indi-vidually and probed by EPR; and for S129A/D, three (27, 56, and 69).Binding to large unilamellar vesicles of 100 nm diameter of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-racglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in a 1 : 1 composition is not affected bythe phosphorylation state of S129. For phosphorylation at S87, local un-binding of aS from the membrane is observed. We speculate that modu-lating the local membrane affinity by phosphorylation could tune the wayaS interacts with different membranes; for example, tuning itsmembrane-fusion activity.[1]Kumar et al. Isr. J. Chem. (2016) DOI: 10.1002/ijch.201600083

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2049-Pos Board B65pH-Induced Frustration in the FREE Energy Landscape Dictate Misfold-ing of the Prion ProteinRoumita Moulick, Rama Reddy Goluguri, Jayant B. Udgaonkar.Biophysics, Biochemistry and Bioinformatics, National Centre ForBiological Sciences, Bangalore, India.Free energy landscapes of proteins have been theoretically predicted to beextremely complex, wherein protein folding/unfolding occurs in a parallelflow process via multiple intermediates separated by small and large energybarriers. However, not only have such complexities been rarely observed inensemble measurements of folding/unfolding of proteins, how such underlyingcomplexities in free energy landscapes modulate their aggregation propensitieslack detailed understanding.Here, a comparative study of the folding pathways of the mouse prion pro-tein was carried out at two solvent conditions: low pH where the protein isdestabilized and aggregation-prone; and in physiological conditions wherethe protein does not normally aggregate, unless destabilized. In physiolog-ical conditions, the protein utilizes a single pathway to fold via two obliga-tory intermediate states. However, kinetic folding studies in aggregation-prone solvent conditions reveal an unusually complex energy landscape.Folded and unfolded protein molecules are observed to traverse different,distinct regions of the energy landscape of the protein. The thermodynamicstates accessible on the folding pathway are not accessible to the unfoldingpathway and vice versa as they are separated by large energy barriers thatrender the two folding pathways inaccessible to each other. Consequently,the folding/unfolding pathway utilized depends on the initial folding condi-tions i.e. on where on the energy landscape the folding/unfolding com-mences from. This energetic frustration channelizes the native protein tounfold to the denatured state via an intermediate previously identified tobe the monomeric precursor conformation initiating misfolding of the prionprotein.This study proposes a plausible mechanism by which frustration and rugged-ness in a protein’s free energy landscape determines the aggregation propensityof the protein.

2050-Pos Board B66The Physical Factors Governing Tensile Force Generation by Co-translational Protein FoldingSarah E. Leininger, Edward P. O’Brien.Chemistry, Penn State University, State College, PA, USA.Tensile forces generated from co-translational protein folding can affect thetranslation elongation rate, which will in turn modify the protein’s ability tofold and function properly. Here, we use computational methods to determinethe magnitude and scaling of the tensile forces generated by the co-translational folding of several protein domains. We also use these domainsto determine how the magnitude of the tensile force depends on physical fac-tors, especially domain stability and topology. These results show that co-translational folding can exert a force which can influence the translationelongation rate, including rescuing a stalled sequence. This force is biologi-cally significant because it can change the orientation of the PTC, whichcan change translation elongation rates, which affects co-translational foldingand other downstream processes.

2051-Pos Board B67Probing the Effect of the Ribosome on the Protein Folding Pathway usingSingle-Molecule Chemo-Mechanical FoldingEmily Guinn1, Susan Marqusee2.1Institute for Quantitative Biosciences, University of California, Berkeley,Berkeley, CA, USA, 2Institute for Quantitative Biosciences, Department ofMolecular and Cell Biology, University of California, Berkeley, Berkeley,CA, USA.In vivo, proteins function in a complex environment where they are subject tomany stresses that can modulate the protein’s energy landscape. Using a com-bination of force and chemical denaturant (chemo-mechanical unfolding), wehave demonstrated that a simple, two-state folding protein can fold throughmultiple parallel pathways and that seemingly small changes in force, dena-turant concentration and protein sequence can strongly modulate the flux be-tween the pathways. This result suggests that in vivo, the heterogeneouscrowded cellular environment could strongly influence the mechanisms of pro-tein folding and unfolding. Another perturbant that could affect the foldingpathway in vivo is the ribosome since many proteins fold while they are beingtranslated. To explore how the ribosome affects the folding mechanism, we usechemo-mechanical unfolding to characterize folding pathways both on and offthe ribosome.

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Posters: Protein-Small Molecule Interactions III

2052-Pos Board B68Characterizing the Direct Influence of a Small Molecule on a Ras-RelatedProtein InteractionDjamali Muhoza1, Alix Montoya-Beltran1, Paul D. Adams2.1Chemistry and Biochemistry, Fayetteville, AR, USA, 2Chemistry andBiochemistry, University of Arkansas, Fayetteville, AR, USA.Ras-related Protein-Protein Interactions (PPIs) are critical to eventsinvolved in cellular proliferation, inhibition of cell death, and cell transfor-mation. It is therefore important to characterize molecular features of Rasprotein interactions that dictate their function as ‘timing switches’ in signaltransduction pathways. This is an important step towards targeting the inhi-bition of cell transformation as well as controlling abnormal signalingactivity of tumor-causing Ras variants. Cell division cycle 42 (Cdc42) isa Ras-related protein, and previous studies from this laboratory have out-lined differences in conformational dynamics in an important region that in-teracts with various effectors, known as the Switch 1 region. Thesedifferences resulted in an increase rate of GTP hydrolysis in the presenceof an effector protein that inhibits GTP hydrolysis when bound to wildtype Cdc42. These findings suggest that induced conformational changesin key effector-binding regions of Ras-related proteins may serve to altereffector protein interactions. As such, the targeting of a small moleculetowards this Switch 1 region of Cdc42 could facilitate changes in localconformation as well as dynamics without affecting the overall stabilityof the protein. We are currently using biochemical and biophysical ap-proaches to characterize the influence of a small molecule ZCL278 on theinteraction between Cdc42 and an effector protein that inhibits Cdc42-stimulated GTP hydrolysis. Preliminary results on the Cdc42-ZCL278 inter-action are presented.

2053-Pos Board B69A Combretastatin Analogue C12 Binds to Colchicine Site in Tubulin, In-hibits Spindle Microtubule Dynamics, Activates Mitotic Checkpoint andInduces Apoptosis in Cancer CellsAnuradha Kumari1, Shalini Srivastava1, Shweta Shyam Prassanawar1,Shailendra Sisodiya2, Sankar K. Guchhait2, Dulal Panda1.1Department of Biosciences and Bioengineering, Indian Instituteof Technology Bombay, Mumbai, India, 2Department of MedicinalChemistry, National Institute of Pharmaceutical Education and Research,Punjab, India.Several combretastatin derivatives are currently undergoing clinical trialsfor various types of cancer. In this work, we elucidated the antiproliferativemechanism of action of a highly potentcombretastatin analogue, C12 (5-Quinolin-3-yl and 4-(3,4,5-trimethoxyphenyl) substituted imidazol-2-amine). The compound displayed much stronger activity than CA-4 againstdifferent types of cancer cells. Further, C12 inhibited the proliferation ofdifferent cancer cells more effectively compared to their non-cancerouscounterparts. The binding of C12 to tubulin was examined using surfaceplasma resonance and the intrinsic fluorescence of C12. Using modifiedDixon plot, C12 was found to competitively inhibit the binding of podophyl-lotoxin, a colchicine-site binding agent, to tubulin. A molecular dockinganalysis also supported the finding that C12 shares its binding site in tubulinwith colchicine. C12 displayed preferential binding to GTP-tubulin thanGDP-tubulin indicating that it may bind to the end of microtubules. Consis-tent with this idea, C12 inhibited the binding of a microtubule plus-endtracking protein, EB1, to microtubules. C12 depolymerized microtubulesboth in in-vitro and in cells. Using fluorescence recovery after photobleach-ing technique, C12 was found to strongly suppress the dynamics of spindlemicrotubules in live HeLa cells. C12 treatment induced multipolar spindleformation, increased the chromosome compactness index, activated mitoticcheckpoint protein BubR1 and ultimately induced apoptotic cell death inHeLa cells.

2054-Pos Board B70Role of Electrostatic Interactions in Ligand Recognition by Orotidine-5’-Monophosphate Decarboxylase (ODCase)Jesi Lee, Trevor Gokey, Weiming Wu, Anton B. Guliaev.Chemistry and Biochemistry, San Francisco State University, San Francisco,CA, USA.The calculation of electrostatic potential (EP) maps on enzyme-ligand interac-tions provides critical information regarding substrate affinity and specificity.

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Here we probed the electrostatic interactions between the orotidine-5’-phos-phate decarboxylase (ODCase) active site and the substrate orotidine-5’-mono-phosphate (OMP), the product uridine-5’-monophosphate (UMP) and twoinhibitors, 6-hydroxyuridine-5’-monophosphate (BMP) and xanthosine-5’-monophosphate (XMP), using density functional theory calculations. All fourligands are structurally similar. However, BMP is the strongest known inhibitorof ODCase. The charge distribution in XMP and BMP shared clear similaritieswith OMP but not UMP. We propose that the resemblance of the EP maps ofXMP and BMP to OMP is likely to account for their ability to effectively bindto ODCase and act as potent inhibitors. Using molecular dynamics simulations,we observed that the binding of BMP induced changes in the active site leadingto a distribution of additional positive charge not observed in the apo form. Inaddition, the inhibitor was stabilized by hydrogen bonding interactions betweenS154 of a loop adjacent to the active site and the N3 and O4 atoms of the hy-droxyuridine ring. Based on the EP map calculations, these atoms have themost negative partial charges in comparison to other ligands investigated inthis work. The S154/BMP interactions also reduced flexibility of the loopwhich helped to form a well-defined binding cavity around the ligand. Our cal-culations showed that the ability of BMP to increase overall positive charge atthe enzyme active site combined with the interaction with S154 could explainwhy BMP is such a potent inhibitor. This work showed how specific electro-static interactions could improve ligand binding and will aid in the develop-ment of future therapeutic inhibitors.

2055-Pos Board B71An Efficient Cell Model for Screening Small Molecule Agonists of GLP-1ReceptorNi Pi, Xiyao Cheng, Yongqi Huang, Zhengding Su.Hubei University of Technology, Wuhan, China.The glucagon-like peptide-1 receptor (GLP-1R) belongs to the class B G-pro-tein-coupled receptor family and is an important drug target for the treatmentof type-2 diabetes. The therapeutic transformation of GLP-1 analogs for thetreatment of type-2 diabetes mellitus has enlightened us to search for smallmolecule agonists of GLP-1 receptor. Here we reported a cell model thatthe GLP-1R was firstly expressed on the surface of H1299 cells and its extra-cellular domain (ECD) was then removed by protease digestion. The cellmodel was utilized to screen small molecule library for GLP-1R agonistswith interaction with ECD. As results, we selected 20 leads that exhibited sig-nificant activity to stimulate the cells to produce cAMP, compared with GLP-1, a positive control. These compounds may lead to the design of orally activeGLP-1 agonists.

2056-Pos Board B72Determination of Effector Binding Affinities Using PhotoacousticCalorimetryJovany J. Betancourt, Jaroslava Miksovska.Department of Chemistry & Biochemistry, Florida International University,Miami, FL, USA.Determination of binding affinity and thermodynamic parameters of interac-tions between two molecules is a common procedure in biochemical contexts.Isothermal titration calorimetry and steady-state fluorescence titration arecommonly used to determine the binding affinity of small effector compoundsand ligands to large biomolecules such as proteins. These methods howeverhave several disadvantages, such large quantity of protein in case of ITC andthe presence of a fluorescence probe in case of fluorescence. Photoacousticcalorimetry (PAC) is often used to monitor volume and enthalpy change asso-ciated with a photo-triggered reaction. Here we have tested the application ofPAC to measure equilibrium binding constant and thermodynamic parametersfor small organic molecules binding to hemoglobin. This technique does notrequire a fluorescent probe to be present and instead takes advantage of thefact that the association of small organic molecules to hemoglobin modulatesthe amplitude of PAC signal for O2 photo-dissociation from this protein.From the temperature dependence of the equilibrium constants, the enthalpychanges were determined using Van’t Hoff plots. So far, we have determinedthe dissociation constant (KD) and enthalpy change (DH) for binding of inositolhexakisphosphate (IHP) (KD = 73.5 mM at 20 �C, DH = �9.8 kJ mol�1) and 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine) (KD = 46.7 mM at 20 �C, DH =13.2 kJ mol�1) to fully-oxygenated adult human hemoglobin; and the deter-mined parameters are in excellent agreement with those published previously.Being able to determine the binding affinity and thermodynamic parameters forligand binding to proteins is extremely useful for understanding the environ-ment of the ligand binding site, potentially allowing us to create new andmore effective drugs. The use of PAC may provide a new approach of evalu-ating ligand protein interactions quickly and with smaller quantities of pro-tein.

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2057-Pos Board B73Discovery of Zika NS5 Polymerase InhibitorsAnthony F.T. Moore, Eda Koculi.Chemistry, University of Central Florida, Orlando, FL, USA.Zika virus (ZIKV) is a mosquito-borne Flavivirus implicated in neurologicaldisorders including microcephaly in infants, and Gillian-Barr�e syndrome.There are currently no vaccine or drug therapies for treating those infectedwith ZIKV. Due to the continued geographical spread of the virus, the devel-opment of Zika therapeutics is of great public health interest. The C-terminalof Zika non-structural protein 5 (NS5) is an RNA dependent RNA polymeraseessential for Zika replication. Hence, this protein has been poised as a ZIKVdrug target. Herein, two high-throughput assays have been designed to investi-gate the effect of small-molecule inhibitors on Zika polymerase activity. Thefirst assay is a malachite green assay, which detects the pyrophosphate releasedin solution as a consequence of the addition of nucleotide to the growing RNAchain. The second assay is a fluorescence assay that detects the formation ofdouble-stranded RNA. Natural product compounds obtained from the NationalCancer Institute’s Developmental Therapeutics Program will be screened formodulators of Zika NS5 RNA polymerase activity. Once a modulator is found,its specificity for Zika RNA polymerase will be investigated by studying theeffect of the inhibitor on T7 DNA dependent RNA polymerase. Moleculesthat modulate the activity of both Zika NS5 RNA polymerase and T7 RNA po-lymerase will not be further investigated. On the other hand, the effect of spe-cific NS5 RNA polymerase inhibitors on the promoter binding, transcriptioninitiation, and elongation will be measured by several biochemical techniques.The effect of most potent inhibitors on halting Zika infections in different hu-man cell lines will be investigated by Quantitative PCR. The small-moleculeinhibitors will both be used as probes to investigate the Zika viral cycle andas lead compounds for drugs that cure Zika infection.

2058-Pos Board B74Reversible Covalent Binding as Concept for Allosteric Inhibition of HostCell Invasion by Malaria ParasitesJanna Ehlert1, Julia Weder1, Matthias Preller1,2.1Institute for Biophysical Chemistry, Hannover Medical School, Hannover,Germany, 2Centre for Structural Systems Biology (CSSB), German ElectronSynchrotron (DESY), Hamburg, Germany.More than 200 million people worldwide are infected with malaria with nearlyhalf-million deaths per year. Malaria is caused by apicomplexan parasites of thePlasmodium species. A major problem is the rapid development of resistancemechanisms by the parasites towards existing drugs, increasing the need fornew treatments. A critical step in the complex life cycle of the parasites isthe invasion of human hepatocytes and erythrocytes, driven by a sophisticatedmotor machinery - the so-called glideosome. This key role during host cell in-vasion renders this dynamic structure an attractive and innovative target forcombating malaria infection. Here, we exploit the concept of reversible cova-lent binding for the development of small molecule inhibitors targeting theparasitic glideosome. Protein-ligand binding and the underlying mechanismof action were determined using a combination of biophysical methods, leadingto highly potent inhibitors against the target protein. Significant antiparasiticactivities against malaria parasites demonstrate the high potential of reversiblecovalent binding inhibitors as a novel concept for treating malaria.

2059-Pos Board B75Interactions of Neuronal Calcium Sensor Dream with ZincMaria D. Santiago.Chemistry & Biochemistry, Florida International University, Miami, FL,USA.Maria J. Santiago Estevez and Jaroslava MiksovskaDepartment of Chemistry and Biochemistry, Florida International UniversityNeuronal calcium sensors regulate several physiological processes in the brainand are linked to numerous pathological conditions such as autism, Parkinsonand Alzheimer disease. Downstream regulatory element antagonist modulator(DREAM) is among the neuronal calcium sensor family, it is an EF-hand pro-tein highly expressed in the central nervous system in various areas such as thehippocampus and the neural cortex. It is responsible for regulating the kineticsof potassium channels, gene expression, calcium homeostasis and enzymaticactivity of presenilin. The characterization of the metal-binding properties ofDREAM and how it affects DREAM structure and DREAM interactionswith effector proteins is fundamental to understand its biological function.Essential metals like Mg2þ, Zn2þ and Ca2þ play extremely important rolesin biological processes through direct interactions with proteins. DREAMcarries out two Ca2þ binding EF hands and one Mg2þ binding EF hand, how-ever, DREAM binding to other biologically significant metals is not very wellknown. Interactions of DREAM with Zn2þ have been monitored by using

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intrinsic and extrinsic fluorescent probes. Data shows that Zn2þ associates toDREAMwith Kd� 200 mM. Zn2þ and it does not compete for the same bindingsite as Ca2þ as the changes in the protein tertiary structure are distinct fromthose observed upon Ca2þassociation. Considering increased concentrationof Zn2þ in neuronal tissue (150 - 200 mM), these results point towards the po-tential role of Zn2þ in modulating DREAM interactions with other intracellularproteins.

2060-Pos Board B76Combination Therapies with Antimicrobial Peptide LL-37 and Conven-tional AntibioticsMehrnaz A. Siavoshi1, Federico I. Prokopczuk2,Nathan-Alexander Del Rosario2, Lannah Abasi1, Sattar Taheri-Araghi3.1Chemistry and Biochemistry, California State University, Northridge,Northridge, CA, USA, 2Biology, California State University, Northridge,Northridge, CA, USA, 3Physics and Astronomy, California State University,Northridge, Northridge, CA, USA.The emergence of antibiotic resistance is one of the largest problems in currentpublic health. A potential solution is combination therapy, where two or moredrugs are combined to fight resistant bacteria. In this work, we evaluate harness-ing the power of antimicrobial peptides (AMPs), which are part of the innate im-munity of virtually all eukaryotic life. The evolutionary stability of AMPs hintsthat it is difficult for bacteria to develop resistance against AMPs’ mechanism ofaction, which is based on membrane perturbation. We conducted a battery ofcombination therapy tests to determine the outcome of combining AMPs withmore than 20 conventional antibiotics representing a range of drug classes. Tothis end, we employed a serial dilution method to test the effect of combiningvarying dosages of AMPs and antibiotics on growth of Escherichia coli cultures.A robust statistical analysis of the experimental data allows us to quantify growthrates and any extended delay in initial growth to discern if and to what extent anyinteractions occur. We discovered that AMPs’ and antibiotics’ action influenceeach other. Certain drugs, like chloramphenicol, experienced heightened antibac-terial activity while other drugs, like streptomycin, were suppressed. Ultimately,our results give insight into how antibiotic effectivity in a clinical setting can beimpacted by these naturally occurring biomolecules.

2061-Pos Board B77Solvation Thermodynamic Properties of Cleanser Surfactants and theirSkin HarshnessManori Jayasinghe.Mathematics Physics and Computer Science, University of Cincinnati BlueAsh College, Blue Ash, OH, USA.The skin irritation potential of cleanser surfactant sodium dodecyl sulfate(SDS), C12H25 OSO3Na, and the ethoxylated derivatives of SDS, sodium laurylether sulfates (SLES), C12H25 (OCH2CH2)xOSO3Na, where x = 1 (SLE1S), x =2 (SLE2S), and x = 3 (SLE3S), has been investigated in the past using exper-imental methods. These studies show that the higher the degree of ethoxylation,the lower the skin irritation potential, with SLE3S being the mildest. In thisstudy, we have investigated the solvation thermodynamic properties of SDSand SLES compounds. With this in mind, we have investigated how the degreeof ethoxylation affects the solvation thermodynamic properties, such as solva-tion entropy, enthalpy, and free energy of this series of surfactants. ExtensiveMD simulations and free-energy calculations were performed over a range oftemperatures to determine the electrostatic and van der Waals contributionsto the hydration entropies and enthalpies. The van der Waals component wasfurther broken down into repulsive and dispersive components by using theWeek-Chandler-Anderson criteria. The electrostatic contribution of the entropyis negative and small in magnitude compare to that of the enthalpy, which isnegative as well. The electrostatic contribution of the entropy and enthalpyvary significantly as a function of the degree of ethoxylation. Our results indi-cate that the higher the degree of ethoxylation, the stronger the electrostatic hy-dration. The van der Waals contribution to the hydration is also examined, andthis component of the hydration enthalpy is significantly lower than the electro-static counterpart for all the compounds in our study, as expected from the well-known fact that hydrophobic hydration is primarily entropic. The van derWaals component of the hydration entropy is negative and shows a variationas a function of the degree of ethoxylation.

2062-Pos Board B78Biophysical Characterization of Interactions of Heparin with HIV-1 TatPeptide 47-57 and ITS Perturbation by Cationic Small MoleculeNeha Tiwari.School of Physical Sciences, Jawaharlal Nehru Univerisity, NewDelhi, India.Tat is a regulatory protein released by HIV infected cells, which is responsiblefor viral replication. GAGs such as heparan sulphate, are required for their en-try inside the new cells. In our present study, heparin, a highly sulphated gly-

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cosaminoglycans, was used as a model compound for the interaction study ofGAGwith HIV-1 tat peptide (47-57). We characterized the interactions throughdifferent biophysical methods like isothermal calorimetry, atomic force micro-scopy, circular dichroism, surface plasmon resonance, fluorescence as well asthrough molecular docking by using ClusPro Server. This investigation wasfocused on understanding the type of interactions involved and how smallcationic molecule can be used as inhibitor of peptide-heparin complex. Weobserved distinct biphasic isotherm for peptide-heparin interaction in ITC,which exhibits different affinities in peptide-heparin complex formation. Bind-ing was mainly driven by non-ionic interactions with small contribution fromionic interactions. ITC as well as ClusPro docking studies suggests one tat pep-tide binds to a chain of 4-5 saccharide molecules. Complex formation betweentat peptide and heparin was confirmed by AFM images. Quinacrine, a smallcationic molecule used to test perturbation of tat peptide-heparin interactions,exhibited successful inhibition of these interactions in ITC and SPR. CD studiesconfirmed binding of quinacrine to heparin, blocking its peptide binding sites.These results demonstrate the possibility of exploitation of small cationic mole-cule in inhibiting disease relevant heparin-protein interaction, which could beeffective strategy for designing future therapeutic drugs.

2063-Pos Board B79Isothermal Titration Calorimetry and Oxygen Binding Studies betweenInositol Hexakisphosphate and Human HemoglobinAntonio Tsuneshige1, Takashi Yonetani2.1Frontier Bioscience, Hosei University, Tokyo, Japan, 2Biochemistry andBiophysics, University of Pennsylvania, Philadelphia, PA, USA.When the naturally occurring effector 2,3-bisphosphoglygerate binds to humanhemoglobin (Hb), the unligated, low affinity state for oxygen of Hb is stabi-lized. In a similar manner, a powerful yet non-natural effector, inositol hexaki-sphosphate (IHP), is known to exert a much stronger effect on Hb, by not onlybinding and stabilizing the low affinity state, but apparently, the high affinitystate, as well. As a result, both ligated and unligated forms are altered, andthe overall oxygen affinity drops dramatically. Previously, oxygenation exper-iments of Hb in the presence of IHP had suggested that this effector might bebinding in two ways. However, as IHP has been customarily used in excess, adetailed characterization of each binding process has not been carried out. Inthis study, we have investigated the interactions between IHP and humanHb -both in the liganded and unliganded forms- by isothermal titration calorim-etry (ITC) and oxygen binding measurements in the equimolar range at pH 7.0and 15 �C. For the liganded ‘‘R’’-structure, we chose the cyanmetHb derivative(HbþCN-), and for the unliganded ‘‘T’’-structure, the nickel-porphyrin Hb(NiHb). ITC experiments showed that IHP binds to both tetrameric derivativesin equimolar amount and with relatively high affinity, but with clear differ-ences. Isothermal titration curves for both derivatives could not be fitted witha single, but rather a two-binding site model. Tertiary/quaternary structural per-turbations introduced systematically into HbþCN- by removal of specific aminoacid residues suggested that the IHP binding site is identical to that exhibited byNiHb, i.e., between the beta subunits. Under these conditions, we have notfound any evidence that suggests that IHP binds between the alpha subunits,or in the central cavity, as recent reports have suggested

2064-Pos Board B80Modulation of the Circadian Period: Searching for Isoform-SelectiveCyclophilin InhibitorsAli Yousefi,Kiernan Kringen, Ryan Noland, AndrewMcShan, Scott Lokey,Carrie L. Partch.Chemistry and Biochemistry, UC Santa Cruz, Santa Cruz, CA, USA.A dynamic, transcription-based oscillator coordinates physiological and behav-ioral processes with specific times in the 24-hour day, ultimately acting as acellular clock. The transcription factor complex known as CLOCK:BMAL1is at the core of this oscillator that generates circadian (about a day) rhythms.BMAL1 possesses a transactivation domain (TAD) that acts as a molecularswitch to control the activation or repression state of CLOCK:BMAL1 andmodulate circadian period. Recent findings from our lab have demonstratedthat cis/trans isomerization about a Trp-Pro imide bond in the TAD is neces-sary for proper 24-hour circadian timekeeping. Members of the cyclophilinfamily, which possess peptidyl-prolyl isomerase (PPIase) activity, typicallycatalyze this intrinsically slow process in vivo. Use of the broad specificity cy-clophilin inhibitor Cyclosporin A lengthens circadian rhythms and its persistentuse as an immunosuppressant in transplant recipients is associated with dereg-ulation of circadian rhythms. We found that a number of nuclear-localized cy-clophilins exhibit robust catalytic activity on the BMAL1 TAD in vitro, yet theabsence of isoform-selective inhibitors has made it difficult to tease out theirrespective contributions to circadian timekeeping. Here we present our initialstudies on three nuclear-localized PPIases: PPIA, PPIE, and PPIH.

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Fluorescence polarization binding assays have allowed us to explore substratebinding in conjunction with 15N/1H ZZ NMR exchange assays that probe cat-alytic activity. Utilizing in silico ligand docking against high-resolution x-raystructures and NMR-based fragment-based drug discovery (FBDD), we aimto find isoform-selective inhibitors to better understand their role in circadianrhythms.

2065-Pos Board B81Nanoscale Encapsulation for Fragment Based Drug DiscoveryBrian Fuglestad1, Nicole E. Kerstetter2, Sabrina B�edard1,A. Joshua Wand1,2.1Department of Biochemistry and Molecular Biophysics, University ofPennsylvania, Philadelphia, PA, USA, 2Graduate Group in Biochemistry andMolecular Biophysics, University of Pennsylvania, Philadelphia, PA, USA.Despite tremendous technical advances in drug discovery, rational de novodevelopment of small molecule drugs remains difficult. High-throughputscreening (HTS) of large natural product libraries thus remains the bedrockapproach in the classical pharmaceutical industry. HTS usually employs detec-tion strategies based on biochemical assays, which inherently require relativelyhigh affinity binding. The discovery of hits is random and infrequent and ob-tained hits are often ill-suited for elaboration into drug-like molecules. Thisis unsatisfactory in many ways. A more recent screening strategy utilizing frag-ment libraries, which offers greater potential for elaboration, has not penetratedthe discovery space to the extent anticipated. This is largely because such mol-ecules are generally weak binders. Weak binding renders activity-based assaysunreliable. Here we describe a new approach for the detection of weak but site-specific binding to proteins. The method takes advantage of solutions of indi-vidual protein molecules encapsulated within the nanoscale water core ofreverse micelles. Using the reverse micelle as a confined space for NMR-based screening, we find 1) weak yet specific binding can be efficiently de-tected; 2) conditions can be defined to reduce non-specific binding; 3) theamount of ligand and target required is significantly reduced; and, most impor-tant, 4) the method allows entry into a region of chemical space that is highlydesired but difficult to access with existing strategies (i.e. weak hydrophilicbinders). Preliminary results demonstrate the basic elements of the approach.An unusually high hit rate from a polar subset (230 compounds) of a commer-cial fragment library gave over one-third coverage of a target protein surface.This level of coverage has the potential to invert the paradigm for hit discoveryand would provide unprecedented flexibility in drug development. Supportedby the NIH.

2066-Pos Board B82Consistency Criterion for Particle Sorting in Single-Particle Cryo-EMDaniel Asarnow, Yifan Cheng.Biophysics, University of California, San Francisco, San Francisco, CA,USA.Current cryo-EM datasets contain tens of thousands to millions of high qualitysingle-particle images, representing snapshots of all states present in solution.They also contain many ‘‘bad’’ particles - denatured protein or other contam-inants. High-resolution reconstructions hinge on identifying and eliminating‘‘bad’’ particles, separating ‘‘good’’ particles into distinct classes, and accurateorientation estimation within each class. Particle sorting attempts to differen-tiate ‘‘good’’ and ‘‘bad’’ particles, but typically does not address persistentmisalignment of ‘‘good’’ particles.To further high-resolution structure determination of macromolecules inher-ently difficult for image alignment because of lack of obvious structural fea-tures, such as purely transmembrane proteins embedded in lipid nanodiscs,we describe an approach differentiating ‘‘misaligned’’ and ‘‘well-aligned’’ par-ticles based on consistency of alignment across multiple de novo 3D refine-ments with different random initializations. Orientation alignment differencesof the same particles are calculated using geodesic distance in SO(3), and con-sistency is defined as having similar final orientations across all refinements.We applied this approach to the published cryo-EM dataset of the TRPV1 ionchannel, and to a synthetic dataset that contains only the transmembranedomain of TRPV1. The entire channel has well-defined soluble domains, facil-itating image alignment. On the contrary, particles of the synthetic dataset lackwell-defined molecule shape. Compared to the whole TRPV1, the proportion ofparticles with inconsistent orientation assignment increases significantly(�24% vs �70%) for the synthetic dataset. We additionally found that a 3Dreconstruction calculated from the subset of particles with consistent orienta-tion assignment can reach a higher resolution using the same resolution crite-rion. Interestingly, a 3D reconstruction calculated from the excludedparticles with inconsistent angular assignment has generally worse features,but still reached a relatively high resolution. This suggests such particles aremisaligned ‘‘good’’ particles, rather than residual ‘‘bad’’ particles.

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2067-Pos Board B83Interactions of Antibodies with HIV 1 Protease: Towards Identification ofNew Small Molecules for TherapySuchetana Gupta, Sangeetha Balasubramanian, Sanjib Senapati.Biotechnology, Indian Institute of Technology Madras, Chennai, India.HIV Protease plays a significant role in the life cycle of the virus. As a result, ithas been studied extensively for developing therapeutics to prevent the prolif-eration of the virus. Presently there are ten FDA approved drugs against prote-ase. However, long term usage of these drugs induce mutations that lead to drugresistance. A newer approach to anti retroviral therapy is to design allostericinhibitors against protease. In this context, complexes of protease with mono-clonal antibodies are important starting points. The dynamics of these com-plexes coupled with the detailed interactions at the residue level may be vitalin designing novel non active site inhibitors against protease. Presently thereare a few structures of protease-monoclonal antibody complexes, however, inall the cases, the epitopes constitute only a small fragment of protease (ear-flap and N terminal regions). The binding of the mAb 1696 antibody to theN terminal of the protease monomer disrupts its cleavage from gag-pol andhence this complex was chosen in the present study. To obtain the completestructure of monomeric protease and antibody complex, we have docked themonomer to the antibody using the crystal structure as reference to generateseveral possible conformers. To further increase the sampling of conformersand to identify the most favourable starting structure, we have performedsteered molecular dynamics simulation. From our results, we have identifieda probable complex structure where the monoclonal antibody occupies the di-merisation interface of the protease. The interactions in this complex are beingstudied extensively to use as template for pharmacophore based virtualscreening to identify novel allosteric inhibitors.

2068-Pos Board B84Interaction of Clitoria ternatea L. Flower Extract with Alpha-Amylase byPhoton Streaming Time-Resolved FluorescenceGraham Hungerford1, Rachael Divers2, M. Adilia Lemos2,Boon-Seang Chu2.1Horiba Scientific, Glasgow, United Kingdom, 2University of Abertay,Dundee, United Kingdom.The rising prevalence of chronic diseases related to high caloric food intake,especially the rise in type 2 diabetes is proving a critical health concern. Thecosts of treating diabetes and the associated complications are an increasingdrain on health system resources. As well as an approach tackling the causeof the problem, lowering the postprandial hyperglycaemic response providesa means to prevent the development of type 2 diabetes. One method to achievethis is to modulate starch digestion using natural enzyme inhibitors. We havedemonstrated the potential of an extract from the flower of C. ternatea(commonly known as the butterfly pea) to inhibit a-amylase during starchdigestion. The extract of this dark-blue tropical flower is used as a food colorantand is a mixture of anthocyanins; the composition of which can influence thedegree of enzyme inhibition. Thus these compounds have potential as func-tional foods. Anthocyanins can be addressed using light and their photophysicsand stability are known to be pH dependent. We have previously studied antho-cyanins using time-resolved fluorescence spectroscopy (TCSPC) to study theirlocation and form by FLIM (fluorescence lifetime imaging) and determinationof decay associated spectra.In order to fully elucidate the inhibitory potential of the C. ternatea flowerextract we further explore its interaction with the digestive enzyme a-amylaseusing time-resolved fluorescence. A high repetition rate excitation laser (up to100MHz), along with a high quantum efficiency detector were employed toassess the binding of the extract with a-amylase using a ‘‘photon streaming’’approach. This was performed by simultaneously monitoring two differentwavelengths enabling different forms/ locations of anthocyanin binding to beascertained concurrently.

2069-Pos Board B85Effect of the Flexible Regions of the Oncoprotein Mouse Double Minute Xon Inhibitor-Binding AffinityXiyao Cheng, Jingjing Zhou, Yongqi Huang, Zhengding Su.Hubei University of Technology, Wuhan, China.The oncoprotein MdmX (Mouse double minute X) is highly homologous toMdm2 (Mouse double minute 2) regarding their amino acid sequences andthree-dimensional conformations, but Mdm2 inhibitors exhibit very weak affin-ity for MdmX, providing an excellent model for exploring how protein confor-mation distinguishes and adapts inhibitor-binding. The intrinsic conformationflexibility of proteins plays pivotal roles in determining and predicting the bind-ing properties and the design of inhibitors. Although the molecular dynamicssimulation approach enables the understanding of protein-ligand interactions,

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the mechanism underlying how a flexible binding pocket adapts an inhibitor isless explored experimentally. In this work, we have investigated how theintrinsic flexible regions of the N-terminal domain of MdmX (N-MdmX) affectthe affinity of the Mdm2 inhibitor nutlin-3a using protein engineering. Guidedby heteronuclear NOEs measurements, we identified the flexible regions thataffect inhibitor-binding affinity around the ligand-binding pocket on N-MdmX. A disulfide-engineering mutant, N-MdmXC25-110/C76-88, which incor-porated two staples to rigidify ligand-binding pocket, allowed high-affinityfor nutlin-3a-binding than the wild-type N-MdmX (Kd � 0.48 vs. 20.3 mM).Therefore, this mutant provides not only an effective protein model forscreening and designing of MdmX inhibitors, but also a valuable clue toenhance the intermolecular interactions of the pharmacophores of ligandwith pronounced flexible regions. In addition, our results revealed an allostericligand-binding mechanism of N-MdmX that the ligand initially interacts with acompact core, followed by augmenting intermolecular interactions withintrinsic flexible regions. This strategy should be also applicable to many otherprotein targets to accelerate drug discovery.

2070-Pos Board B86Comparison of Calmodulin Ligand Interactions by High Pressure X-Rayand Neutron ScatteringClaus Czeslik, Roland Winter, S€uleyman Cinar.Physical Chemistry, Technische Universit€at Dortmund, Dortmund, Germany.Calmodulin (CaM) is a small Ca2þ binding protein that is involved in numeroussignaling pathways in eukaryotic cells. In this project, we have explored andcompared the interactions of two different ligands of CaM by high pressure ex-periments that characterize the structure and dynamics of the CaM - ligandcomplexes. The ligands used are the hypervariable region of the K-Ras4B pro-tein composed of a polylysine sequence and a farnesyl residue. This lipidatedpeptide is playing a key role in the so-called ERK signaling pathway and is anatural binding partner of CaM. On the other hand, the antagonist trifluopera-zine (TFP) has been studied, which is a clinically used drug that inhibits CaM.Our results show that Ca2þ saturated CaM (holo-CaM) has an open, dumbbell-shaped conformation at ambient conditions and binds both ligands under for-mation of a closed, globular conformation. Upon pressure and temperature in-crease, the closed holo-CaM - TFP complex turns out to be very stable, whereasthe closed holo-CaM - K-Ras4B complex dissociates at about 2500 bar suggest-ing some void volumes and pressure sensitive ligand interactions. The sub-nsdynamics of both holo-CaM complexes have also been studied as a functionof pressure. It has been found that both ligands alter the dynamics of holo-CaM in a similar way. Surprisingly, the mean squared displacement of the pro-tein H-atoms is slightly increased, when the ligands are bound. The secondarystructures of holo-CaM, holo-CaM - TFP, and holo-CaM - K-Ras4B are domi-nated by alpha-helices and change only in a minor way up to several kbar. How-ever, the Ca2þ affinity of CaM is strongly increased at higher pressures by bothligands.

2071-Pos Board B87Monitoring Protein-Ligands Interactions by Single-Molecule LysozymeNanocircuitsJames Froberg.North Dakota State University, Fargo, ND, USA.A hybrid single-molecule nanocircuit technique is applied to interrogate dy-namic interactions between a single lysozyme and various ligands includingpeptidoglycan substrates and synthetic peptide inhibitors. We have directlyobserved the different ligands and their interactions with lysozyme, character-izing conformational state transitions and how they are affected by inhibitors.In addition, we have identified several kinetic parameters to determine reac-tivity and efficiency of inhibitors. These results provide understanding of themolecular mechanism governing lysozyme’s interaction with inhibitors aswell as insight potentially leading to more effective drugs, such asmechanism-based inhibitors. This research was supported by the NIGIM/NIHunder Award No. R15GM122063.

2072-Pos Board B88Structure-Activity Relationship and Characterization of Novel InfluenzaInhibitorsGregory Mohl1, Nathan Liddle1, David Michaelis2, David Busath1.1Physiology and Developmental Biology, Brigham Young University, Provo,UT, USA, 2Chemistry and Biochemistry, Brigham Young University, Provo,UT, USA.Resistance makes the Influenza virus a difficult target for antiviral develop-ment. Amantadine and rimantadine are now ineffective against most circulating

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strains due to point mutations in the M2 channel. Neuraminidase inhibitors arealso vulnerable to viral resistance. This highlights the need for the discovery ofantivirals that inhibit infection by a new mechanism. Novel compounds thatinhibit Influenza protein synthesis were identified using a high-throughput vir-tual screen of a protein-protein interaction inhibitor library. Top compoundswere validated in an immunofluorescence assay to determine compound bioac-tivity. Three compounds inhibited Influenza protein synthesis at low micro-molar concentrations. Variants of the top compound were synthesized andcharacterized, and several compounds were identified with nanomolar inhibi-tion of Influenza protein synthesis. The top compound was effective at protect-ing MDCK cells in the CPE assay at low-micromolar concentrations. Viralresistance develops slowly, suggesting that these inhibitors target a key,conserved binding site. We tested these compounds against the Influenza poly-merase complex directly using a Luciferase reporter assay and sequenced resis-tant strains to better understand the mechanism of action.

2073-Pos Board B89Reactivity of Hexacoordinated HEME Proteins on Ultrafast TimescalesAntonija Tangar1, Shiori Yamazaki2, Sophie Bernard3, Valerie Derrien3,Pierre Sebban3,4, Amy M. Scott2, Jaroslava Miksovska1.1Florida International University, Miami, FL, USA, 2University of Miami,Miami, FL, USA, 3Univerist�e Paris-Sud, Orsay, France, 4University ofScience and Technology of Hanoi/USTH, Hanoi, Viet Nam.Neuroglobin (Ngb) and cytoglobin (Cygb) are among the newest members ofthe vertebrate globin family characterized by a classical 3-over-3 a helicalfold and a heme prosthetic group capable of reversibly binding small ligandssuch as O2, CO and NO. These hemoproteins differ from hemoglobin (Hb)and myoglobin (Mb) in two distinct features: heme iron coordination and disul-fide bond formation. In contrast to pentacoordinated Hb and Mb, Ngb and Cygbare internally hexacoordinated by a distal histidine (HisE7) residue, whosedissociation from heme iron is the rate limiting step in binding of small gaseousligands. Although hexacoordination is widespread within eukaryotic species,especially nonsymbiotic hemoglobins, the physiological role of this feature re-mains unknown. Additionally, both Ngb and Cygb can form intramolecular di-sulfide bond, which induces conformational changes that regulate ligandmigration within protein matrix and modulates ligand binding affinity, indi-cating that the role of these proteins may be dependent on the intracellularredox potential. In this study, we employed femtosecond transient absorptionspectroscopy to determine the role of disulfide bond, internal (HisE7) andexternal (CO) ligands on heme reactivity and ligand rebinding on subnanosec-ond timescales. Our results indicate that heme vibrational cooling mechanism ismore efficient in CO-bound Ngb and Cygb, then in Mb and Hb. Additionally,geminate rebinding on picosecond timescales was observed in Ngb-CO, whichis up to four times faster than observed for other globins in this study. In com-parison, distal histidine mutation did not have a significant impact on geminaterebinding of CO, indicating that HisE7 does not rebind to heme on subnanosec-ond timescales.

2074-Pos Board B90Distinct Mechanism of Oxygen and Carbon Monoxide Interactions withHEME ProteinJaroslava Miksovska1, Ruipeng Lei1, Sophie Bernad2, Valerie Derrien3.1Chemistry, Florida International University, Miami, FL, USA, 2Chemistry,University of Paris XI Orsay, Orsay, France, 3University of Paris XI Orsay,Orsay, France.Interactions of heme proteins with gaseous ligands (CO and O2) have beencharacterized to provide insight into the structure function relationship invarious heme protein with CO being often used as a model compound due tothe stability of the Fe(II)-CO complexes towards oxidation. Here we haveused photoacoustic calorimetry to determine the thermodynamic profiles forO2 photo-dissociation from several heme proteins (human hemoglobin, rise he-moglobin, neuroglobin and cytoglobin) and compare them to the thermody-namic profiles for CO photo-release. Our data indicate a distinct structuralchanges in terms of reaction volume and enthalpy changes association withthe transition between the oxygen bound, six-coordinate heme protein andligand free, five-coordinate protein. Interestingly, thermodynamic parametersfor O2 release from human hemoglobin and neuroglobin demonstrate a strongtemperature dependence that has not been observed for the CO photorelease.These results are discussed in term of the role of distal histidine and a hydrogenbond network involving the heme propionate groups and surrounding aminoacid residues in modulating energetics and dynamics of structural changes inheme proteins.

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Posters: Protein Dynamics and Allostery III

2075-Pos Board B91Analyzing Allostery with Long-Range Rigidity Propagation Across ProteinNetworksAdnan Sljoka.Kwansei Gakuin University, Kwansei Gakuin University, Kyoto-Shi, Japan.Allostery can be viewed as an effect of binding at one site of the protein to asecond, often significantly distant functional site, enabling regulation of theprotein function. In majority of cases, the molecular mechanisms and net-works mediating distant communications that give rise to allostery arepoorly understood. We have developed a rigidity-transmission allostery(RTA) algorithm, a computational method based on mathematical rigiditytheory. Starting with an X-ray crystal structure or an ensemble of threedimensional snapshots, we model the protein as a constraint network (graph)consisting of vertices (atoms) and edges (i.e., constraints including covalentbonds, electrostatic bonds, hydrogen bonds, and hydrophobic contacts).RTA algorithm provides a mechanical interpretation of allosteric signalingand is designed to predict if mechanical perturbation of rigidity (mimickingligand binding) at one site of the protein can transmit and propagate across aprotein network and in turn cause a transmission and change in conforma-tional degrees of freedom at a second distant site, resulting in allosterictransmission. Presence of rigidity-based allostery means that a change inshape (conformation) at one site (i.e. mechanically change the shape asbinding might) would lead to rearrangement and change of shape andconformation of the second site. We will describe the RTA method andillustrate predictions of allosteric interactions on several PDB structuresincluding GPCRs, an enzyme and others, and show how these predictionsare in agreement to NMR chemical shift changes caused by allosteric prop-agations. RTA algorithm is computational very efficient (takes minutes ofcomputational time on standard PC) and is useful in quantification of allo-steric signals, mapping out of allosteric pathways and identification of novelallosteric sites.

2076-Pos Board B92Using Current-Flow Scheme to Capture the Protein-Protein BindingAllostericityYun Luo, Wesley M. Botello-Smith.Pharmaceutical Sciences, Western University of Health Sciences, Pomona,CA, USA.Protein-protein interactions usually encompass several communication sub-optimal paths that are often difficult to capture using conventional geomet-ric based network analysis. Here we developed a new method to capture thecomplex protein-protein communication and how the communication istransmitted from binding interface to the protein functional domain. Forinstance, serine/threonine kinase receptor I (STKR1) represents an impor-tant family that controls the TGF-beta and BMP signaling. In the absenceof ligand, STKR1 kinase activity is physiologically inhibited by bindingof a negative regulator protein FKBP12 to the GS domain. Several gain-of-function mutations in Activin-Like Receptor 2 (ALK2), one of the sevenSTKR1, are associated with various diseases such as heterotopic ossifica-tions and cancer. Previously, we have reported that all those mutationsbreak a conserved salt-bridge near the ATP-binding site (PLOS Comput.Biol. 13(8): e1005711). Surprisingly, those mutations do not necessarilyreduce the protein-protein binding affinity between ALK2 and FKBP12,but allosterically alter the kinase catalytic domain. Therefore, we use thisALK2-FKBP12 system as examples to illustrate how to capture theprotein-protein communication using current-flow betweeness score in thenetwork analysis. Our results can explain how a single mutation at theprotein-protein binding interface changes the protein-protein communica-tion landscape and allosterically shift the kinase to catalytic competentconfiguration.

2077-Pos Board B93Evolution of Caspase Allostery and Enzyme SpecificityClay Clark, Robert Grinshpon, Melvin E. Thomas III, Liqi Yao,Suman Shrestha.Biology, University of Texas at Arlington, Arlington, TX, USA.We used phylogenetic, structural, and biophysical studies to examine theevolution of enzyme specificity and allosteric regulation in caspases.Caspase-3 activation and function has been well defined during programmedcell death, but caspase activity, at low levels, is also required for cell devel-opment. We established a database, the CaspBase, for phylogenetic andancestral reconstruction of caspases, which consists of >1,500 caspasegenes. From this database, we resurrected ancestors of the caspase-3, �6,

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�7 subfamily, and the common ancestor displays enzyme specificity similarto the initiator caspase subfamily from which it evolved. The specificities ofthe caspase-3/-7 and caspase-6 enzymes evolved early, after the two lineagessplit. Structural studies of the ancestral enzymes identify changes in theactive sites that result in changes in substrate selection. In contrast toenzyme activity, several allosteric sites evolved later in the lineages. Wedefine interaction networks that facilitate the allosteric regulation incaspase-3, and we show that, within a conserved loop, one site of phosphor-ylation evolved with the apoptotic caspases, while a second site is a morerecent evolutionary event in mammalian caspase-3. Localized changes inthe loop propagate to the active site of the same protomer and disrupt sub-strate hydrolysis by facilitating transient ionic interactions with the catalytichistidine. In contrast, a cluster of hydrophobic amino acids in the dimerinterface connects the conserved loop to the active site of the second proto-mer. The presence of the second modification in the conserved loop intro-duces a ‘‘kill switch’’ in mammalian caspase-3. The data reveal howevolutionary changes in a conserved allosteric site result in both a commonpathway for lowering activity during cell development as well as introducinga more recent cluster-specific switch to abolish activity.

2078-Pos Board B94Getting Allosteric Control over Protein Activity: New DevelopmentsEnrico Guarnera.Bioinformatics Institute - ASTAR, Singapore, Singapore.Allosteric regulation of protein activity via effector binding at distinct remotesites is under an unprecedented focus in current drug research. Potentiallydruggable allosteric sites are ubiquitous in most if not all dynamic proteins.The key advantages of targeting allosteric sites include the prospect of non-competitive remote fine-tuning of protein activity. As a result, the intereston simple quantitative models to uncover the mechanisms behind allostericsignaling is constantly increasing. We proposed a structure-based statisticalmechanical model of allostery to quantify causality and energetics in allostericsignaling. The model considers binding as a perturbation of the protein dy-namics and quantifies allosteric effects in terms of a per-residue free energy.The model was also extended to account for the allosteric effects induced bysequence mutations and it was recently included in a web server: ‘‘Allo-SigMA’’. AlloSigMA allows users to quantify the allosteric effect inducedby binding and/or mutations. It also provides a rationale for the selection ofallosterically relevant binding sites and mutations, which facilitate the designof experiments.Given the observation that perturbation of allosteric sites propagates signalsto distal catalytic regions, we assumed the reversibility of allosteric signalingand hypothesized the detection of allosteric sites via perturbing the catalyticones. The reverse signaling hypothesis was tested on a diverse set of 13 allo-steric enzymes. We found that reverse signaling from catalytic sites allowsthe identification of the protein regions that most likely include allostericsites. The predictive power of our method is positively correlated with theseparation between allosteric and catalytic sites, consolidating the notionthat allosteric regulation acts at distance. We conclude that reversible allo-steric communication provide the foundation for the high-throughputscreening of druggable allosteric sites via reverse signaling from the cata-lytic site.

2079-Pos Board B95Theoretical Analysis of Allosteric and Operator Binding for Cyclic-AMPReceptor Protein MutantsTal Einav1, Julia Duque2, Rob Phillips1.1California Institute of Technology, Pasadena, CA, USA, 2University Collegeof London, London, United Kingdom.Allosteric transcription factors undergo binding events both at their operatorbinding sites as well as at distinct allosteric sites, and it is often difficult todisentangle the structural and functional consequences of the two types ofbinding. In this work, we compare the ability of two statistical mechanicalmodels – the Monod-Wyman-Changeux (MWC) and the Koshland-N�emethy-Filmer (KNF) models of allostery – to characterize the multi-stepactivation mechanism of the cyclic-AMP receptor protein (CRP). We firstanalyze the allosteric transition, where cyclic-AMP binds to CRP, then modelhow CRP binds to its operator, and finally investigate the ability of CRP toactivate gene expression. We examine data from a recent beautiful experimentthat created a single-chain version of the CRP homodimer, thereby enablingeach subunit to be mutated separately. Using this construct, six mutantswere created using every possible combination of the wild type subunit, aD53H mutant subunit, and an S62F mutant subunit. We show that both theMWC and KNF models can simultaneously characterize the cyclic-AMPand DNA binding of all six CRP constructs based solely on their subunit

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compositions, thereby tying together the behavior of the mutants to a small,self-consistent set of parameters.

2080-Pos Board B96Thermodynamic Coupling Function Analysis of Allosteric Couplingbetween NaD Release and Inward-Opening in the Human DopamineTransporterMichael V. LeVine, Michel A. Cuendet, Asghar M. Razavi,George Khelashvili, Harel Weinstein.Department of Physiology and Biophysics, Weill Cornell Medical College ofCornell University, New York, NY, USA.Allostery plays a crucial role in the mechanism of neurotransmitter-sodiumsymporters, such as the human dopamine transporter (hDAT). To investigatethe molecular mechanism that couples transport-associated inward release ofNaþ from the Na2 site to conformational changes associated with inward-opening, we applied a novel combination of our recently developed thermody-namic coupling function (TCF) theory of allostery and Markov State Model(MSM) analysis to a 50-microsecond dataset of Molecular Dynamics trajec-tories of hDAT, in which multiple spontaneous Naþ release events wereobserved. Our TCF approach reveals a complex landscape of thermodynamiccoupling between Naþ release and inward-opening, and identifies diverse,yet well-defined roles for different Naþ-coordinating residues. In particular,we identify a prominent role in the allosteric coupling for the Naþ-coordinatingresidue D421, where mutation has previously been associated with neurologicaldisorders. Our results highlight the power of the TCF theory and analysis toelucidate the molecular mechanism of complex allosteric processes in largebiomolecular systems.

2081-Pos Board B97Leveraging Cooperativity for Pocket DetectionJustin R. Porter, Gregory R. Bowman, Katelyn E. Moeder.Biochemistry & Molecular Biophysics, Washington University in St. Louis,St Louis, MO, USA.Cryptic pockets—transient pockets that are invisible to conventional structuraltechniques—are the subject of considerable interest, as they are appealing fordrug design against difficult targets. A nontrivial first step is identifying thepocket, as they are by definition difficult to find. Although molecular dynamicscan sample these open states, traditional pocket identification algorithms searchfor concavities at a protein’s surface, yielding tens or hundreds of pockets perframe. Consequently, synthesizing predictions from that multitude of hits re-quires numerous subjective choices.To overcome this limitation, we operationalize the notion of a pocket differ-ently: we define a pocket to be a group of residues that demonstrate cooperativesolvent exposure. Our method then only requires choices of the solvent probesize, which can be used to tune the desired pocket size, and the extent of solventexposure that constitutes the exposed state. Using these parameters, wecompute the mutual information of all pairs of amino acid sidechains’ expo-sure/burial states and use that as input to affinity propagation clustering. Theresulting clusters are pockets. Combined with the pocket definitions, we canleverage Markov state models over the featurized space to make comparisonswith experiment.This method could serve as a simple, nearly turn-key first step in a drug devel-opment pipeline targeting cryptic allosteric sites.

2082-Pos Board B98The Rheostatic Response of Dynamic Allosteric Residue Couples (DARC)Spot MutationsPaul Campitelli1, Liskin Swint-Kruse2, Banu Ozkan1.1Center for Biological Physics, Arizona State University, Tempe, AZ, USA,2Department of Biochemistry and Molecular Biology, University of KansasMedical Center, Kansas City, KS, USA.Distinguishing causal from non-causal relationships between sequence varia-tion and functional consequence lies central to disease prediction and presentsa major challenge in biology and genomics. The need to better define the linkbetween variation and functional impact has grown dramatically as unprece-dented advances in sequencing complete exomes have yielded tens of thou-sands of non-synonymous single nucleotide variants (nSNVs), leading tomissense variants (i.e mutations) on the human proteome. Currently, nomethods consistently predict effects of missense mutations at non-conservedamino acid positions. Here, we present a method to aid in this prediction calledthe dynamic coupling index (dci). This technique analyzes molecular dynamicstrajectories to quantify the coupling strength between selected amino acids. Wehave applied our approach to variants selected from a set of>1000mutations ofLacI/GalR homologues for which experimental outcomes are known. In thisdataset, mutations at many non-conserved positions produce ‘‘rheostatic’’, orprogressive effects on function spanning several orders of magnitude. This con-

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trasts with mutations at evolutionarily conserved positions, which producetoggle-switch (on/off) behavior in these proteins. We show that dci providesinsight regarding how different amino-acid substitutions at dynamic allostericresidue coupling (DARC) spots induce different dynamic responses, leading tochanges in conformational dynamics of DNA binding sites. Moreover, dci canaid in the ability to predict a toggle site versus a rheostatic site, particularlywhen coupled with machine learning and evolutionary data.

2083-Pos Board B99Dynamic Communities in Proteins: Allosteric Hotspots and FunctionalModulesSambit Kumar Mishra1, Gaurav Kandoi2, Robert L. Jernigan1.1Biochemistry, Biophysics and Molecular Biology, Iowa State University,Ames, IA, USA, 2Department of Electrical and Computer Engineering, IowaState University, Ames, IA, USA.Dynamic Communities in Proteins: Allosteric Hotspots and FunctionalModulesDynamic communities in proteins are cohesive units that exhibit rigid body mo-tions. These communities can model allostery in proteins. Previous studies haveshown that mutations to key community residues can hinder the transmission ofallosteric signals among communities. Previouslymolecular dynamic simulations(� 100 ns or longer) were used to obtain these communities - a demanding task forlargemulti-domainproteins. In the present study,wepropose amethodwhich usescoarse-grained Gaussian Network Model (GNM) and hierarchical clustering toobtain protein dynamics communities. We evaluate our method by comparingthe communities obtained fromGNMwith those fromMD, for a set of 45proteins.At certain clustering levels, we observe strong correspondence between the com-munities from the twomethods.Wehypothesize that these can informus about thenumber of functional communities in proteins. In another study we identified theallosteric hotspots for effector ligand binding for the same proteins with a previ-ously trained predictive machine learning model.

2084-Pos Board B100Transient Pocket Identification and Evaluation of their Role for AllosteryDenis Schmidt1, Christopher Pfleger1, Susanne M.A. Hermans1,Markus Boehm2, Holger Gohlke1.1Institute of Pharmaceutical and Medicinal Chemistry, Heinrich HeineUniversity D€usseldorf, D€usseldorf, Germany, 2Pfizer Worldwise Research &Development, Cambridge, MA, USA.It is widely accepted that allosteric modulators have a huge potential to over-come typical hurdles in drug design. Our group recently showed that the‘‘Constraint Network Analysis’’ (CNA) approach is capable of identifying allo-steric pathways by means of rigidity analysis. CNA hence has the potential topredict the allosteric effect of new ligands. Even in the absence of a defined bind-ing site, allosteric regulation might be possible via so-called ‘‘cryptic sites’’(‘‘transient pockets’’). Such cryptic sites are closed in the apo state due to theirlipophilicity. Consequently, they are hard to identify, although they are predictedto exist in many proteins. Molecular dynamics simulations have been proposedas promising approach to sample cryptic sites in their open states. Here, we sys-tematically investigated the influence of different organic solvents on the open-ing of cryptic sites duringmolecular dynamics simulations, starting from the apostate. We identified phenol as the best solvent to foster the opening of transientpockets. In five out of seven test cases, we not only measured the opening of thecryptic site but we were also able to validate the pocket conformations by re-docking the crystal ligand. Unlike perturbation-based approaches, equilibriumsimulations do not require an a-priori knowledge of the location of the bindingsite. After their identification, we employmolecule surrogates (‘‘fuzzy ligands’’)to fill the cryptic sites. This way, CNA is able to quantify the allosteric potentialof a ligand in this binding site. The generated fuzzy ligands subsequently serve asseed for virtual screening. The cryptic site identification in combinationwith the‘‘fuzzy ligand’’ approach and CNA yields a powerful workflow to prospectivelyidentify new sites, evaluate their allosteric potential and ultimately predict newallosteric compounds.

2085-Pos Board B101Weak Domain Stability and Higher Ca2D Binding Affinity Contribute toAllostery between the D/E Linker and N-Helix of Cardiac Troponin CMayra A. Marques1, Adolfo H. Moraes2, Jerson L. Silva1, Jos�e R. Pinto3,Guilherme A.P. de Oliveira1.1Medical Biochemistry Institute Leopoldo de Meis, Federal University of Riode Janeiro, Rio de Janeiro, Brazil, 2Institute of Exact Sciences, ChemistryDepartment, Federal University of Minas Gerais, Minas Gerais, Brazil,3Department of Biomedical Sciences, College of Medicine, Florida StateUniversity, Tallahassee, FL, USA.Hypertrophic cardiomyopathy (HCM) is an inherited myopathy caused bythe production of anomalous sarcomeric proteins that can lead to severe

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cardiac dysfunction. Here, we used structural and biophysical approaches tobetter understand the pathogenesis of a cardiac troponin C (cTnC) C84Y mu-tation located in the D/E linker, first reported in a 17-year-old proband, pre-senting with left-ventricular hypertrophy. Despite the relevance of HCMdisease, little is known concerning the function of the D/E linker and allo-steric phenomena governing cTnC Ca2þ affinity. Monitored by bis-ANSfluorescence, Ca2þ-titrations reveal that C84Y exhibits enhanced Ca2þ-bind-ing affinity in both domains and conformational changes compared to WT.Although WT and C84Y display distinct Ca2þ-binding behaviors, the overalldimensional values and molecular envelopes generated by small-angle-X-rayscattering data remains similar. Using circular-dichroism, C84Y revealedsignificantly lower thermostability in non-Ca2þ-bound form compared toWT. Most of our understanding of the molecular mechanisms underlyinghow troponin and troponin peptides switch muscle contraction ‘‘on’’ and‘‘off’’ has been derived using experimental NMR techniques. Currently,no experimental techniques are available that allow the understanding ofprotein regulatory/dynamic processes at the molecular level of large,multi-domain protein complexes. To further unravel molecular changes inC84Y, three-dimensional NMR experiments were performed for backboneassignment. The largest chemical shifts were observed in N-Helix residuesand at the end of D-helix and D/E linker. NMR-derived backbone amidetemperature-coefficients indicate different temperature-dependent conforma-tional changes exist between WT and C84Y Carr-Purcell-Meiboom-Gillrelaxation dispersion (CPMG-RD) and R1/R2 experiments were used toprobe the population and exchanging rates of C84Y compared to WT.This work sought to elucidate: main structural components underlying thispathological mutation, novel allosteric mechanisms, and the role of D/Elinker in cTnC.

2086-Pos Board B102Role of Lys Residue at Position 87 of DREAM in Allosteric Regulation ofDREAM’s Interactions with KV ChannelSamiol Azam, Jaroslava Miksovska.Florida Int University, Miami, FL, USA.Downstream regulatory element antagonist modulator (DREAM) is a 29kDaprotein, which interacts with diverse intracellular partners and is involved inmany biological processes, namely, pain sensation, gene apoptosis, and mod-ulation of Kv4 voltage channels. Previous research in our group and else-where demonstrated that DREAM interacts with the helix-9 of presenilin-1(PS1HL9), the residue 2-22 (site-1) peptide of Kv4.3, and the residue 70-90 (site-2) peptide of Kv4.3 in a calcium-dependent manner. Molecular dy-namics data suggests that Lys at the position 87 forms a salt bridge with Asp165 and directly involved in the propagation of calcium-triggered structuralchanges between the C- terminal and N- terminal domain. To determine theimpact of Lys 87 on the interdomain communication as well as to charac-terize its contribution to DREAM stability, Lys 87 was mutated to Ala,and the effects of the mutation on DREAM’s interaction with ‘‘PS1HL9’’,‘‘site-1’’ peptide, and ‘‘site-2’’ were determined. The results show that Trpresidue in DREAM(K87A) is more solvent exposed compared with wild-type suggesting that the absence of the salt bridge destabilizes the proteinstructure. The emission maximum of 1,8-ANS in the presence ofDREAM(K87A) is about 10 nm redshifted indicating that the mutation influ-ences the hydrophobic cavity between the N- and C-terminal domain. Thelifetime of Trp and 1,8-ANS are significantly altered in DREAM(K87A)compared with wild-type form, which in agreement with the steady-statedata. Fluorescence anisotropy titration data suggest that mutation of Lys toAla at position 87 of DREAM completely inhibit DREAM’s interaction withsite-2. We do plan to investigate DREAM(K87A)’s interactions with site-1and PS1HL9. Data presented here provide insight into the role of Lys residueat position 87 of DREAM regulating DREAM’s interaction with intracellularpartners.

2087-Pos Board B103Classification of Allostery in Proteins: A Deep Learning ApproachGirik Malik1, Andrzej Kloczkowski2.1Nationwide Children’s Hospital, Columbus, OH, USA, 2NationwideChildren’s Hospital and The Ohio State University, Columbus, OH, USA.Protein fluctuational dynamics is a key element of protein functioning. One ofthe most interesting manifestations of protein dynamics is allostery. Binding aneffector molecule at one site often results in long-range conformational changesin protein structure. Allosteric communication mechanism plays a pivotal rolein the natural regulatory processes. Our recent work [1] attempted to decipherthe characteristics of residues forming the Allosteric Communication Paths, bystudying the annotated proteins from the AlloSteric Database (ASD) [2],

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belonging to four classes (kinases, nuclear receptors, peptides, transcriptionfactors). Central aim of the study was to decipher consistent patterns inherentin the allosteric communication subsystem (ACSS). The underlying graph-theoretic approach unveiled interesting patterns in terms of individual and col-lective effects. Furthering our analysis in this study we tried to automate theentire process by developing an algorithm that could find some patterns inthe complex networks generated earlier and classify the dataset into the fourdifferent classes presented. Using the learned graph representations we aimto classify unknown graphs, in one of the four classes using convolutional neu-ral network. Preliminary data obtained seems encouraging and the methodcould further improve by the growth in the size of ASD and using other deepneural network approaches.[1] Malik, G., Banerji, A., & Kloczkowski, A. (2017). Deciphering GeneralCharacteristics of Residues Constituting Allosteric Communication Paths. Bio-physical Journal, 112(3), 499a.[2] Huang Z, Mou L, Shen Q, et al. ASD v2.0: updated content and novel fea-tures focusing on allosteric regulation. Nucleic Acids Res. 2014; 42 (Databaseissue):D510-D516.

2088-Pos Board B104An Allosteric Mechanism of Abl Kinase Activation and CatalysisTamjeed Saleh.Structural Biology, St Jude, Cordova, TN, USA.c-Abl kinase plays a critical role in coordinating responses to growth fac-tors, cytokines, cell motility, DNA damage responses and oxidative stress.Bcr-Abl, a fusion oncoprotein and the genetic basis of chronic myeloid leu-kemia (CML) is constitutively active and despite intense research the basisfor this activation remains unclear. c-Abl activity is regulated by the N-ter-minal SH3 and SH2 domains working in concert to lock the kinase domainin an inhibited conformation. However, mechanistic insight into how regu-lated inhibition and activation is achieved is missing. Moreover, the effectof the regulatory domain on the conformation of the kinase domain has notbeen elucidated. Using high resolution NMR and other biophysical tech-niques we characterize allosteric mechanism of regulation of Abl kinaseand identify a dynamic role of the regulatory module in the activationprocess.

2089-Pos Board B105Allostery in NMDA ReceptorsRyan Durham, Drew Dolino, Vasanthi Jayaraman.University of Texas Health, Houston, TX, USA.Ionotropic glutamate receptors, of which the N-methyl-D-aspartate receptor(NMDAR) is one subtype, play a crucial role in excitatory neurotransmissionwithin the mammalian central nervous system. In order to fully understandthe role the NMDAR plays in neurotransmission, the conformational statesand transitions that the receptor undergoes must be understood. Althoughthe structures of the various conformational states of the NMDAR areknown, the conformational changes by which the NMDAR transitionsfrom state to state are not well understood. One question concerningNMDAR dynamics that remains to be answered is that of the nature of allo-steric communication between the glycine binding sites and glutamate bind-ing sites on the various subunits of the heterotetrameric receptor. In order tounderstand this allosteric communication, we utilized both electrophysiologyand single molecule Forster Resonance Energy Transfer (smFRET) toexamine the changes in the ligand binding domain (LBD) brought aboutby the binding of glycine and/or glutamate. The electrophysiological studiesshowed that there is negative cooperativity between the glycine and gluta-mate binding sites. The smFRET studies allowed the observation of thevarious states that a single receptor adopts under various ligand conditions.These single molecule experiments demonstrated that the conformational dy-namics of the NMDAR LBD change when the receptor is in the presence orabsence of various ligands; the NMDAR exhibits altered conformational dy-namics when it is bound to glycine, glutamate, both, or neither. This dataillustrates a potential mechanism for the negative allosteric effects betweenthe glycine and glutamate binding sites that was observed in the electrophys-iological studies.

2090-Pos Board B106Allostery Advocates in Monoclonal Antibody Engineering towards Anti-gen BindingChinh Su.Bioinformatics Institute, A*STAR, Singapore, Singapore.Current therapeutics antibodies, such as Trastuzumab and Pertuzumab, havemarked significant success in disease treatment, particularly for Her2 positive

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cancers. However, side effects are sometimes associated with such targetedtherapies due to the nature of the antigen binding, e.g. autoimmune diseasesor cardiac failure. To address these issues, our work focuses on antibody struc-tural modifications such as manipulating heavy-chain constant region to affectlocalization. In addition, we also explore both light-chain constant and frame-work (VL-FWR) manipulation to induce allosteric effects onto the antigenbinding regions (i.e. CDRs).By modulating VL-CDR3 exposure on the scaffold through serendipitousdeletions in the VL-FWR3 of a Trastuzumab model, we found selectivesynergic effects of the mutations on abolishing the Her2 binding. Interest-ingly, we also found an allosteric effect on the Protein L binding site (im-pacting purification and superantigen binding) exerted by these distantdeletions.Similarly, in our work on the antibody constant engineering, we foundallostery-driven structural changes elicited by substitution mutations in the con-stant region to also affect antigen binding in both Trastuzumab and Pertuzumabmodels.In conclusion, our work studies allosteric effects of various often-neglected re-gions (constant and framework) of the antibodies in overall antigen recognition.With such knowledge, it may be possible to engineer therapeutic antibodies intheir localization as well as antigen binding capabilities for mitigating systemicside effects and for fine-tuning antigen binding.

Posters: Membrane Protein Structures II

2091-Pos Board B107Specific Interactions of Protein-Protein Interaction between Human Pro-grammed Death 1 (PD-1) and its Ligand 1 (PD-L1) with AB Initio Frag-ment Molecular Orbital MethodHo Cheol Lim1, Jung Ho Chun1, Sung Bo Hwang1,2, Jong Wan Kim1,2,Kyoung Tae No1,2.1Department of Biotechnology, Yonsei University, Seoul, Republic of SouthKorea, 2Bioinformatics & Molecular Design Research Center, YonseiUniversity, Seoul, Republic of South Korea.Immunologic checkpoint pathways, including Programmed Death 1 and its li-gands, are crucial in mediating self-tolerance and preventing autoimmunity, sothey protect tissues from self-damage. It is now clear that significant numbersof cancers manipulate the signal pathways to evade immune surveillance andefficacy of the blockade of immune checkpoints in cancer therapy was vali-dated in preclinical studies targeting programmed death 1. Several therapies,including small molecules, to interfere interactions between PD-1 and PD-L1have been developed. Nevertheless, this is obstructed by the imperfect struc-ture information about these proteins, even after the crystal structures weresolved. Here, we applied ab initio fragment molecular orbital method towild-type PD-1/PD-L1 complex at MP2/6-31G* level with PCM. Further-more, we also applied the FMO method to two complexes of small moleculeinhibitors targeting PD-L1 at MP2/6-31G** level with PCM. Based on calcu-lated pair interaction energies (PIEs), we revealed key interactions and quan-tified the strength of the interactions with Pair Interaction EnergyDecomposition Analysis (PIEDA) and a visual representation of 3D scatterplot of PIEs, a so-called 3D SOP. As a result, we investigated specific inter-actions in previously reported three hot spots on a basis of the FMO results.This approach provides an effectual tool to understand the protein-protein in-teractions at the quantum mechanical level.

2092-Pos Board B108Interaction between A-Synuclein and VAMP2 Promotes SNARE-Ependent Vesicle Docking and FusionBrenden Hawk, Ryan Khounlo, Yeon-Kyun Shin, Julien Roche.BBMB, Iowa State University, Ames, IA, USA.Despites its central role in the pathogenesis of Parkinson disease, thenormal biological function of a-synuclein remains largely unknown. Previ-ous studies have shown both in-vitro and in-vivo that a-synuclein specif-ically binds to anionic membrane and could serve as a chaperone for theSNARE complex that mediates the docking and fusion of synaptic vesicles.Using a combination of solution NMR spectroscopy and single-molecule as-says, we show here that a-synuclein promotes SNARE-dependent dockingand fusion through its interaction with v-SNARE synaptobrevin 2(VAMP2). We show that while the amphipathic N-terminal domain of a-synuclein binds to the surface of negatively charged vesicles, its C-terminaltail directly interacts with the C-terminal region of VAMP2. Our studyhighlights the critical role VAMP2 in recruiting a-synuclein in order tocatalyze the SNARE-dependent vesicle fusion process and suggests thatin non-pathological conditions a-synuclein is an important mediator of syn-aptic vesicle trafficking.

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2093-Pos Board B109Probing Plexin A3 Dimerization and the Importance of the Near Mem-brane Extracellular ResiduesPouyan Khakbaz, Jeffery B. Klauda.Chemical and Biomolecular Engineering, University of Maryland, CollegePark, MD, USA.Plexins are functional transmembrane proteins involved in signal transduc-tion, which they are widely recognized as receptors during many biologicalpathways including cancer metastasis. Plexins have a large extracellular(EC) domain that binds to other proteins to send signals to inside the celldue to homodimerization. This dimerization is modulated in transmembrane(TM) and juxtamembrane (JM) domains. The role of the glycine-rich region,which is known as small-x3-small motif in TM domain, and hydrophobicheptad repeat in plexin dimerization and activation still needs to be investi-gated. Our previous molecular dynamics (MD) simulations along with exper-imental results suggested mutations in small-x3-small motif of Plexin A3enhance dimerization (Barton et al., 2015). Same event was seen, where mu-tations were done for cytosolic JM heptad repeat that damage dimerizationprocess even in the presence of TM mutations. In this study, more residues(up to 30) were included in extracellular domain of Plexin A3 to betterconsider extracellular effect on dimerization process, and MD simulationswere carried out on wild-type Plexin A3 (residues 1211-1310 with someof the EC domain, TM helices, and the JM domain). Results indicated stron-ger interaction in TM domain, which was not observed in previous MD sim-ulations with shorter sequence (Barton et al., 2015). Strong hydrophobicmutations were performed in the EC domain to prove the effect of ECdomain on stronger interaction in TM domain. Overall, our results providedmore insight of different domains of Plexin A3 and their role in signal trans-duction at the molecular level.

2094-Pos Board B110The Structure of KRAS4B-FME at the Lipid MembraneFrank Heinrich1, Que Van2, Mathias Losche1, Andrew Stephen2.1Department of Physics, Carnegie Mellon University, Pittsburgh, PA, USA,2Frederick National Laboratory for Cancer Research, Frederick, MD, USA.Ras genes are the most frequently mutated oncogenes in human cancer. Rasexists as three isoforms (H, N and K-Ras). These Ras isoforms have differentmutational frequencies, with KRas being the most frequent. The Ras proteinsfunction as a molecular switch that transmit mitogenic signals across the cellmembrane. When Ras is in the active GTP state, it binds to a series of effectorproteins, with CRAF kinase being one of the best characterized. The hydroly-sis of GTP to GDP results in a conformational change such that Ras no longerinteracts with effector proteins. KRas has proven to be an elusive drug target,in part because the membrane-bound state of the protein, particular in com-plex with effector proteins, is unknown. We present structural studies of far-nesylated KRas by itself and in complex with partial constructs of CRAF atthe membrane using neutron reflectometry. In contrast to structural modelsbased on NMR and MD simulations, the G-domain of KRas is found to belocated on average about 30 A away from the membrane while the hypervar-iable region of KRas serves as a flexible membrane tether. Binding to CRAF-RBD (Ras Binding Domain) does not affect this conformation, whereas pre-liminary data indicates that binding to CRAF-RBD-CRD (Cysteine RichDomain) initiates a close engagement of the G-domain with the membrane.This work constitutes first steps toward a structural characterization of theentire membrane-bound KRas/CRAF complex and is validated againstNMR-PRE data.

2095-Pos Board B111High Resolution Cryoem Structure of a Mycobacterial Glycosyl-transferaseYong Zi Tan1,2, Jos�e Rodrigues3, Oliver B. Clarke1, Clinton S. Potter1,2,Bridget Carragher1,2, Margarida Archer3, Filippo Mancia1.1Columbia University, New York, NY, USA, 2New York Structural BiologyCenter, New York, NY, USA, 3Instituto de Tecnologia Quımica e BiologicaAntonio Xavier, Universidade Nova de Lisboa (ITQB NOVA), Oeiras,Portugal.Tuberculosis has been, and still is, a huge health problem for the world, beingthe 9th most deadly disease in the world, responsible for 1.3 million deaths in2015. With the rise of fully drug resistant variants of Mycobacterium tubercu-losis, the causative agent for tuberculosis, there is a real need to seek out newdrug targets againstM. tuberculosis. TheM. tuberculosis cell wall is a commontarget for many existing antibiotics, and it is unique in structure because of thepresence of additional lipid-sugar moieties, arabinogalactans and lipoarabino-mannoses, which are essential for mycobacterium survival and virulence.Membrane-bound glycosyltransferases build these essential lipid-sugar

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moieties. Here, we present the full-length membrane-bound structure of amycobacterial glycosyltransferase solved to 3.3 A resolution using single-particle cryo-electron microscopy. The structure provides insights into the cat-alytic activity and sugar binding capability of these proteins, and serves as atool for future structure-based drug design efforts.

2096-Pos Board B112Mechanism of Catalysis and Inhibition in DGAT1Lie Wang, Yin Nian, Ming Zhou.Biochemistry and Molecular Biology, Baylor College of Medicine, Houston,TX, USA.Acyl-CoA: diacylglycerol acyltransferase, commonly known as DGAT, trans-fers an acyl chain onto diacylglycerol to form triacylglycerol. DGAT1 is amembrane-embedded protein and required for the absorption of dietary fattyacids and the storage of triacylglycerol in adipocytes. Our understanding ofDGAT1 is limited. The biochemical mechanism of DGAT has not been studiedin any detail and it is unclear how the acyl transfer reaction proceeds andwhether any metal ions are required. In addition, all the known DGAT1 inhib-itors have a similar chemical structure and the mechanism of inhibition issimilar. To address these questions and to identify novel inhibitors forDGAT1, we overexpressed and purified DGAT1s from human and other eu-karyotic species, and we showed that these proteins are stable and enzymati-cally active. We have started to examine the reaction mechanism, and wehave identified Inhibitors that have a different chemical structure.

2097-Pos Board B113Structural Studies of Retinol-Binding Protein Receptor RBPR2Jonathan Kim1, Yong Zi Tan2, Brianna Costabile2, Yunting Chen1,Filippo Mancia1.1Physiology, Columbia University, New York, NY, USA, 2ColumbiaUniversity, New York, NY, USA.Vitamin A (retinol) is a crucial nutrient required for a variety of essential phys-iological processes in living organisms. It is necessary for vision, embryonicdevelopment, reproduction, immunity, cellular differentiation and prolifera-tion. Since retinol is insoluble in water, it is bound by retinol-binding protein(RBP) in the bloodstream in order for it to be transported from the liver to targettissues. The structure of STRA6 (Stimulated by retinoic acid 6), a multi-transmembrane protein that specifically recognizes retinol bound RBP and me-diates cellular uptake of retinol, was recently determined to 4.19A resolution bysingle-particle cryo-electron microscopy. Mutations in STRA6 gene have beenimplicated in numerous genetic diseases including anophthalmia, congenitalheart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypopla-sia, and mental retardation. A second retinol-binding protein receptor (RBPR2)was discovered and predicted to have structural and functional resemblance toSTRA6. While STRA6 is broadly expressed, RBPR2 is primarily expressed inthe liver and intestine where the uptake, storage, and distribution of retinoloccur. Although it has been implied that RBPR2 mediates retinol transport inliver, the mechanism by which RBPR2 functions and interacts with ligandsin and outside of the cell is still unclear. The study aims to understand the mech-anism of retinol transport by RBPR2 using a structural coupled to functionalapproach.

2098-Pos Board B114Crystal Structure of a Bacterial ABC HEME Exporter in the APO FormMd. Mahfuzur Rahman1,2, Tamao Hisano2, Hiro Nakamura3,Yoshitsugu Shiro1.1Graduate School of Life Science, University of Hyogo, Ako, Japan, 2RikenSPring-8 Center, Sayo, Japan, 3Riken Center for Life Science Technologies,Yokohama, Japan.Many pathogenic bacteria acquire heme from the host as a source of iron fortheir growth and virulence. Bacteria regulate the intracellular heme concentra-tion strictly as heme is toxic at high concentrations. Corynebacterium diphther-iae, which is the causative agent of the disease diphtheria, possesses an ABC(ATP-Binding Cassette) heme exporter HrtBA for resistance to high levels ofheme. The hrtBA genes are regulated by the ChrAS two-compartment systemin a heme-dependent manner. Biochemical analyses show that heme bindingstimulates the ATPase activity and ATP binding reduces heme affinity ofHrtBA. In the present study, we have determined the structure of HrtBA inthe apo form using an X-ray crystallographic technique. The crystals weregrown by vapor diffusion and the structure was solved by molecular replace-ment using the structure of the ATP analog-bound form. HrtBA is a tetramerof two copies of the transmembrane subunit (HrtB) and the ATPase subunit(HrtA). Compared to reported exporter structures, it is unique that HrtB con-tains four transmembrane helices (TMHs 1–4) accompanied by a large extra-cellular domain between TMHs 1 and 2, while the ATPase subunit is wellconserved. The packing interaction in the HrtB dimer association of the apo

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form is distinct from that of the ATP analog-bound form. The HrtB dimer isstabilized through interactions of TMH 2 from each monomer, providing anopen conformation, accountable for heme entry. The structural comparison be-tween apo and ATP analog-bound form gives insights into the heme exportmechanism of HrtBA.

2099-Pos Board B115Structure and Mechanism of Bacterial HEME ExporterTamao Hisano1, Hiro Nakamura2, Yoshitsugu Shiro3.1Riken SPring-8 Center, Sayo-cho, Japan, 2Riken Center for Life ScienceTechnologies, Yokohama, Japan, 3Graduate School of Life Science,University of Hyogo, Ako, Japan.Dietary heme is a major source of iron, an important element for living organ-isms, while excess of free heme is known to be cytotoxic. Pathogenic bacteriautilize host’s heme via uptake systems, eliminating its toxicity by a sophisti-cated manner. The hrtBA genes which encode a heme exporter are well-conserved and responsible for heme detoxification in Gram-positive pathogens.Amino acid sequence analysis indicates that HrtBA is an atypical ATP-bindingcassette (ABC) transporter: the HrtB subunit is unique in the number of trans-membrane helices and the size of a hydrophilic region, while the HrtA subunitis a conserved ATPase. We have shown that heme binding enhances theATPase activity and that ATP binding decreases the heme binding affinity inthe reconstituted HrtBA system. In order to deepen the understanding of theheme efflux mechanism, we have undertaken a crystallographic study on thisprotein. Crystals of HrtBA complexed with ATP analogs or a heme analogwere successfully obtained by vapor diffusion. Diffraction data were collectedup to resolutions of 2.9 - 3.7 A at the beamline BL26B1/B2 in SPring-8, Har-ima, Japan, and processed with HKL2000. Phases for one ATP analog complexdata were obtained by Mn-SAD, and phases for other data sets by molecularreplacement. Structural analyses were conducted using PHENIX. Structuralfeatures of HrtA share with ATPase subunits/domains of other ABC trans-porters. HrtB represents a novel two-domain structure; one domain containsfour transmembrane helices in a bundle, and the other builds a large extracel-lular domain consisting of ten b-strands and five a-helices inserted between thefirst and second transmembrane helices. Based on the observation of largeconformational differences between ATP analog-bound and heme analog-bound forms, we propose a novel mechanism of this class of the ABC trans-porter family.

2100-Pos Board B116Revealing the Subunit Architecture of NAD(P)H Dehydrogenase Type-1from Cyanobacteria through Cryo-EMThomas G. Laughlin1,2, David F. Savage1, Karen M. Davies2.1Molecular & Cell Biology, University of California, Berkeley, Berkeley,CA, USA, 2Molecular Biophysics and Integrated Bioimaging, LawrenceBerkeley National Laboratory, Berkeley, CA, USA.The coupling of electron transfer reactions to proton-pumping in order to estab-lish an electrochemical gradient, which can be either leveraged directly or uti-lized to synthesize ATP, is a conserved aspect of cellular energy production.This process is typically depicted in the context of Complex I, or NAD(P)H de-hydrogenase type-1 (NDH-1), of the respiratory chain in mitochondria and bac-teria. In cyanobacteria and chloroplasts, NDH-1 is implicated in the lightreactions of photosynthesis. Moreover, the complex does not displayNAD(P)H oxidase activity in vitro and likely utilizes a distinct electron donormediated by additional oxygenic photosynthetic specific (OPS) subunits. Herewe present the near-atomic structure of the cyanobacterial NDH-1 obtainedthrough cryo-EM single-particle analysis. The structure reveals the spatialarrangement of the novel OPS subunits and how they may enable the complex’sdivergent activity from the canonical Complex I.

2101-Pos Board B117Structural and Functional Studies of Antiviral Protein IFITM3Emma H. Garst1, Avital Percher1, Hang Hoang2, Howard Hang1.1Rockefeller University, New York, NY, USA, 2Bard College, Annandale-on-Hudson, NY, USA.Interferon-induced membrane protein 3 (IFITM3) is a key antiviral proteininvolved in the restriction of a number of viruses including influenza,dengue, ebola, and HIV. Despite its significance in the host innate immuneresponse, very little is known about IFITM3’s mechanism of action. Proteo-mic studies have determined IFITM3 is S-palmitoylated at multiple sites andthis modification is necessary for its proper function. The S-palmitoylation ofIFITM3 can change its subcellular localization; however, the effect it has onIFITM3 structure and function has not been characterized. An understandingof the structure, topology, and oligomeric state of lipidated IFITM3 wouldlead to a better understanding of its mechanism of action. To address thesequestions, we have optimized the expression, purification and site-specific

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lipidation of recombinant IFITM3 for in vitro reconstitution and structuralstudies. Through structural analysis and in vitro fusion studies, we may eluci-date how this family of IFN-effectors prevents virus translocation acrosscellular membranes.

2102-Pos Board B118HEME Trafficking by the Cytochrome C Biogenesis PathwaysMolly C. Sutherland, Joshua M. Jarodsky, Robert G. Kranz.Biology, Washington University, St. Louis, MO, USA.Trafficking of heme is critical for cellular function (e.g. oxygen transport,energy production), however the tools to directly investigate this processare rudimentary. Here we describe a novel method to trap heme as it is traf-ficked through the cell, allowing for identification of heme binding domainsand heme transporters. The approach was developed using prokaryotic cyto-chrome c biogenesis pathways as model systems. Cytochrome c biogenesisrequires the covalent attachment of heme, via two thioether bonds, to aconserved CXXCH motif and is dependent on the trafficking of heme tothe holocytochrome c synthetase. In prokaryotes, two pathways, System Iand System II, mature cytochrome c. System I is composed of 8 membraneproteins, CcmABCDEFGH, and System II is composed of two integralmembrane proteins, CcsBA. These pathways contain a conserved,tryptophan-rich domain called the WWD domain that is proposed to interactwith heme. Here, we crosslink heme in the WWD domains of CcmC andCcsA, demonstrating for the first time that the WWD domain is a hemebinding domain. Subsequently, a heme binding domain was defined inCcmE, delineating 2-vinyl and 4-vinyl pockets for the heme vinyl groups.The stereospecific position of heme in these domains was determined anda two-step model for System I heme trafficking is proposed. CcmABCDtrafficks heme to the WWD domain of CcmC where it is stereospecificallypositioned and attached to the heme binding pockets of CcmE (step 1).CcmE transports heme to the holocytochrome c synthetase, CcmF/H, for ste-reospecific attachment to apocytochrome c (step 2). We envision that theheme crosslink approach will be applicable to other putative heme trans-porters to prove that they are directly involved in heme transport. Moreover,we believe that these purified integral membrane proteins are excellent can-didates for cryoEM structural studies.

2103-Pos Board B119Dynamics of Ternary Redox Complex Influencing Cytochrome P450Metabolon: An NMR StudyKatherine Gentry, Ayyalusamy Ramamoorthy.Biophysics, University of Michigan, Ann Arbor, MI, USA.Cytochrome P450s (cytP450s) are a ubiquitous superfamily of enzymes thatare responsible for the metabolism of a plethora of substrates including�75% of the drugs in the current market. Each turn of cytP450 catalytic cyclerequires two electrons donated by either Cytochrome P450 Reductase (CPR)alone or the second electron by cytochrome b5 (cytb5). In this study, we inves-tigate how CPR and cytb5 compete for binding to cytP450, the dynamic struc-tural basis for this competition, and the many roles of lipid membrane andsubstrates. In addition to various biophysical/biological approaches, cutting-edge NMR techniques and peptide-based lipid nanodiscs are used to charac-terize the interplay between cytP450, CPR, and cytb5 in the ternary complex.Our results show how substrates differently modulate the interaction betweencytP450 and its redox partners and the factors enhancing the preference ofcytP450 for one of the redox partners. The cationic residues of P450 andthe anionic residues from one of the redox partners form the hot-spot of theprotein-protein complex. Our previous studies have shown the role of electro-static interaction and dynamics nature of the complex. Structural interactionsbetween membrane-bound redox partners in the presence and absence of sub-strates, electron transfer pathways, and protein-membrane interactions deter-mined at high-resolution will be presented. This study starts to define acomplete picture of how this ternary complex functions together to metabolizevarious substrates.

2104-Pos Board B120Cytochrome-P450’s Spin State Influences its Binding Affinity to its RedoxPartnerNirupama Sumangala, Mukesh Mahajan, Thirupathi Ravula,Ayyalusamy Ramamoorthy.Biophysics, University of Michigan, Ann Arbor, MI, USA.Cytochrome P450s comprise a superfamily of membrane-bound heme mono-oxygenases responsible for the metabolism of �75% of drugs in the currentmarket. Electron transfer from Cytochrome P450 reductase (CPR) is a keystep to the enzyme catalysis, and there exists an equilibrium between high-spin and low-spin ferric iron that determines the enzymatic function ofP450. In the ground state the heme iron is in low-spin ferric with a loosely

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coordinated water molecule in the distal position. Binding of a substrate shiftsthe ferric heme iron equilibrium to the high-spin form, enabling one electrontransfer from CPR to reduce the iron to the ferrous state. The factors thatdetermine the heme spin equilibrium state is still under debate. In this study,we probed the spin state of P450’s heme in the presence of lipid membrane,redox partner, and substrate using biophysical experiments and peptide-basedlipid nanodiscs. Our NMR results reveal the effect of the membrane-P450interaction induced change in the binding affinity of P450 to its redox partner(cytochrome b5 or CPR). We believe that these results would provide us adeeper understanding on the mechanism of P450 catalytic and electron trans-fer mechanisms.

2105-Pos Board B121Structural Studies of TREM2, a Central Sensor Linked to AlzheimersDiseaseYounghee Park, Charles R. Sanders.Dept. of Biochemistry and Center for Structural Biology, VanderbiltUniversity, Nashville, TN, USA.Alzheimer’s disease (AD) is now major cause of death worldwide and themost common cause of dementia in the elderly, marked by the appearanceof amyloid-b (Ab) plagues in the central nervous system. In recent years,‘‘triggering receptor expressed in myeloid cells 2’’ (TREM2) protein vari-ants have been identified as risk factors for AD and other neurodegenera-tive diseases. TREM2 is an immunomodulatory receptor composed of anextracellular V-type Ig domain followed by a short stalk, a single trans-membrane helix, and a short cytoplasmic tail. TREM2 has been implicatedin a wide array of functions related to microglial activation, survival, clus-tering, and phagocytosis. It signals as a complex with DNAX-activationprotein 12 (DAP12). Moreover, TREM2 variant R47H impairs the micro-glia barrier function leading to decreased amyloid compaction and severaxonal dystrophy. The Ig domain of TREM2 is shed from the cell surfaceby an ADAM protease. The remaining transmembrane domain is thencleaved by g-secretase releasing the soluble TREM2 (sTREM2) c-terminalfragment (CTF) and intracellular domain (ICD) which could potentially bebiomarkers for AD. A mechanistic understanding of TREM2 regarding howit engaged its ligand(s), however, remains to be determined. In this study,we highlight structural features of TREM2 and its interaction with ligandsusing nuclear magnetic resonance (NMR) spectroscopy. This will provideinsight of how different ligands might interact with the receptor and howthese interactions alter TREM2 structure and trigger signaling. We alsodescribe purification of the TREM2 transmembrane (TM) domain, itsreconstitution into model membranes, and subsequent structural studies.Finally, we also describe efforts to characterize the TREM2-DAP12 com-plex by NMR methods.This work is supported by Bright Focus Foundation grant A2015565S.

2106-Pos Board B122In Silico and In Vitro Analysis of ITGB1 Binding to its Partners ITGA5and ITGA6Maia Isabella R. Leyretana, Marie Angeline M. Francisco,Kevin Benedict O. Cristobal, Andre Rhey C. Haro, Kim Ivan A. Abesamis,Neil Andrew D. Bascos.National Institute of Molecular Biology and Biotechnology, University of thePhilippines Diliman, Quezon City, Philippines.Integrins are cell adhesion molecules that mediate cell-cell and cell-matrix in-teractions, forming heterodimers which affect their interaction specificity. Inparticular, the integrin (ITG) b1 subunit heterodimerizes with the a5 subunitin various extracellular matrix, blood, and cell surface proteins but not inepithelial cells, where the a6 subunit is its preferred binding partner. Theimportance of a6b1 and a5b1 in maintaining normal cell activity is observedin cases of abnormal levels a6b1 and a5b1 heterodimer expression that leadto metastasis. As the type of integrin expressed (i.e. ITGa5b1 or ITGa6b1) de-pends on the ITGb1 binding partner (i.e a5 or a6), the current study focuses oninvestigating factors that affect binding partner preference. In-silico structuralanalysis investigated the electrostatic potential of the integrin heterodimer (e.g.a5b1) interface, quantified the bonds and accessible surfaces present, andanalyzed the contributions of electrostatic complementarity to binding. Steeredmolecular dynamics (SMD) simulations were used to compare the relativebinding affinities of the different heterodimer pairings. In vitro analysis willbe conducted to verify the in silico predictions. Current progress with in vitrostudies has allowed the recombinant expression and purification of the b-pro-peller domain of ITGa6. The putative recombinant protein was purified byfast protein liquid chromatography (FPLC) using a HisTrap metal affinity col-umn. SDS-PAGE and Anti-His Western Blot analysis indicated the presence ofthe successfully expressed, His-tagged recombinant ITGa6 ligand binding

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domain. For both ITGa6b1 and ITGa5b1, equilibrium molecular dynamics arein progress to determine factors that stabilize their interactions.

2107-Pos Board B123Structural Characterization of Peripheral Myelin Protein 22 and itsMutants in Model Membrane by NMR SpectroscopyGeoffrey Li, Manuel Castro, Charles R. Sanders.Center for Structural Biology and Department of Biochemistry, VanderbiltUniversity, Nashville, TN, USA.Peripheral myelin protein 22 (PMP22) is a tetraspan integral membrane pro-tein that is highly expressed in myelinating Schwann cells. It is known toregulate Schwann cell proliferation and myelin production. Missense muta-tions or gene duplication of PMP22 cause severe dysmyelination of peripheralnerves, a condition known as Charcot-Marie-Tooth Disease (CMTD), forwhich no treatment is yet available. To date, there is no experimentally deter-mined structure of PMP22 except for a homology-based model based on thecrystal structure of claudin-15. However, previous nuclear magnetic reso-nance (NMR) spectroscopy data showed that the first transmembrane helixhas higher propensity to dissociate from the rest of the transmembrane helixbundle (Sakakura-M and Sanders-CR, Structure 2011). A long term goal re-mains to determine the structure of PMP22 in model membrane usingNMR. We acquired NMR spectra for PMP22 in different micelles, bicelles,and nanodiscs to screen for optimal conditions for structure determination.We also compared the NMR spectra of two CMTD mutant forms ofPMP22: L16P (Trembler-J phenotype) and G150D (Trembler phenotype)with that of the wild type protein. Our findings will illuminate structural basesboth for the function of properly folded PMP22 and for the misfolding of themutant forms of PMP22 that promote CMTD. This work is supported by USNIH grant RO1 NS095989.

2108-Pos Board B124Architecture and Dynamics of the Autophagic ATG2-ATG18 ComplexGoran Stjepanovic1,2, James H. Hurley1,2.1University of California Berkeley, Berkeley, CA, USA, 2MolecularBiophysics and Integrated Bioimaging Division, Lawrence BerkeleyNational Laboratory, Berkeley, CA, USA.Autophagy is a highly regulated process of degradation and recycling ofcellular components and pathogens. Hallmark of autophagy is formation ofa double-membrane cytosolic vesicles, termed autophagosomes, whichsequester cytoplasm and delivers it to the lysosome where it is degraded andrecycled. Nucleation and expansion of autophagic membranes require recruit-ment of autophagy related protein 2 (ATG2) and phosphatidylinositol-3-phosphate (PI3P) effector protein ATG18 to the pre-autophagosomal structure(PAS). We used single-particle electron microscopy, structural proteomics andbiochemical analyses to characterize S. cerevisiae ATG2 in complex with theATG18. ATG2 has an elongated, rod-shaped structure and can exist as an anti-parallel dimer or as a monomer with the ATG18 bound at one of the ends.Liposome binding experiments have been used to characterize the affinity ofthe ATG2-ATG18 complex for different lipids and cooperativity betweenthe subunits as a possible mechanism for membrane recruitment and vesicletethering, shedding light on its targeting to the PAS. Furthermore, weperformed hydrogen-deuterium exchange mass spectrometry (HDX-MS) andidentified a localized change in conformational dynamics upon complexformation. This provides one of the first structural insights into the ATG2-ATG18 complex and its membrane interaction mechanism and exploresmolecular mechanisms involved in the early steps of autophagosomebiogenesis.

2109-Pos Board B125Structure and Function of Mammalian Stearoyl-COA DesaturaseJiemin Shen1,2, Gang Wu3, Ah-Lim Tsai3, Ming Zhou2.1Graduate Program in Quantitative and Computational Biosciences, BaylorCollege of Medicine, Houston, TX, USA, 2Verna and Marrs McLeanDepartment of Biochemistry and Molecular Biology, Baylor College ofMedicine, Houston, TX, USA, 3Division of Hematology, Department ofInternal Medicine, University of Texas - McGovern Medical School,Houston, TX, USA.Membrane bound stearoyl-CoA desaturase (SCD) is an iron-containing met-alloenzyme. It catalyzes the rate-limiting step in the conversion of saturatedfatty acids to monounsaturated fatty acids. The formation of a double bond iscatalyzed by a diiron center in the protein, and is coupled to NAD(P)Hoxidation via cytochrome b5 and cytochrome b5 reductase. In mammals,SCD1-deficiency leads to resistance against diet-induced obesity and dia-betes because of elevated levels of lipid metabolism and insulin sensitivity.Many efforts have been made to develop effective SCD1 inhibitors for thetreatment of obesity, ectopic fat deposition, diabetes and other metabolic

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syndromes. SCD1 is also a potential drug target for cancers because its ac-tivity is upregulated and required for propagating cancer cells. Crystal struc-tures of mouse and human SCD1 have been reported recently, however, inboth structures the presumed diiron center is occupied by zinc ions. Themis-incorporation of zinc ions during heterologous overexpression in insectcells renders the purified SCD1 protein biochemically inactive, and raisesthe question of whether the structures faithfully represent the iron-containing SCD1. The inactive SCD1 also hinders our ability to unravelthe mechanism of catalysis and identification of inhibitors. Therefore, wedeveloped a procedure to remove zinc ions and reconstitute ferrous ionsinto the mouse SCD1 protein. After confirming the presence of irons inSCD1, we then crystallized and solved the structure of the iron-loadedSCD1 to 3.5 A. Initial analysis showed that the iron containing SCD1 struc-ture is similar to that of the zinc containing SCD1, indicating that the previ-ous structure was not altered significantly by the presence of zinc ions. Theiron containing SCD1 also enabled us to conduct further experiments todissect the redox reaction mechanism.

2110-Pos Board B126The Structural Layers of the Nuclear Pore ComplexJoseph S. Glavy.College of Pharmacy, University of Texas at Tyler, Tyler, TX, USA.The human nuclear pore complex (hNPC) is a 110 MDa structure of �30different proteins that occur in multiple copies. Proteins that compose thehNPC are themselves built into subcomplexes, which are arranged witheight-fold rotational symmetry to build the overall assembly. The three-dimensional structure of purified subcomplex was determined by single particleelectron microscopy. By crosslinking of subcomplexes engaged within thehNPC and the subsequent identification of crosslinked peptides by crosslinkingmass spectrometry (XL-MS), we uncover their dynamic interacting layers ofthe NPC. These data, along with a high resolution structure of the hNPCwere integrated to determine how the layers of the NPC come together formingreticulated rings of the hNPC. Our approach to elucidate NPC structure inte-grates biochemistry and several structural biology techniques that span lengthscales and may be widely applied to the study of large, complex molecular or-ganizations and will contribute to the field of structural biology as a whole. Weused molecular modeling combined with XL-MS and cryo-electron tomogra-phy to obtain a composite structure. This architectural map explains the vastmajority of the electron density of the scaffold.

2111-Pos Board B127Studying Hofmeister Ion Induced Effects in Model Lipid Drug DeliverySystemsJenny Skubal.Chemistry and Biochemistry, James Madison University, Harrisonburg, VA,USA.Caffeine consists of two fused aromatic rings with four nitrogens and a va-riety of functional groups, and its structure is similar to many biomolecules.In previous studies, the Hofmeister anion series was shown to affect the sol-vation and aggregation of caffeine. In this study, the effects of the Hofmeis-ter series are expanded to examine the cation and anion induced interactionswith caffeine, lipid, and solvent. ATR-FTIR was used to monitor both Hof-meister anion and cation induced changes in the caffeine and lipid absorp-tion spectra, as well as isolated caffeine and lipid interactions. Hofmeisterions effects on caffeine-caffeine and caffeine-lipid interactions will bediscussed.

Posters: Intrinsically Disordered Proteins (IDP)and Aggregates II

2112-Pos Board B128Conformational Flexibility of HIV-1 Vif in Complex with Recruited HostCell ProteinsLieza M. Chan1, Elise Tierney1, Sampriti Thapa1, John Gross2,Katherine Ball1.1Chemistry, Skidmore College, Saratoga Springs, NY, USA, 2University ofCalifornia, San Francisco, San Francisco, CA, USA.HIV-1 Vif is an intrinsically disordered protein (IDP) that is responsible forthe ubiquitination of the APOBEC family antiviral proteins. Vif gainsstructure through binding with an E3 ubiquitin ligase complex includingElongin-B (EloB), Elongin-C (EloC), CBF-b and Cullin5 (Cul5) creatingthe Vif-CBFb-EloB-EloC-Cul5 (VCBC-Cul5) complex. Understandinghow Vif interacts with the ubiquitin complex is essential for creating ther-apeutics. This complex has been crystallized but contains a truncated C-ter-minus EloB tail. We have collected NMR data on VCBC without Cul5

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which showed that with the full-length EloB tail, there was improvement inthe signal. To understand the intermolecular interactions, MolecularDynamics (MD) simulations characterize the conformational changes ofthe VCBC complex with and without the full-length EloB C-terminus tailto explain the differences in the NMR data. Principal Component Analysis(PCA) was used to observe global movements based on correlated motionsof the VCBC complex. The additional EloB C-terminal residues allowedfor better NMR signal by decreasing fluctuations in EloB, but did not affectthe global conformational dynamics sampled by the VCBC complex. Wethen ran MD simulations of VCBC with and without Cul5 to characterizethe effect of Cul5 on VCBC dynamics. Vif contains a flexible linker regionlocated at the hinge of the VCBC-Cul5 complex, which is involved inglobal motions. PCA and dihedral angle analysis showed that the VCBCcomplex is more flexible than the VCBC-Cul5 complex allowing formore alternate state sampling centered around changes in the linker region.VCBC-Cul5 samples states similar to the crystal structure but VCBC ap-peared to sample more conformational changes involving the linker regionof Vif. Cul5 interactions give VCBC a more structurally defined state insideinfected cells.

2113-Pos Board B129Structural Insights into AggregationMechanism of Immunoglobulin LightChain Variable DomainPinaki Misra, Luis Blancas Mejia, Marina Ramirez-Alvarado.Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA.Light chain (AL) or systemic amyloidosis is an amyloid disease resulting froman over production of immunoglobulin light chain (IgG) due to abnormal pro-liferation of monoclonal plasma B cells. These abundant circulating lightchains misfold and gets deposited in the form of insoluble fibrils in the extra-cellular space of various organs leading to their impairment. AL amyloidosis isfurther complicated by the fact that each patient involving a similar or differentorgan often tend to present a slightly different protein due to somatic hyper mu-tation that might aggregate following different kinetics.Little is known about the mechanism of amyloid assembly in AL amyloid-osis. Investigation of the aggregation pathways and stages is especiallyimportant as many of these involve intermediates structures that could becytotoxic and play a critical role in the initiation of amyloid assembly. Inthis work, we discuss the mechanism of in vitro fibril formation in germline(wild type/control) protein kI O18/O8 and its amyloidogenic mutants AL-09and AL-12. Both amyloidogenic proteins aggregate significantly faster ascompared to germline protein with similar rates of aggregation for bothAL-09 and AL-12. We also observed that during early stages of aggregation,AL-09 rapidly forms transient low molecular weight oligomers that poly-merize into mature fibrils. The single restorative mutation AL-09 H87Y(restoring the amino acid in the germline sequence) alters the aggregationkinetics of AL-09. As compared to AL-09, the rate of aggregation in AL-09 H87Y drastically decreases and the low molecular weight oligomersdisappear completely. On the contrary, AL-12 appears to aggregate by poly-merization of soluble protein without involving any oligomeric intermedi-ates. The single restorative mutation of AL-12 (AL-12 R65S) to itsgermline sequence had no effect and appears to have very similar rates ofaggregation compared to AL-12.

2114-Pos Board B130Exploring the Hendra Virus Replicative Complex using Thiocyanyte IRProbes and Docking SimulationsJohn Halifax, Maryna Khromava, Casey H. Londergan.Department of Chemistry, Haverford College, Haverford, PA, USA.The Hendra virus is an extremely pathogenic virus with serious potentialhealth concerns due to its severity, wide global reach, and the lack of cur-rent defenses against it. The viral polymerase self-assembles after an intrin-sically disordered part of the nucleocapsid known as NTAIL binds to the X-Domain (XD) of the viral phosphoprotein. Research into possible drugtarget sites has sought to better understand this binding interaction, in whichit appears that NTAIL retains a large degree of structural disorder. Severaldifferent single cysteine variants of NTAIL were created and then modifiedto add a thiocyanate infrared reporter group. IR spectroscopy was thenused to determine NTAIL’s shape upon binding, and isothermal titration calo-rimetry experiments on the bound complex were performed to determinewhether the thiocyanate tag did not perturb the binding. CABS-flex pep-tide-protein docking simulations were performed on both free and boundNTAIL to independently determine a library of possible structures and givecontext to findings from IR experiments. Discussion will include a compar-ison of our IR experiments, prior work from others, and the simulated dock-ing structures.

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2115-Pos Board B131Intrinsically Disordered HAX-1 Regulates SERCA in a Calcium-Dependent MannerErik K. Larsen1, Cristina Olivieri2, Seth Robia3, Evangelia Kranias4,Gianluigi Veglia1.1Chemistry, University of Minnesota, Twin Cities, Minneapolis, MN, USA,2Biochemistry, Molecular Biology, and Biophysics, University of Minnesota,Twin Cities, Minneapolis, MN, USA, 3Physiology, Loyola Univiversity,Maywood, IL, USA, 4Pharmacology & Cell Biophysics, University ofCincinnati, Cincinnati, OH, USA.Hematopoietic-subtrate-1 associated protein X-1 (HAX-1) is a 279 aa proteinexpressed ubiquitously. In the cardiac muscle, HAX-1 was found to inhibit thesarco-endoplasmic reticulum calcium ATPase (SERCA) by shifting the pCa tohigher Ca2þ concentrations. Co-immunoprecipitation assays suggest thatHAX-1 binds to phospholamban (PLN), enhancing its inhibitory action.HAX-1 function is reversed upon PLN phosphorylation by cAMP-dependent protein kinase A. Our activity assays, show that HAX-1 shiftsSERCA’s pCa to lower values, inhibiting the ATPase activity even in theabsence of PLN. Using a combination of NMR and CD spectroscopy, wefound that HAX-1 is intrinsically disordered in the absence of a binding part-ner. Chemical denaturation experiments show only a few portions of the pro-tein might adopt a defined secondary structure conformation, while theremainder is essentially unfolded. In agreement with previous reports, wefound that HAX-1 binds Ca2þ ions and PLN’s cytoplasmic domain. We pro-pose that HAX-1 regulates the SERCA/PLN complex in a Ca2þ-dependentmanner. This would add another layer of control in Ca2þ homeostatic balancein the heart muscle.

2116-Pos Board B132Structural Characterization of the Mechanism of Aggregation andDisaggregation of HuntingtinSilvia A. Cervantes Cortes1, J. Mario Isas1, Janine Kirstein2, Ralf Langen1,Ansgar B. Siemer1.1Biochemistry and Molecular Medicine, University of Southern California,Los Angeles, CA, USA, 2Leibniz-Forschungsinstitut f€ur MolekularePharmakologie im Forschungsverbund Berlin e.V. (FMP), Berlin, Germany.Huntington’s disease (HD) is a fatal neurodegenerative disorder affecting thephysical, mental, and emotional state of approximately 1 in 10,000 individ-uals. The disease is caused by a mutation in exon 1 of the gene coding forHuntingtin (Htt), and results in a protein that contains an over-extended poly-glutamine (polyQ) tract. For reasons that remain unknown, an expansionbeyond 36Q results in protein misfolding, aggregation and toxicity to nervecells. The focus of my research is on investigating the mechanism underlyingthis association. Specifically, I aim to investigate how a polyQ expansion fa-cilitates disease onset and progression through the use of solid-state NuclearMagnetic Resonance (ssNMR) and Electron Paramagnetic Resonance(EPR). ssNMR and EPR are powerful spectroscopic tools that allow forsite-specific characterization of Htt exon1 (Htt_ex1). Using these tools, Iplan to characterize the interaction of Htt_ex1 fibrils with DnaJB1, a mamma-lian chaperone. Fibrils represent key conformers in the misfolding process andare believed to be prominent sources of cellular toxicity. Elucidation of thestructural features of these conformers and how they are recognized bycellular chaperones will, therefore, allow for greater insight into molecular or-igins of protein misfolding, aggregation, and disaggregation Huntington’sdisease.

2117-Pos Board B133Tight Binding through Structural Disorder: Mechanism and ApplicationQingliang Shen1, Jie Shi2, Pingwei Li1, Wonmuk Hwang2, Jae-Hyun Cho1.1Biochemistry and Biophysics, Texas A&MUniversity, College Station, TX,USA, 2Biomedical Engineering, Texas A&M University, College Station,TX, USA.We present molecular mechanisms by which the 1918 Spanish influenza A vi-rus (IAV) hijacks host signaling proteins and the application of the viral hijack-ing mechanism to design a potent protein-protein interaction inhibitor. The1918 Spanish flu caused one of the most serious pandemics in history. Thenonstructural protein 1 (NS1) of the 1918 IAV hijacks the interaction of humanCrkII with cAbl kinase and c-Jun-N-terminal kinase (JNK1), suppressing thehost antiviral immune response. Little is, however, known about its molecularmechanism. We discovered that NS1 uses its disordered tail to hijack CrkII andsuppresses the interactions of CrkII with cAbl and JNK1. Remarkably, thedisordered tail of NS1 binds to CrkII with strikingly rapid kinetics and nM af-finity, which is 3,300-fold higher affinity compared to that for the CrkII-JNK1interaction. We performed X-ray crystallography, NMR relaxation dispersionexperiment, and molecular dynamics simulation to determine the structural,

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kinetic, and thermodynamic mechanisms underlying the hijacking of CrkII by1918 IAV NS1. We found that the structurally disordered C-terminal tail ofNS1mediates long-range electrostatic interactions to enhance the binding affin-ity to CrkII. Furthermore, we applied the viral hijacking mechanism to designan inhibitor for CrkII-cAbl interaction to block the CrkII-phosphorylation bycAbl kinase, which plays a critical role in chronic myeloid leukemia (CML).Biochemical assays demonstrated that the inhibitor suppresses the cAbl-dependent CrkII-phosphorylation more strongly than imatinib, which is a front-line therapy for CML. Moreover, we found that the inhibitor suppresses theCrkII-phosphorylation by T315I gatekeeper mutant of cAbl kinase, which isresistant to imatinib and all second-generation tyrosine kinase inhibitors. Ourstudy provides the first example of using structural disorder to design a potentprotein-protein interaction inhibitor as a means of overcoming the drug-resistance.

2118-Pos Board B134The Cytosolic Domain of the Human ZIP4 Zinc Transporter is Intrinsi-cally DisorderedElizabeth Bafaro, Robert Dempski.Worcester Polytechnic Institute, Worcester, MA, USA.The human (h)ZIP4 protein is the primary zinc uptake protein in the intes-tine, the main location of zinc uptake. hZIP4 encodes eight transmembranedomains as well as a large cytosolic domain. The cytosolic domain containsa histidine rich region. Previously, we have demonstrated that two zinc ionscan bind to this cytosolic domain in a sequential manner. It has been pro-posed that zinc binding within the cytosolic domain regulates the surfaceexpression of hZIP4. Here, we have used NMR spectroscopy to quantifythe structural characteristics of this cytosolic domain in the absence andpresence of zinc.

2119-Pos Board B135Quantifying Disorder of an Intrinsically Unstructured Domain in EstrogenReceptorYi Peng, Shufen Cao, Matthias Buck, Sichun Yang.Case Western Reserve University, Cleveland, OH, USA.The N-terminal domain (NTD) of estrogen receptor is a hallmark of the re-ceptor’s transactivation function. While it is viewed as intrinsically disor-dered, the molecular knowledge about its disorderness is still limited.Here, we examine the NTD unfolding by chemical denaturants using circulardichroism (CD), small-angle X-ray scattering (SAXS), nuclear magneticresonance (NMR), and molecular dynamics (MD) simulation. The changeat 222 nm of CD signals and disappearance of N15-NMR peaks show theincreased unfoldedness of the already unstructured NTD, while SAXS dem-onstrates its molecular size in Rg increased from 32 A to 58 A induced by6-M guanidine hydrochloride, accompanied by substantial broadening ofpair-wise distance distribution. Combination of SAXS data with replica ex-change MD simulations further reveals that the NTD undergoes a large-scalestructural change from a relatively compact to an elongated conformation.Collectively, these results provide an alternative view of the NTD structuraldisorder as being as an unstructured, yet relatively compact structure-ensemble.

2120-Pos Board B136Deciphering Protein-Rich Domains Formed by Non-structured Proteins inthe Nuclear Pore ComplexHide A. Konishi.Kyoto University, Kyoto, Japan.Central channel of the nuclear pore complex (NPC) is occupied by a largeamount of non-structured polypeptides with high contents of Phe-Gly (FG)motifs. The protein-rich domain formed by such FG-rich polypeptides actsas an entropic barrier across the nuclear envelope, as well as binding sitesfor transport receptors such as karyopherins. In this study, we utilized a novelcrowding-sensitive FRET probe (GimRET) to reveal spatial distributions ofprotein-rich domains within the NPC, and characterized the entropic barrier.More than 10 different nucleoporins were fused with GimRET and expressedin HeLa cells. In vivo quantitative imaging revealed that the central channelof the NPC contains at least two distinct protein-rich domains; in the cyto-plasmic and nucleoplasmic peripheries, but not in the central cavity. Trans-port (receptor-cargo) complex did not largely affect protein crowding ofthese two domains. These results suggest a two-step mechanism of molecularpassage through the NPC; reaching to the central cavity over the first barrierand escaping from the cavity over another barrier to reach to the opposite sideof the NPC.Time-lapse observations of protein crowding in late-mitosis alsorevealed an important role of protein-rich domains in the NPC reassemblyprocess. The reassembly starts from nucleoporins located in the nucleo-

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plasmic domain, which is followed by the formation of the central cavity.The formation of the nucleoplasmic protein-rich domain seems to be neces-sary for the following formation of the central cavity. After the nucleoplasmicdomain and central cavity has been properly assembled, the cytoplasmicprotein-rich domain is constructed. Interestingly, FG-fragments of nucleo-plasmic nucleoporins show strong tendency to self-assemble into liquid drop-lets in vitro. These results suggest that protein-rich domains play importantroles not only in the barrier formation but also in the post-mitotic reconstruc-tion of the NPC.

2121-Pos Board B137Structural Disorder in Action in a Bacterial Toxin: Secretion, Folding andHost Cell HijackingDarragh P. O’Brien1, Dominique Durand2, Sara Cannella3, Alexis Voegele1,Patrice Vachette2, Julia Chamot Rooke1, S�ebastien Brier1, Daniel Ladant1,Alexandre Chenal1.1Structural Biology and Chemistry, Institut Pasteur, Paris, France, 2I2BC,CNRS, Paris, France, 3Department of Biochemistry, University of Oxford,Oxford, United Kingdom.The adenylate cyclase toxin (CyaA, 1706 residues) is an RTX protein thatplays an essential role in the early stages of respiratory tract colonizationby Bordetella pertussis, the causative agent of whooping cough. Its cell intox-ication process, however, is still poorly understood. After its secretionthrough a dedicated type 1 secretion system, CyaA intoxicates human cellsvia a unique mechanism of translocation of its catalytic domain (AC) directlyacross the plasma membrane of target cells. Once in the cytosol, AC interactswith calmodulin (CaM) and produces supraphysiological levels of cAMP,leading to cell death. Our results, based on SAXS, HDX-MS and SR-CDdata, illustrate how the structural flexibility of this bacterial toxin contributesto various biological processes, including its secretion, its folding, its translo-cation into target cells, and its activation by CaM. All of these steps involvedisorder-to-order structural transitions that are finely tuned to the specificenvironmental conditions that the toxin successively experiences along itsjourney from the host bacterium to the eukaryotic target cell. Finally, CyaAis known for its propensity to aggregate into multimeric forms in the absenceof a chaotropic agent in vitro. We have recently shown that calcium and mo-lecular confinement are required for CyaA folding into a monomeric and func-tional species. This opens up new avenues for both basic science, as well asbiotechnological applications of recombinant CyaA as an antigen deliveryvehicle, and as a potential protective antigen in the next-generation ofpertussis vaccines.

2122-Pos Board B138Mechanisms of Selective Transport through the Nuclear Pore ComplexLaura Maguire1, Michael Stefferson1, Meredith Betterton1, Loren Hough1,2.1Dept. of Physics, University of Colorado Boulder, Boulder, CO, USA,2Biofrontiers Institute, University of Colorado Boulder, Boulder, CO, USA.Few cellular processes require such intricate active control as transportthrough the nuclear envelope. The nuclear pore complex (NPC) facilitatesall transport, preventing most macromolecules from crossing the envelopewhile allowing the passage of transport factors (TFs) and their cargo. Whilethe basic biochemical interactions of transport are well-understood, thedetailed mechanism remains a topic of significant debate. We model trans-port using reaction-diffusion equations. The results suggest that (1) the flex-ible nature of the disordered FG nups and (2) the transient, multivalentnature of FG nup - TF interactions are together sufficient for selectivity.Our model makes directly-testable predictions of the mechanisms ofselectivity.

2123-Pos Board B139On the Origins of Regulated Disorder within the C-Terminus of P53Carlos X. Hernandez1, Hannah Wayment-Steele2, Vijay S. Pande1.1Biophysics, Stanford University, Stanford, CA, USA, 2Chemistry, StanfordUniversity, Stanford, CA, USA.The role of intrinsic disorder in protein-protein interactions remains poorlyunderstood, even as it has become increasingly clear that many critical biolog-ical processes rely upon it. An understanding of how these interactions haveevolved might help exploit intrinsically disordered regions in proteins asnovel therapeutic targets. In this work, we identify multiple post-translational switches in the C-terminus of p53, which are relevant to its as-sociation with Sirt2, using ancestral sequence reconstruction. Using MarkovState models constructed from milliseconds of aggregate atomistic simula-tions, we characterize the role of each switch in regulating disorder-to-order transitions of the C-terminus in solution and how it might affect bindingto Sirt2.

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2124-Pos Board B140Polyphosphate-Induced Aggregation-Prone Conformations of TauHope E. Merens1, Sanjula Wickramasinghe2, Justine Lempart3,Ursula Jakob3, Elizabeth Rhoades1.1Chemistry Department, University of Pennsylvania, Philadelphia, PA, USA,2Biochemistry and Molecular Biophysics Graduate Group, University ofPennsylvania, Philadelphia, PA, USA, 3Department of Molecular, Cellular,and Developmental Biology, University of Michigan, Ann Arbor, MI, USA.Tau is an intrinsically disordered protein expressed in the axons of neurons,where it functions to stabilize microtubules. In neurodegenerative diseasesknown as tauopathies, tau forms aggregated, cytosolic inclusions consistingof paired helical filaments. In vitro, a variety of anionic polymers have beenshown to induce aggregation of tau,including polyphosphate. However,whether or not these inducers share a common mechanism is unknown.Here we used single molecule FRET to characterize conformational changesin tau upon binding to polyphosphate, contrasting the 2N3R and 2N4R iso-forms. Our measurements determined that the positively chargedmicrotubule-binding region of tau and the proline-rich region preceding itcompact in the presence of polyphosphate, suggesting that these regionsinteract electrostatically with polyphosphate. The negatively charged N-ter-minal region was extended in the presence of polyphosphate, resulting in theloss of long-range contacts found in solution. These findings largely corrob-orate with a model for the aggregation-prone state of tau in the presence ofheparin; comparison between the two models may elucidate generalizablefeatures of tau’s aggregation-prone state. Furthermore, as the ratio of2N3R and 2N4R isoforms is imbalanced in some tauopathies, their compar-ison may provide insight into how this perturbation contributes to aggregateformation.

2125-Pos Board B141Capturing Conformational Changes of the Tau Protein Upon AggregationYann Fichou, Neil Eschmann, Songi Han.Chemistry and Biochemistry Department, University of California SantaBarbara, Santa Barbara, CA, USA.Many neurodegenerative diseases (NDs) are associated with abnormal accu-mulation of protein aggregates in the brain. The intrinsically disordered tauprotein, which does not possess a well-defined 3D structure in normal con-ditions, is involved in several NDs, including Alzheimer diseases and fron-totemporal dementia. In pathological conditions, tau can aggregate intostructured aggregates, in so-called neurofibrillarly tangles, which are deadlyto neurons. Different fibers supra-structures, referred to as strains, have beenshown to be associated with different diseases, thereby suggesting that thearrangement of tau molecules in the fibers is pathologically relevant. Strik-ingly, tau has recently been termed as a prion-like protein because of the ca-pacity of one tau strains to effectively propagate from neuron to neuron. Theability of a strain to convert naıve disordered monomers into specific struc-tured aggregates is still not understood, but conformational changes of tau isthought to be a critical factor. Here we have characterized tau structure bycombining electron paramagnetic resonance spectroscopy and spin-labelling techniques. In particular, double electron-electron resonance spec-troscopy (DEER) allowed us to measure intra-molecular distances atdifferent time points along the aggregation process, in particular at earlytimes before the formation of beta-sheets structures. We have used differentaggregation triggers including heparin cofactors and fiber seeds extractedfrom model mouse brains. We will show some key conformational changesoccurring during aggregation, and relate them to the seeding capacity of taumisfolded species. A better view of tau disorder-to-structure transition willhelp understand the aggregation mechanisms, and pave the way to newdrug development strategies that aim at limiting propagation of deleteriousaggregates in NDs.

2126-Pos Board B142Fibrillation of N-Terminal Prion Protein Fragment in Presence of ZincIonsMaciej B. Gielnik1, Micha1 Nowakowski2, Aneta Szyma�nska3, Igor YuZhukov4,5, Wojciech Maria Kwiatek2, Maciej Leszek Kozak1,6.1Macromollecular Physics, Adam Mickiewicz University, Pozna�n, Poland,2Department of Experimental Physics of Complex Systems, Institute ofNuclear Physics, PAS, Krakow, Poland, 3Department of Medical Chemistry,University of Gda�nsk, Gda�nsk, Poland, 4Institute of Biochemistry andBiophysics, PAS, Warszawa, Poland, 5NanoBioMedical Centre, AdamMickiewicz University, Pozna�n, Poland, 6Joint Laboratory for SAXS studies,Adam Mickiewicz University, Pozna�n, Poland.Abnormal (misfolded) form of human prion protein displays high propensitytowards self-association which may result in formation of insoluble fibrillar

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deposits in the brain, associated with the development of fatal neurodegener-ative disorders, transmissible spongiform encephalopathies (TSE) [1]. Theformation of such deposits can be driven by many intra- and extracellular fac-tors. One of them can be disrupted homeostasis of metal ions. As reported pre-viously, Zn ions bonded to N-terminal fragment regulate the interactionsbetween N- and C-terminals [2]. Here we present structural analysis of thefragment of PrP N-terminal domain in the presence of Zn ions. Our experi-ments demonstrate that Zn-saturation leads to formation of N-terminaldomain fibrillar structures. The fibrils were analyzed by atomic force micro-scopy (AFM), electron microscopy (EM), X-ray fiber diffraction and X-rayabsorption spectroscopy (XAS). AFM and EM images indicated, that in thepresence of zinc, PrP peptide forms long fibrillar structures. The presenceof two characteristic reflections at 1.12 nm and 0.36 nm in the X-ray diffrac-tion pattern confirmed the fibrillar nature of the studied PrP peptide. These re-flections corresponded to characteristic distances in the fibrillar structure ofthe studied PrP peptide. Zn XAS K-edge 1s->4p transition in the edge regionindicates, that Zn occupies a mononuclear site and is coordinated by lightatoms (N, O). Fourier transform of c(k) signal indicates the presence of imid-azole groups in the nearest Zn shell. Our findings may shed new light on therole of zinc in TSE disorders. This work was supported by the funds from theNational Science Centre (Poland) granted on the basis of decision no. 2014/15/B/ST4/04839.[1] Kovacs, G. G., & Budka, H. (2008). The American Jour-nal of Pathology, 172(3), 555-565. [2] Spevacek, A.R., et al. (2013) Structure,21, 236-246.

2127-Pos Board B143Polymorphism of Prion Protein Amyloid-Like FibrilsTomas �Sneideris, El�zbieta Kulicka, Vytautas Smirnovas.Department of Biothermodynamics and Drug Design, Vilnius UniversityInstitute of Biotechnology, Vilnius, Lithuania.Prions - infectious protein aggregates that are thought to be the main cause oftransmissible spongiform encephalopathies (TSEs) in mammals. Prions areentirely composed of misfolded prion protein (PrP), which is prone to aggregateand can form insoluble, partially protease resistant and exceptionally stable am-yloid fibrils. The picture is complicated by the ability of the same prion proteinto adapt slightly different pathogenic structures (prion strains) leading todifferent disease phenotypes. There is still no clear understanding what leadsto formation of wide variety of strains, or how many different strains canPrP form.We studied aggregation of recombinant prion protein in vitro under differentenvironmental conditions in order to understand the variety of formedamyloid-like structures. We used Thioflavin T fluorescence assay to follow ki-netics of spontaneous and seeded aggregation, Fourier Transform Infraredspectrometry to detect differences in the secondary structure of aggregates,Atomic Force Microscopy to compare morphology of amyloid-like fibrils,and chemical denaturation in guanidine thiocyanate to evaluate their stability.Our findings may help to take a deeper insight into process of prion strainformation.

2128-Pos Board B144Amyloid-b Peptide Interaction with Lipid Bilayer Promotes PeptideAggregation on the Surface and Modulates Lipid BehaviorJacob Usadi, Arthur Vale, Sashin Natesh, Karl Freed, Esmael Haddadian.University of Chicago, Chicago, IL, USA.Alzheimer’s disease (AD) is an important, increasingly prevalent neurolog-ical disorder. Aggregation of Amyloid-b (Ab) is important in etiology ofAD and the interaction of these peptides with cell membranes is probablyone of the key pathological events responsible for neuron cell death inAD. However, the molecular mechanism underlying the interaction is notknown. The slow nature of conformational changes of protein and mem-brane lipids contributes to this void in the literature. To accelerate themembrane contribution to the conformational changes of Ab without theloss of atomistic detail, we used the novel high mobility mimetic membrane(HMMM) model to investigate the Ab-membrane interactions using molec-ular dynamic simulations. These membrane models increase lateral lipidmovement by an order of magnitude while maintaining atomistic detailby modeling the lipid tails nearest the membrane center as a fluid organicsolvent while maintaining the atomic description of the lipid head-groups.We built three systems: clusters of two and three unfolded Ab40 peptidesinside a membrane bilayer and an Ab40 dimer with one peptide anchoredto the membrane surface. The HMMM lipid composition used wasPC:pPE:SM:CHOL 38:27:6:29. The transmembrane Ab-peptides demon-strated rapid clustering (<50ns) with subsequent b-sheet formation betweenresidues 18-22 of the monomers, concording with experimentally observedAb fibril structures and computationally determined dimer structures. The

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b-sheet formation can potentially lead to creation of a pore inside the mem-brane that can affect cellular function. The anchored dimer demonstratedincreasing inter-peptide b-sheet content and subsequent movement towardthe membrane surface, implying a mechanism for nucleation and aggrega-tion of Ab oligomers. The strong Ab dimer-membrane interaction loweredthe fluidity of the lipid molecules.

2129-Pos Board B145Modulating Amyloid-Beta Aggregation to Reduce the Toxicity of itsOligomeric AggregatesRyan Limbocker1, Benedetta Mannini1, Sean Chia1, Francesco S. Ruggeri1,Michele Perni1, Roberta Cascella2, Catherine Xu1, Johnny Habchi1,Janet R. Kumita1, Fabrizio Chiti2, Tuomas P.J. Knowles1,Michele Vendruscolo1, Christopher M. Dobson1.1University of Cambridge, Cambridge, United Kingdom, 2Department ofExperimental and Clinical Biomedical Science, University of Florence,Florence, Italy.The misfolding and aggregation of the amyloid-b (Ab) peptide plays a centralrole in the pathogenesis of Alzheimer’s disease (AD). Targeting the generationor structure of the highly cytotoxic oligomeric species that form during thedeposition process represents a promising therapeutic strategy to reduce thetoxicity associated with Ab aggregation. Through an integrative approachcombining in vitro techniques, including chemical kinetics, atomic force mi-croscopy, and other biophysical measurements, with in vivomethods, includingneuroblastoma cells and a C. elegans model of neurodegenerative disease, wehave investigated modes by which oligomeric aggregates can be targeted withsmall molecules. We thus report compounds that show that the cytotoxicityrelated to Ab aggregation can be reduced both by the promotion of fibril forma-tion and by the direct modification of oligomer structure. Indeed, both ap-proaches were revealed to generate aggregates with unique tinctorialproperties and reduced cytotoxicity. These results provide insight into therole of oligomers in the induction of cellular toxicity, and suggest that novelmechanisms of modulating the aggregation process, in addition to inhibitingoligomer production, may provide a viable therapeutic route for the treatmentof AD.

2130-Pos Board B146Probing Synaptic Amyloid-Beta Aggregation Promoted by Copper ReleaseBogachan Tahirbegi1, Alastair J. Magness2, Aurelien Boillat3,Keith R. Willison1, David R. Klug1, Thomas Knopfel3, Liming Ying2.1Department of Chemistry, Imperial College London, London, UnitedKingdom, 2National Heart and Lung Institute, Imperial College London,London, United Kingdom, 3Department of Medicine, Imperial CollegeLondon, London, United Kingdom.Whether or not the metal ions released during synaptic transmission induceamyloid-beta oligomer formation in the vicinity of synapses is a centralquestion pertinent to the molecular mechanism of Alzheimer’s disease.Recently, through a combination of experimental kinetics studies andcoupled reaction-diffusion simulations, we predicted that Cu(II) ratherthan Zn(II) plays an important role in the very early stages (i.e., dimer for-mation) of Ab aggregation in the synapse. Single molecule photobleachinganalysis is a powerful tool to determine the stoichiometry of amyloid-betaoligomers which enables us to examine the time course of small amyloid-beta oligomer formation in solution, immobilised to a solid-phase substrateor artificial lipid membrane, and in live neurons in the presence of Cu(II).Preliminary results indicate that small amyloid-beta oligomers can be lockedin their oligomeric state without dissociation on a poly-lysine coated surfaceand that Cu(II) increases the diversity and abundance of amyloid-betaoligomers.

2131-Pos Board B147Gas Phase Studies of the Amyloid-b PeptideNicklas Osterlund1, Sebastian K.T.S. W€arml€ander1, Leopold L. Ilag2,Astrid Gr€aslund1.1Department of Biochemistry and Biophysics, Stockholm University,Stockholm, Sweden, 2Department of Environmental Science and AnalyticalChemistry, Stockholm University, Stockholm, Sweden.The amyloid-b (Ab) peptide is an amphiphilic peptide that exhibits self-aggregating properties forming the amyloid fibrils that can be found in thebrains of Alzheimer patients. Today it is believed that that it is the solubleAb oligomers rather than the mature fibrils that are the main neurotoxic spe-cies. These small peptide-assembles are known to associate with membranesand form pores that can transport Ca2þ into the cell, which in part could beresponsible for their neurotoxic properties. However, most biophysicalmethods that have been developed to study Ab target either the monomer

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or the mature fibrils, and not the low abundance and polydisperse oligo-mers.We have developed a soft ionization mass spectrometry (MS) methodthat retains and detects these non-covalent oligomer interactions in the gasphase. And since MS is a non-averaging technique the aggregation kineticsfor different oligomeric species, as well as reduced and oxidized peptides,could be followed simultaneously. The high-resolution MS was also coupledto an ion mobility spectrometer that separates ions of the same m/z accordingto collisional cross section, yielding information about peptide conformationsin the gas phase. Additionally, peptide interactions with other molecules, suchas membrane mimicking detergents can be monitored. Large complexes be-tween Ab(1-40) and detergent micelles was shown to survive in the MSand could be identified. Detergent head group charges seem to be essentialfor peptide-micelle interactions, both in the gas phase and in solution, as nostructure induction is observed upon addition of non-ionic detergent. Thedeveloped methodology is promising for future detection of pore-formingoligomers in micelles, and probably also in other more sophisticated mem-brane models.

2132-Pos Board B148Quantitative Hydroxyl Radical Footprinting Study Reveals StructuralDetails of the Disorder-to-Order Transition in Amyloid-Beta (1-42)OligomerizationJanna Kiselar1, Andrew Nix2, Anant Paravastu3, Terrone Rosenberry2,Alexandra Klinger4.1Case Western Reserve University, Cleveland, OH, USA, 2Mayo Clinic,Jacksonville, FL, USA, 3Georgia Institute of Technology, Atlanta, GA, USA,4Decipherbio, Philadelphia, PA, USA.This work presents a novel integrated analysis approach to characterize theintrinsically disordered 42 amino acid long b-amyloid peptide (Ab42) and toelucidate mechanisms driving its misfolding and aggregation into neurotoxicoligomers. We describe this approach, which uses synchrotron based hy-droxyl radical footprinting (HRF) results to determine residue specific sol-vent accessibilities for each of seventeen individual amino acids inmonomer, multimer (2-4mer), and oligomer (�150 kDa) Ab42 sample prep-arations. Results complement published solid state NMR constraints with so-lution state structural details for the oligomers, and provide much neededexperimental support for molecular dynamics (MD) computational studiesof the monomer and 2-4mer species, where solid state NMR cannot beused. The pattern of protection from hydroxyl radical attack in the Ab42monomer (relative to the same residues in the fragmented unstructured pep-tide) shows partial folding in the monomer. The sequence location of thispartial folding is in agreement with MD predictions of transient b-strand for-mation in the hydrophobic core and C-terminal regions. Sequence-specificprotection (i.e. reduced side chain solvent accessibility) upon further assem-bly into the 2-4mer and oligomer species suggest specific conformers thatare selected from the intrinsically disordered monomer ensemble to driveoligomerization. Results of the current study are also consistent with generalmodels developed from previously published solid state NMR oligomerstudies from this and other laboratories.

2133-Pos Board B149AMulti-scale Study of b-AmyloidWild-Type andMutant Peptides: Mono-mers, Oligomers, FibrilsArthur O. Vale1, Jacob Usadi1, Sachin R. Natesh1, Sarida Pratuangtham2,Karl F. Freed3, Esmael J. Haddadian1.1University of Chicago, Chicago, IL, USA, 2California Institute ofTechnology, Pasadena, CA, USA, 3Department of Chemistry and JamesFrank Institute, University of Chicago, Chicago, IL, USA.Alzheimer’s Disease affects millions of people and is becoming more prev-alent. It is believed to be caused by aggregates of b-amyloid (Ab) peptides.The end-points of aggregation are amyloid fibrils, though is it widelybelieved that soluble, oligomeric precursors of the fibrils are the more impor-tant neurotoxins. Ab is known to form highly polymorphic fibrils, with manymutant forms observed in patients. Some mutants, such as the Iowa (D23N)and the Osaka (E22D), are known to have increased neurotoxicity and fasteraggregation in relation to wild-type peptides. To investigate the differencesbetween wild-type and mutant Ab-peptides, we modeled their behavior atfour levels: monomer, oligomer, finite and infinite fibrils; using long allatom molecular dynamics simulations. The two wild-type fibril structuresin our simulations were pdb entries 2LMN and 2LMP (having two- andthree-stacks), the Osaka mutant structure was 2MVX (having two-stacks)and the Iowa mutant structures were 2LNQ and 2MPZ (having one- andthree-stacks). The Osaka mutant demonstrated the most structural stabilityevident from its higher average b-content (that is known experimentally tobe strongly correlated to stability in amyloid fibrils). We attribute this to

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strong inter-backbone hydrogen-bond network and a strong salt-bridge be-tween residues E3-R28 unique to the Osaka mutant. We also observed alarger number of sodium ions accumulated on the interior pockets of theOsaka-mutant fibril. These can explain the very fast aggregation of theOsaka mutant and possibly its higher neurotoxicity. The Iowa mutant struc-ture (2MPZ) and wild-type structure (2LMP) had similar structural stabilityas well as aqueous-pore like behavior, which might disrupt cell function ifinserted through a membrane. The one-stack Iowa mutant showed the leaststructural stability that may imply the need for more than one-stack for astrong fibril integrity.

2134-Pos Board B150The Functional Amyloid Orb2A Interacts with Lipid BilayersMaria A. Soria1, Silvia A. Cervantes1, Thalia H. Bajakian2,Ansgar B. Siemer3.1Programs in Biological and Biomedical Sciences, University of SouthernCalifornia, Los Angeles, CA, USA, 2Drexel University, Philidelphia, PA,USA, 3Biochemistry and Molecular Medicine, University of SouthernCalifornia, Los Angeles, CA, USA.Orb2 is a CPEB homologue and translational regulator found in D. mela-nogaster, which is able to form functional amyloid-like aggregates in thefruit fly brain. Both isoforms A and B are found in these aggregates, butit is isoform A that is thought to initiate the aggregation process. IfOrb2A loses the ability to aggregate, the fruit fly loses the ability to retainlong-term memory. This mechanism must be highly regulated to avoid toxicamyloid-related species, such as those that occur in Alzhiemer’s, Parkin-son’s, and other amyloid-related diseases. Previous data suggested thatOrb2A might be able to bind lipid membranes. As membrane binding affectsthe aggregation of many other amyloid-forming proteins, we investigate thepotential for membrane binding of Orb2A, as well as how lipid membranebinding affects amyloid aggregation. We use circular dichroism and electronparamagnetic resonance (EPR) to identify how and where Orb2A binds lipidvesicles. We also use transmission electron microscopy and EPR to trackamyloid formation over time with and without lipid vesicles present. Weshow that Orb2A binds to anionic small unilamellar vesicles (SUVs) usingan N-terminal amphipathic helix, and that charge is important for vesiclebinding as well as a high degree of membrane curvature. We also showthat the presence of anionic SUVs inhibits amyloid formation, which opensthe door for a regulatory role for membranes in the formation of functionalOrb2A amyloid fibrils.

2135-Pos Board B151Fibrillation of A-Beta Peptides in Presence of Phenolic Inhibitors: Coarse-Grained SimulationsCarol K. Hall, Yiming Wang.Chemical and Biomolecular Engineering, North Carolina State University,Raleigh, NC, USA.Alzheimer’s Disease (AD) is a neurodegenerative disease that causes de-mentia, nervous system degradation, and death. Currently there are no ther-apeutic agents available for the treatment of AD despite great efforts by theresearch community. Many small phenolic compounds have been shown inexperiment to be effective at preventing the formation of Ab fibrils. In thiswork we use discontinuous molecular dynamics (DMD) simulations tolearn how four naturally-occurring phenolic compounds, resveratrol,vanillin, curcumin, and epigallocatechin-3-gallate (EGCG), bind toAb(17-36) monomers and affect oligomerization and fibrillation. We usea coarse-grained model/force field for inhibitors which has geometric andenergetic parameters that are compatible with the coarse-grained proteinmodel PRIME20. Preliminary results show that the U-shaped protofibrilstructure formed by Ab(17-36) is similar to the corresponding part of theAb(1-42) fibril model, based on solid state NMR data. The order of peakheights in the peptide-inhibitor radial distribution function shows that thestrength of the inhibitor binding affinity is resveratrol > curcumin andEGCG > vanillin. Simulations of 8 Ab(17-36) peptides aggregating inthe presence of 30 inhibitors show that EGCG, resveratrol and curcumininhibit Ab(17-36) fibril formation while vanillin only retards the lag phase.Instead of forming an ordered b sheet structure (as occurs in the absenceof inhibitors), the peptides remain random coils and form complexeswith the inhibitors, binding mainly to the hydrophobic residues near thepeptide termini. The relative ability to inhibit Ab(17-36) fibrillation isEGCG > resveratrol > curcumin > vanillin, consistent with experimentalfindings on full length Ab(1-42) aggregation. Our simulations providemolecule-level insights into the mechanisms by which small moleculesinhibit Ab aggregation.

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2136-Pos Board B152Atomic-Level Insights into the Dynamics of Enzymes and IntrinsicallyDisordered Proteins within Sea Spray Aerosol ParticlesJamie Schiffer, Rommie Amaro.Chemistry and Biochemistry, UC San Diego, San Diego, CA, USA.Sea spray aerosols (SSA), liquid particles emitted from the ocean upon wavebreaking, represent a major source of atmospheric aerosols. Understandingthe chemical complexity within these aerosols will inform of the mechanismsby which aerosols impact climate and the environment. Here, we use computa-tional modeling strategies to build realistically complex models of sea sprayaerosols. These models contain enzymes and intrinsically disordered proteins,sugars, salts, and lipids in ratios informed by measurements from mesocosmwave flume experiments undertaken through the center for aerosol impactson the chemistry of the environment (CAICE). The dynamic atomic-level mo-tions of the components within the computational models of SSA particlereveal (1) the effects that enzymes can have on particle chemistry, (2) howin turn the SSA particle chemistry can affect the chemical environment andstructure of enzymes, and (3) the atomic-level dynamics of intrinsically disor-dered, ice-nucleating proteins in different chemical environments that are com-mon within SSA particles.

2137-Pos Board B153Modeling Intrinsically Disordered Proteins and Amyloid Fibrils inPyrosettaJohn Ferrie1, Abhinav Nath2, E. James Petersson1.1Chemistry, University of Pennsylvania, Philadelphia, PA, USA, 2MedicinalChemistry, University of Washington, Seattle, WA, USA.Intrinsically disordered proteins (IDPs) are an expanding class of proteinswhose conformation placisticity can result in aggregate formation. More-over, the conformational heterogeneity of IDPs can be influenced byvarious buffer conditions and chemical modifications. Therefore, the pro-duction of models which effectively describe these structurally diverse en-sembles has been the focus of much effort. Much of this work has utilizedmolecular dynamics (MD) simulations, specially designed tools, like CAM-PARI, or experimental constraints develop structural models. Previously,others have demonstrated that experimentally constrainted simulationswithin Rosetta are capable of producing accurate structural models of fibrilsand disordered protein regions. Here we focus on improving both of thesecapabilities within PyRosetta. First, we have focused on using experimentaldata from our lab and others to optimize simulations of the disorderedensemble of a-synuclein. Following the development of an effective simu-lation protocol, we have demonstrated the efficacy of this method byapplying it to several other IDPs, which have published experimentaldata from a variety of techniques. Furthermore, we have improved thealready existing framework for simulating fibrils using Rosetta. By utilizingthe intrinsic geometric constraints of fibrils to influence both sampling andscoring, we have enhanced Rosetta’s ability to generate fibrillar structuresde novo. Lastly, we demonstrate the utility of this method in producingstructures of ab.

2138-Pos Board B154Coarse-Grained Simulations of Intrinsically Disordered Proteins in theContext of Liquid-Liquid Phase SeparationGregory L. Dignon1, Wenwei Zheng2, Young C. Kim3, Jeetain Mittal1,Robert Best4.1Lehigh University, Bethlehem, PA, USA, 2Arizona State University,Temple, AZ, USA, 3Naval Research Laboratory, Washington, DC, USA,4National Institutes of Health, Bethesda, MD, USA.Cellular assembly of highly concentrated RNA and proteins into phase sepa-rated liquid- or hydrogel-like phases can provide cellular compartmentaliza-tion in the absence of lipid membranes. The details of the interactions, anddriving forces which promote phase separation into these types of assembliesare still not well understood and may require difficult and expensive experi-mental techniques, or impractically large atomistic simulations. To addressthis, we have proposed a novel framework for conducting coarse-grained sim-ulations of the phase coexistence using knowledge-based interaction poten-tials and sampling techniques capable of mapping the phase coexistencebehavior[1]. Using this approach, we are also able to determine intermolec-ular contacts and diffusion of chains in the dense phase to help determinethe properties of these high-density phases, and the driving forces of theircondensation.Presently, we have successfully determined the effects of mutations to highlyinteracting regions of commonly studied peptide sequences including TDP-43, FUS, hnRNPA2 and LAF-1. Additionally, we show that small, transient

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helical structures can also have an influence on the ability of the peptide tophase separate through the use of rigid constraints, or more flexible Go-like po-tentials. Our goal is to provide researchers with the tools to predict the effects ofmutations, post-translational modifications, changes to solution conditions, andmany other factors on the protein’s ability to phase separate under physiolog-ical conditions. Much effort is being directed toward improved nonbonded pair-wise potentials, and the design of efficient sampling methods and othertheoretical models to allow for high-throughput screening of a large numberof protein sequences.[1] Dignon, G. L.; Zheng, W.; Kim, Y. C.; Best, R. B.; Mittal, J.PLoS Compu-tational Biology (under review).

2139-Pos Board B155Increasing the Accuracy in All-Atom Simulations of Intrinsically Disor-dered Proteins based on the Absinth ModelMartin J. Fossat, Tyler S. Harmon, Ammon E. Posey, Jeong-Mo Choi,Rohit V. Pappu.Biomedical Engineering, Washington University in Saint Louis, Saint Louis,MO, USA.Sequence-specific conformational preferences of intrinsically disorderedproteins (IDPs) are governed by the overall charge content, the linearpatterning of oppositely charged residues as well as proline and charged res-idues. Physical insights generated using the ABSINTH implicit solvationmodel and forcefield paradigm assume fixed charge states of þ1e for Lysand Arg, �1e for Asp, and Glu, and electroneutrality for His. However,the sequences of IDPs and putative IDPs encode rich possibilities for alter-ations in sequence-to-conformation relationships via charge regulation. Thisrefers to protonation / deprotonation of titratable groups and / or the additionor deletion of charges via post-translational modifications. We have devel-oped a general strategy to capture the effects of charge regulation, specif-ically pKa shifts, within the ABSINTH framework. This is achieved usinga generalized thermodynamic integration approach. The effects of pKa shiftson conformational equilibria and reversible order-to-disorder transitions arecalibrated using data from spectroscopic and potentiometric measurements.Additionally, we have uncovered the context dependence of reference freeenergies of solvation to account for non-additive contributions that aremissing when we coopt data for free energies of solvation of model com-pounds. We are also refining the original forcefield parameters utilizingexperimental results on a diversity of IDPs. These refinements are performedusing an entropy maximization procedure that optimizes the agreement be-tween simulated and experimentally derived conformational ensembles.Taken together, the generalizations contribute to improved accuracy in sim-ulations of IDPs using the ABSINTH model.

2140-Pos Board B156Combining Prediction of Protein Aggregation Propensities with Predictionof Other One-Dimensional PropertiesAndrzej Kloczkowski1, Maksim Kouza2, Girik Malik3, Irina Buhimschi4,Eshel Faraggi5.1Battelle Center for Mathematical Medicine, Nationwide Children’s Hospitaland The Ohio State University, Columbus, OH, USA, 2Battelle Center forMathematical Medicine, Nationwide Children’s Hospital, Columbus, OH,USA, 3Nationwide Children’s Hospital, Columbus, OH, USA, 4NationwideChildren’s Hospital and The Ohio State University, Columbus, OH, USA,5Nationwide Children’s Hospital and IUPU, Columbus, OH, USA.

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Protein and peptide aggregation leads to the development of several debilitativedisorders, such asAlzheimer’s and Parkinson’s diseases or pregnancy-related pre-eclampsia disorder. Misfolded proteins can aggregate forming amyloids that leadto the formation of fibrils and then plaques that disrupt the normal functioning ofcells. Because of this the development of accuratemethods to predict the aggrega-tion of proteins and peptides is of the utmost importance. There are several knownmethods for prediction of aggregation propensity of a protein from its sequence,such as AGGRESCAN, FoldAmyloid, FISH, AMYLPRED, and others. Recentlywe used GOR method originally developed for prediction of protein secondarystructure from sequence to predict protein aggregation propensities. We haveshown in the past that accurate prediction of protein secondary structure and otherone-dimensional structure features is essential for accurate sequence alignment,three-dimensional structure modeling, and function prediction. In the presentwork we extend this approach by combining prediction of aggregation propensitywith predictions of secondary structure, and other one-dimensional properties,such as intrinsically disordered regions, solvent accessible area, and dihedral an-gles by using amultistep neural-network algorithm.Our preliminary studies showthat this method is highly promising and efficient alternative to other known ag-gregation predicting tools, and leads to improved accuracy of prediction of proteinaggregation propensity from sequence.

2141-Pos Board B157Improved Accuracy and Convergence of Intrinsically Disordered ProteinMolecular Dynamics Simulations Using the ff14IDPSFF Force FieldVy T. Duong, Mahendra Thapa, Ray Luo.University of California, Irvine, University of California, Irvine, Irvine, CA,USA.Vital components in signaling pathways, regulation, and other complexbiochemical networks, intrinsically disordered proteins (IDPs) utilize theirconformational flexibility to perform intricate functions, but their flexible con-formations remain poorly understood. A mounting body of experimental evi-dence – ranging from NMR structural studies to X-ray crystallographyexperiments – has accumulated to probe the conformations of IDPs. However,IDPs remain elusive by standard experimental methods due to their conforma-tional flexibility. Molecular dynamics (MD) simulations can provide detailedinsight into these complex structure-function dynamics if they can reproduceavailable experimental observables. The recently developed ff14IDPSFF forcefield developed by Song et. al [2017] corrects dihedral backbone terms of all 20canonical amino acids to achieve this goal. Utilizing this recently-developedforce field, 9 unstructured short peptides and the HIVrev IDP were simulatedextensively to assess the quality of the new force field and to assess the compu-tational challenge of IDP simulations in general. We extended trajectorylengths for at least one microsecond and to at least ten independent trajectoriesto comprehensively evaluate the performance, accuracy, and convergence ofMD simulations in f14IDPSF. All simulated f14IDPSFF values (Ca chemicalshift, J-coupling, RDC) are in close agreement to available NMR-derivedexperimental values than the generic protein force field ff14SB. Simulatedff14IDPSFF values also converged within nanosecond timescales for the 9short peptides whereas simulated ff14SB values converged after 1 ms. HIVrevsimulated values converged after 1 ms for both ff14IDPSFF and ff14SB, whichare expected due to its larger size. Improved accuracy to experimental valuesand timescale of calculation convergence demonstrate the utility offf14IDPSFF to explore IDP conformation space and investigate the structure-function dynamics of IDPs.

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Posters: RNA Structure and Dynamics

2142-Pos Board B158In Vitro Aptamer Selection and Evolution using an Engineered Dual-Ribozyme ComplexMichael Muntifering, David Wendell.Biomedical Engineering, University of Cincinnati, Cincinnati, OH, USA.Small-molecule aptamers are composed of RNA or DNA sequences with intri-cate secondary and tertiary structure allowing them to perform many functions,including ligand binding and catalytic events.Aptamers are selected from a library of random nucleic acids against a specifictarget. The binding sequences are amplified and fed back into the process forsubsequent rounds of selection and amplification. This is a well-described tech-nology called Systematic Evolution of Ligands by Exponential Enrichment(SELEX).We propose a novel SELEX method to characterize and screen new RNA ap-tamers using existing aptamers and randomization of a stem-loop sequence inthe canonical type-I hammerhead ribozyme. As opposed to other reported tech-niques, we hypothesize that ours will allow for rapid and efficient selection ofsmall-molecule ligands in solution and extended to an easy model for expres-sion of these aptamers in vivo. This method reduces the number of selectionrounds required, potentially selecting viable aptamers within one round. Ourmethod utilizes a binary aptamer binding event in which a randomizedsequence is placed in a stem of the hammerhead ribozyme. When the randomsequence binds a ligand, it initiates cleavage of the ribozyme and a second ‘‘se-lecting’’ aptamer folds. The selecting aptamer binds to beads in a column, andthe desired aptamers can be eluted after subsequent washes to remove non- andlow-specificity binders. Further rounds could increase stringency of selectionby means of decreased ligand concentration or increased competing ligands.Advantages of this system are manifested in time saved and resourcesconserved. Additionally, since our ligands are not immobilized on beads noris any substrate required for capturing the sequences once selected, more avail-able moieties for binding will be made available and new aptamers morereadily discovered.

2143-Pos Board B159Identification of an Allosteric Twister Ribozyme for Use as a SyntheticGenetic SwitchSamantha M. Stoupa, Juliane Strauss-Soukup.Chemistry, Creighton University, Omaha, NE, USA.Synthetic biology is a rapidly emerging field focused on engineering biochem-ical systems and cellular functions for a variety of applications, including ther-apies for the treatment of infectious diseases and cancer, as well as tactics forvaccine development, microbiome engineering, cell therapy, and regenerativemedicine. Many of the advances so far have involved engineering syntheticconstructs aimed at bacterial gene regulation, but it is critical that syntheticbiology tools also be designed for use in mammalian systems. One such toolthat shows promise as a ‘‘device’’ for achieving synthetic gene regulation isa class of molecules called allosteric ribozymes, which bind small moleculesand in response undergo self-cleavage resulting in modulation of gene expres-sion. This project investigates whether mammalian gene expression can becontrolled via allosteric ribozymes, namely, the Twister ribozyme.Results from dual luciferase assays indicate that the wild type Twister ribo-zyme undergoes self-cleavage in mammalian HEK293 cells, resulting in thedown-regulation of luciferase gene expression. The exceptional self-cleavage of Twister in vivo led us to investigate a variety of synthesizedTwister ribozymes in vitro. We designed Twister ribozyme constructs withthe aim of making RNA self-cleavage dependent upon the small moleculetheophylline, which has a well-characterized aptamer that makes it suitablefor our purposes. In vitro selection (SELEX) is being used to identify allo-steric Twister ribozymes that are only active in the presence of theophylline.Preliminary results have identified allosteric Twister ribozyme sequences withminimal preference for cleavage in the presence of theophylline. Continuedstudies are ongoing. The results of this project may provide a foundationfor the future use of allosteric ribozymes in modular systems to controlmammalian gene expression.

2144-Pos Board B160Understanding an RNA Helix-junction-Helix Construct by SAXS Refine-ment of MD ModelsYen-Lin Chen1, Tongsik Lee2, Lois Pollack1, Ron Elber2.1Applied & Engineering Physics, Cornell University, Ithaca, NY, USA,2Chemistry, The University of Texas at Austin, Austin, TX, USA.The varied biological functions of many ribonucleic acids (RNAs) are closelyconnected with their secondary and tertiary structures. Non-helical components

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of RNA molecules, such as kinks, junctions or bulges, are intrinsically flexibleand may not adopt a single conformation in solution. Thus, it is of significantinterest to visualize the structures populated by these molecules to determinehow locally flexible components can impact global shape. We apply smallangle X-ray scattering (SAXS) to measure the conformations of amodel RNA Helix-Junction-Helix (HJH) construct, as a function of salt. Weapply an ensemble optimization method (EOM) to refine possible modelsgenerated by all-atom molecular dynamics simulations (MD). We comparethe MD and EOM ensembles to correlate the HJH global and local geometrieswith conclusions of previous smFRET studies (Biophys J. 2015 Dec 15;109(12):2644-53).

2145-Pos Board B161Time-Resolved SAXS and Ensemble Modelling Reveal MagnesiumOrchestration across an RNA Folding LandscapeAlex Plumridge1, Andrea M. Katz1, George D. Calvey1, Ron Elber2,Serdal Kirmizialtin3, Lois Pollack1.1Cornell University, Ithaca, NY, USA, 2University of Texas at Austin,Austin, TX, USA, 3New York University Abu Dhabi, Abu Dhabi, UnitedArab Emirates.RNA’s unique folds and motions enable many of its newly discovered func-tions, yet RNA self-assembly and conformational dynamics are not well under-stood. Here, we use microfluidic mixing to trigger the Mg induced folding oftP5abc; a representative of the common RNA motif the three helix junction.Time-dependent conformations are measured using time-resolved small anglex-ray scattering (SAXS), and analyzed by pairing ensemble decompositionwith all-atom simulations. Detailed structural ensembles, derived at crucialstages of folding, reveal distinct phases of tP5abcs journey to the native state.Many of these phases rely on the presence of Mg ions, and underscore theirunique and varying role(s) in guiding RNA through a complex and ruggedlandscape.

2146-Pos Board B162Molecular Dynamics Simulations of a Riboswitch Binding AminoglycosideAntibioticsMarta Kulik1,2, Takaharu Mori1, Yuji Sugita1, Joanna Trylska2.1Riken, Wako, Japan, 2University of Warsaw, Warsaw, Poland.Riboswitches are regulatory elements in non-coding regions of mRNAs. Bybinding small ligands, they control the expression of the genes located down-stream of the aptamer. The N1 synthetic riboswitch binds aminoglycoside an-tibiotics from the neomycin family. It consists of a 27-nucleotide-long hairpinwith two flexible regions: a bulge and an apical loop. In N1 riboswitch,different aminoglycosides evoke a varying response: ribostamycin andneomycin inhibit the expression of genes in yeast and paromomycin is inactive,even though the two latter ligands differ by only one functional group in ring I.The NMR structures of the ribostamycin and paromomycin complexes with N1are available. However, the structures of the N1 riboswitch with neomycin andwithout ligands are not known.To explain how such a small difference between neomycin and paromomy-cin affects the interactions and dynamics of the N1 riboswitch, we per-formed all-atom molecular dynamics simulations. Replica exchangemethod helped increase the sampling. The non-Watson-Crick hydrogenbonds differ between the riboswitch with and without ligands even thoughthe secondary structure is similar. Aminoglycosides active in yeast(neomycin and ribostamycin) display significant stabilization of the apicalloop nucleobase A17 upon binding with the riboswitch. In paromomycincomplex, the A17 base is only partially stabilized and creates a frequent,but unstable hydrogen bonding with a terminal hydroxyl group in ring Iof paromomycin. Surprisingly, this hydrogen bond was not captured inrecent NMR experiments. The N1 riboswitch shares several similar patternswith another aminoglycoside binding site in ribosome called A-site. How-ever, A-site does not discriminate between paromomycin and neomycin.Thus, we compare those two aminoglycoside binding sites from the pointof view of their sequence, hydrogen bond network, and sodium ionconcentration.

2147-Pos Board B163Mimicking Co-Transcriptional RNA Folding using a SuperhelicaseBoyang Hua, Subrata Panja, Sarah Woodson, Taekjip Ha.Johns Hopkins, Baltimore, MD, USA.Accurate folding is essential for many RNA functions. The vast majority ofRNA folding studies in the literature, with some important exceptions, havebeen performed with fully transcribed or synthesized RNA molecules. Yet,as a linear polymer synthesized from 5’-to-3’ direction, RNA begins foldingduring transcription. Co-transcriptional folding of a riboswitch, for

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example, allows alternative conformation formation that is otherwise inac-cessible in thermal folding. Here, we developed a superhelicase-based sin-gle-molecule vectorial folding assay to mimic co-transcriptional RNAfolding in vitro. In this assay, a fluorescently labeled RNA is synthesizedand annealed to a complementary DNA strand and thus cannot fold untila helicase releases the RNA strand by unwinding the heteroduplex. In orderto mimic the 5’-to-3’ vectorial synthesis of RNA, we used an engineeredbacterial helicase Rep-X that translocates on and unwinds DNA in the 3’-to-5’ direction. Rep-X was made by chemically stabilizing theunwinding-active conformation. Therefore, a momoner Rep-X unwindsthousands of base pairs of DNA processively. Fluorescent labels on theRNA enabled us to study real-time conformational dynamics throughsingle-molecule fluorescence resonance energy transfer. Using the assay,we observed vectorial folding of the Twister ribozyme. After Rep-X startsunwinding by adding ATP, about 35% of molecules showed tertiary struc-ture dynamics with the same kinetic parameters as the pre-synthesized mol-ecules, validating our approach. We observed distinct signatures ofsecondary and tertiary structure formation, which is not possible using con-ventional folding analysis. We found that the first time the Twister ribo-zyme forms a tertiary structure, it often folds into a misfolded structurewhich subsequently transitions to the native fold. Refolding of fully synthe-sized RNAs did not show this behavior. Superhelicase-based vectorial RNAfolding can be applied to studying other kinetically controlled processessuch as riboswitch folding and RNA-protein assembly.

2148-Pos Board B164High Temporal- and Spatial-Resolution Studies of a Helix-to-Coil Transi-tion that Controls the Switching Mechanism of a RiboswitchJason Hon1, Nathan S. Daly1, Scott M. Trocchia2, Colin Nuckolls1,Kenneth L. Shepard2, Ruben L. Gonzalez Jr1.1Department of Chemistry, Columbia University, New York, NY, USA,2Department of Electrical Engineering, Columbia University, New York,NY, USA.Riboswitches are structural elements that are found in the 5’ untranslatedregions of many messenger RNAs (mRNAs) and that undergo ligand-dependent structural rearrangements that regulate transcription, splicing,translation, and/or stability of the corresponding mRNA. Because theyare typically found in bacteria, and because the majority of bacterial ribos-witches lack counterparts in archaea and eukaryotes, riboswitches remainpromising antibacterial drug targets. Architecturally, riboswitches arecomposed of a ‘switching sequence’ that overlaps with an upstreamligand-binding ‘aptamer domain’ and a downstream ‘expression platform’.Ligand-dependent genetic control is achieved when binding of the ligand tothe aptamer domain modulates the base-pairing interactions of the switch-ing sequence such that the expression platform adopts a secondary structurethat triggers expression or repression of the mRNA. In the prototypicaladenine-responsive riboswitch that regulates transcription of the pbuEgene in Bacillus subtilis, the base-pairing interactions of the switchingsequence are modulated subsequent to transcription of the expression plat-form: in the absence of adenine, the switching sequence pairs with theexpression platform to form a transcription terminator hairpin, whereas,in the presence of adenine, the switching sequence is preventedfrom pairing with the expression platform, allowing transcription toproceed.Using a new and powerful single-molecule biophysical approachemploying single-molecule field effect transistors (smFETs), we investi-gated the dynamics of a helix-to-coil transition that controls the base-pairing interactions of the switching sequence of the adenine-responsivepbuE riboswitch at an unprecedented temporal resolution of 40 msec permeasurement and spatial resolution of a single base pair. Our studieshave allowed us to investigate how base-pairing- and adenine-binding inter-actions that are distal to the switching sequence collaborate to allostericallymodulate the dynamics of the helix-to-coil transition and, consequently,control transcription of the pbuE gene.

2149-Pos Board B165Kinetic Investigation of Natural Products and Extracts for PotentialRiboswitch LigandsHeidi Klem1, Juliane Strauss-Soukup2.1Creighton University, Omaha, NE, USA, 2Chemistry, Creighton University,Omaha, NE, USA.Riboswitches are elements within the noncoding regions of mRNAs thatdirectly bind to cellular metabolites and modulate gene expression. GlmS isa catalytic riboswitch, or ribozyme, found in gram-positive bacteria. This ribo-zyme regulates the expression of Glucosamine synthase, the enzyme respon-sible for production of Glucosamine-6-phosphate (GlcN6P). GlmS will

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initiate self-cleavage once it binds to its natural ligand, GlcN6P, via negativefeedback inhibition. Cleavage results in degradation of the RNA and downregulation of gene expression. This current project investigates a series of nat-ural products and extracts in search of novel riboswitch ligands. Kinetic ana-lyses are performed to identify potential activators and inhibitors of glmSribozyme self- cleavage. Multiple natural extracts have been observed tobind with glmS and produce results comparable to that of the GlcN6P binding.An inhibitory ligand has yet to be discovered. Future results of this project willdetermine which extracts should undergo additional characterization. The abil-ity to affectively target bacterial riboswitches offers hope to oppose antibioticresistance.

2150-Pos Board B166Microsecond Conformational Dynamics and Distinct Folding Mechanismsof PreQ1 Riboswitch Studied by Two-Dimensional Fluorescence LifetimeCorrelation SpectroscopyBidyut Sarkar1, Kunihiko Ishii1,2, Tahei Tahara1,2.1Molecular Spectroscopy Laboratory, RIKEN, Wako, Japan, 2Riken Centerfor Advanced Photonics, Riken, Wako, Japan.Riboswitches are small, gene-regulatory segments in the mRNA of lower or-ganisms, mostly bacteria. Specific metabolite-binding induces conformationalchanges in riboswitches, which alters gene expression at the transcription ortranslation level. Therefore, understanding the ligand recognition mechanismis crucial to utilize a riboswitch as an antibiotic drug target. In this work,weinvestigated the folding dynamics of the ligand-binding ‘aptamer’ domain ofa transcriptional preQ1 riboswitch (from B. subtilis) for elucidating the bindingmechanisms with its cognate ligand (preQ1) and cofactor (Mg2þ). By perform-ing two-dimensional fluorescence lifetime correlation spectroscopy1 (2DFLCS) on a FRET-pair labeled preQ1 aptamer, we obtained the followingresults:(1) We observed �100ms dynamics between the open (hairpin) and folded (H-type pseudoknot) states in the apo-form. Similar sub-ms dynamics wasobserved even after addition of Mg2þ or preQ1.(2) Binding of preQ1 or Mg2þ shifts the equilibrium towards the folded state.However, their binding mechanisms are different: ‘conformational selection’for Mg2þ and ‘induced fit’ for preQ1.(3) Only a small population of molecules (�10%) undergoes this fast �100msdynamics, while others may exhibit slower dynamics (>10ms, as reported pre-viously2). We examined the validity of the reaction scheme by kinetic Monte-Carlo simulation.Considering the bacterial transcription rate (40-80 nt/s), the slower dynamicscan govern the transcription thermodynamically. In addition, the ms-dynamics provides finer regulation through kinetic control. 2D FLCS enabledus to identify this novel ms-dynamics in the preQ1 aptamer. It also helpedus distinguish the two binding modes for a system undergoing sub-ms dy-namics, to the best of our knowledge, for first time in a single moleculemeasurement.1Ishii, K.; Tahara, T. J. Phys. Chem. B 2013, 117, 11414.2Sudala, K. C. et al. J. Am. Chem. Soc. 2015, 137, 14075.

2151-Pos Board B167Analyzing the Monomeric Conformation of the HIV-1 5 Prime-LeaderRNA using Nuclear Magnetic Resonance SpectroscopyMatthew R. Orellana, Joshua Brown, Michael F. Summers.Chemistry and Biochemistry, University of Maryland, Baltimore County,Baltimore, MD, USA.The human immunodeficiency virus-1 (HIV-1) infects millions of individ-uals around the globe and is currently treated with a medication cocktail,where each drug targets a specific viral protein. The high rate of mutationof these non-conserved proteins leads the frequent emergence of resistantstrains. The 50 Leader (50-L) of the HIV-1 RNA genome is a highlyconserved region that is constantly in equilibrium between a monomericand dimeric conformation, which has not been exploited as a therapeutictarget. While the dimeric conformation of the HIV-1 RNA has been studied,there is not much structural studies of the monomeric conformation. In themonomeric conformation, the RNA is translated into proteins necessary forvirus replication while the dimeric conformation is packaged as thegenomic material for the new virion. We have used nuclear magnetic reso-nance spectroscopy to extensively probe the secondary structure of themonomeric conformation with the addition of the native guanosine cap.Making NMR assignments on large RNAs can prove to be challengingdue to numerous signal overlap. In order to confidently assign signalsfrom the 50-L, we have constructed various fragment controls and usednucleotide-specific labeling schemes to collect data that can then be over-lapped with the full length 50-L. Some structural findings we have made

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include data that suggest that the U5:DIS region is in an extended form,where it was previously thought to have been a pseudo-knot; as well asthe polyA region in the monomeric conformation being unstructured, whichwould support the translation of the RNA. We intend to study the mono-meric conformation of the HIV-1 RNA in order to determine its secondarystructure and ultimately its 3-D structure with the native cap.

2152-Pos Board B168Determining the Structure of the HIV-1 5 Prime Leader Dimeric Confor-mationTatiana Rodriguez, Joshua Brown, Michael Summers.University of Maryland Baltimore County, Baltimore, MD, USA.The structure of the 50-Leader (50-L) of the human immunodeficiency virus(HIV-1) viral RNA genome is under intense study, due to its role in deter-mining the fate of the RNA. Within the host cell, the 50-L is in equilibriumbetween a monomeric and dimeric conformation. The monomer is translatedinto the Gag-polyprotein, while the dimer is packaged as the new genomicmaterial for virions. Although, a three-dimensional structure of a truncatedcore dimer 50-L has been solved, the overall conformation of the full-lengthcapped 50-L has yet to be determined due to the size limitation. Using nuclearmagnetic resonance (NMR) spectroscopy, the two-dimensional structure ofmultiple regions of the dimer have been solved, however there has been dif-ficulty assigning regions near the native cap residue. In order to overcome thisobstacle, we use (NMR) spectroscopy techniques including nucleotide-specific deuteration and oligo control overlap. The results for these methodssuggests that a novel end-to-end stacking conformation is formed by adjacenttwo hairpins over forty nucleotides in length each. This interaction sequestersthe cap residue needed for translation initiation and thus attenuating functionsof the monomer.

2153-Pos Board B169RNA End Fluctuations out of a Virus: A Parasite’s Fishing ROD for Maxi-mizing AmplificationRichard Sportsman, Liya Oster, Benjamin Kartub, Charles M. Knobler,William M. Gelbart.Chemistry and Biochemistry, UCLA, Los Angeles, CA, USA.Viruses are some of the simplest known biological entities and yet theirparasitic life cycles offer many intriguing elements for study into how com-plex biological systems work. A current challenge in biology is to deter-mine how the dynamics of a system can be deduced, and virusdisassembly certainly has many challenging dynamics to capture to fullyunderstand its developmental process. There are two main disparate virusgenome release strategies. Most double stranded DNA viruses use apressure-based mechanism to release the genome in a spontaneous fashion,but the process of packaging the DNA into the tight confines of a preformedicosahedral capsid shell requires immense cellular energy and powerful mo-lecular motors. Single-stranded RNA viruses, however, must have workdone on them to release their genome, while in contrast to double-stranded DNA viruses, the process of forming single-stranded RNA viruses,via co-self-assembly of its coat protein and RNA genome, is spontaneous.How does a stable self-assembled virus particle release its genome to itshost to prime the process of viral replication? Our experiments show thatRNA from inside virus like particles formed in vitro can make its 5’ endof the RNA available to probes that cannot enter the virus capsid. Our prob-ing data lends to the hypothesis that RNA viruses like brome mosaic virusmay minimally release only an end of its genome to engage the ribosomalmachinery of its host cell while the rest of the genome remains protectedinside the intact virus capsid. In addition, we posit that the ribosomal ma-chinery is responsible for pulling RNA out of the intact capsid, and usesingle-molecule methods to measure the force necessary to pull RNA outof an intact capsid.

2154-Pos Board B170Benchmarking RNA Force Fields using Hairpin Loop Folding Free EnergyChangeLouis G. Smith, Zhen Tan, Aleksandar Spasic, Alan Grossfield,David H. Mathews.Biochemistry & Biophysics, URMC, Rochester, NY, USA.Calculating experimentally-observable quantities from molecular dynamicssimulation—though challenging—benchmarks simulation performance. ForRNA, UV optical melting experiments can be used to quantify the structuralstability for the folding of small oligonucleotides. We would like to estimatethe folding stability of RNA to benchmark force fields and also to identifyRNA sequences with unexpected stability to study experimentally. We useumbrella sampling molecular simulations of three dodecamer hairpins, for

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which there are experimentally determined free energies of unfolding, to es-timate the free energy change of stretching along the end-to-end (5’ OH to3’ hydroxyl oxygen) distance. We estimate the free energy change of thetransition from the native conformation to a fully extended conformationwith no hydrogen bonds between non-neighboring bases. The fully extendedconformation begins at 45 A for each dodecamer. Each simulation wasperformed four times using the standard AMBER ff10 force field and eachwindow, spaced at 1A intervals, was sampled for one microsecond, resultingin 552 ms of total sampling. We analyzed error along the estimated free energycurves (FEC) with the variance in the derivative of the FECs, a novelapproach that indicates which windows vary in free energy. We compareddifferences in the simulated free energy estimates to analogous differencesin experimental values using thermodynamic cycles. This avoids the problemof sampling the random coil, which provides little sequence specificinformation but is difficult to sample thoroughly. The residuals between dif-ferences in free energy changes estimated by simulation and those measuredexperimentally are 1.5 5 0.6, 0.5 5 0.6, and 1.0 5 0.6 kcal/mol. Thissuggests that further improvements are necessary for the AMBER force field,but that the force field may be accurate enough to guide the choice ofexperiments.

2155-Pos Board B171Enhanced Sampling of LNCRNA Conformational Space for Defining En-sembles of Structures Used in Ensemble Docking and Virtual Screening ofRNA-Focused Small MoleculesMichael Yonkunas, Nathan Baird.Chemistry and Biochemistry, University of the Sciences, Philadelphia,PA, USA.RNA remains an underexplored class of drug targets owing to the limitednumber of RNA-focused experimental and computational screening assays.Importantly, the highly charged phosphate backbone and inherent flexibilityunique to RNA macromolecules present computational challenges to virtualscreening techniques involving conformational sampling and molecular dock-ing. We have developed a computational methodology to examine docking ofsmall molecules to an ensemble of RNA conformations. A pipeline is devel-oped to target a critical triple helix in the oncogenic MALAT1, a long non-coding RNA that promotes metastasis in a large number of cancer types.We use a conformational FRET assay to show that the MALAT1 triple helix(M1TH) samples a large conformational space consisting of both native statesat physiological conditions and inhibitory states when solutions conditions arealtered. Preliminary Small Angle X-ray Scattering (SAXS) experiments showa change in radius of gyration (Rg) consistent with conformational FRETdata. Replica Exchange Molecular Dynamics are being used to calculatetwo ensemble sets (i) native-like conformations and (ii) inhibitory conforma-tions) of M1TH structures. Structural states are clustered based on Rgconsistent with SAXS data. The relatively small size of the 76 nucleotideM1TH simulation structure allows rigorous sampling of conformational space.We hypothesize that small molecule induced disruption of this RNA triplehelix is a novel approach for therapeutic intervention to inhibit progressionof multiple cancer types. M1TH ensembles will be used in ensemble dockingof compounds identified through a shape-based virtual screening methodologyto identify novel M1TH-specific lead compounds that reduce cancerproliferation.

2156-Pos Board B172Characterizing the Folding and Misfolding of the Aquifex aeolicusTMRNA Frameshifting Pseudoknot via Massively Parallel MolecularDynamics SimulationsXavier Martinez.Computer Engineering and Computer Science, CSULB, Long Beach, CA,USA.Massive all-atom molecular dynamics simulations were conducted across theFolding@Home distributed computing network to characterize the folding, un-folding, and misfolding of the Aquifex aeolicus tmRNA ribosomal frameshift-ing pseudoknot PK1, as well as to assess the conformational plasticity inherentto this structural motif. With robust sampling spanning the spectrum from theextended unfolded state to the native fold, our simulations yielded over 100 mi-croseconds of cumulative sampling time. Herein, we quantitatively assess thenative and non-native structural content of each simulated conformation andemploy a novel clustering algorithm to identify the thermodynamic stateswithin this data set and the mechanical and kinetic aspects of folding and mis-folding, the latter of which has only recently been studied via all-atom simula-tion. While in line with previous thermodynamics-based models of a generalRNA folding mechanism, our observations indicate that stem-strand-sequence-separation may serve as an alternative predictor of the order of

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stem formation during pseudoknot folding. Our results contradict a modelof frameshifting based on structural rigidity and resistance to mechanicalunfolding, and instead strongly support more recent studies in whichconformational plasticity is identified as a determining factor in frameshiftingefficiency.XM acknowledges support from the RISE program at CSULB (NIH2R25GM071638-09A1).

2157-Pos Board B173Meso-Scale Modeling for Predicting Properties of RNA ComplexesEckart Bindewald1, Mathias Viard1, Bruce A. Shapiro2.1Basic Science Program, Leidos Biomedical Research, Frederick, MD, USA,2RNA Biology Laboratory, National Cancer Institute, Frederick, MD, USA.The rational design of multi-stranded nucleic acid nanostructures is a chal-lenging task. We present a pipeline of computational approaches that aid inthat process. First, an approach for the computational prediction of the basepairing (secondary structure) of several potentially interacting RNA andDNA strands (including RNA/DNA hybrids) is presented. We show thatthis algorithm is uniquely suited to predict the base pairing as well as meltingtemperatures of nucleic acid rings, cube-like structures and RNA switches.We present computational and experimental results of a novel two-strandedRNA switch that was designed using this approach; this switch is shownto be responsive to the presence of the mRNA of tumor suppressorE-cadherin and may be used for the conditional down-regulation of a targetgene via RNA silencing.Next, a new approach is presented, that can quicklygenerate course-grained 3D structures from a given multi-stranded secondarystructure. This approach is based on a bead-model representation that utilizesdistance constraints. It is shown that this approach can, starting from anunfolded structure, generate topologically complicated RNA complex struc-tures. An example for such RNA nanostructures is a designed 6-strandedRNA complex that folds into a cube-like conformation (RNA cube). Further-more, it is shown that this computational approach successfully and quicklydistinguishes - based on 3D structures predicted by the model using onlysequence and base pairing information - experimentally verified variants ofRNA cube structures (containing single-stranded loop linker regions ofdifferent lengths) from variants that cannot assemble into a cube-like struc-ture. Taken together it is concluded that presented computational modelscan aid in the prediction of a variety of important properties of interacting nu-cleic acid strands.Funded in part under contract HHSN261200800001E.

2158-Pos Board B174In-Silico, In-Vitro, and In-Vivo Studies of Sirna Delivery using CationicBolaamphiphile VesiclesTaejin Kim1, Kirill Afonin2, Mathias Viard1, Eliahu Heldman3,Bruce Shapiro1.1National Cancer Institute, Frederick, MD, USA, 2University of NorthCarolina at Charlotte, Charlotte, NC, USA, 3Ben-Gurion University of theNegev, Beer Sheva, Israel.Utilizing small interfering RNA (siRNA) for gene therapy has been widelystudied based on its gene silencing capability. However, unmodified nakedsiRNA delivery to target cells is challenging because of short half-lives inblood serum and difficulty in uptake due to its negatively charged surface.To bypass these biological barriers in siRNA delivery, we investigated theuse of cationic bolaamphiphile (bola) vesicles, GLH-19, GLH-20, and themixture of two, GLH-19/GLH-20. GLH-19 has an acetylcholine head groupwhile GLH-20 has an acetylcholine ester head group which can be hydrolyzedby choline esterase (ChE) after it penetrates the brain blood barrier (BBB).Using in-silico, in-vitro, and in-vivo studies, we characterized the deliveryof siRNA/bola complexes in terms of stability, protection, transfection, andgene silencing. From our in-vitro experiments and atomic level in silicostudies, we found that the GLH-19 vesicles have not only higher stabilitybut also better protection and higher transfection efficiency than the GLH-20 vesicles. On the other hand, both of the GLH-19/siRNA and GLH-20/siRNA complexes showed similar efficiency of eGFP silencing in MDA-MB-231 cells. We also investigated the in-vivo distribution of siRNA/GLH-19 and siRNA/GLH-19/GLH-20 complexes in the liver, tumor, and brain.We found that the siRNA/GLH-19 complexes were delivered into tumorswhile no major accumulation was observed in the liver. We also found thatsiRNA/GLH-19/GLH-20 complexes were efficiently delivered into the brain.Therefore, our in-silico, in-vitro, and in-vivo studies showed that both siRNA/GLH-19 and siRNA/GLH-20 complexes can be used for siRNA delivery withdifferent characteristics. When the two bolas are mixed together, GLH-19 pro-vides enhanced stability, protection, and transfection, while GLH-20 provides

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efficient BBB penetration and siRNA release due to the hydrolyzation ofGLH-20 head group by ChE.

2159-Pos Board B175An Effective Scoring Function for RNA-RNA Interactions Derived with aDouble-Iterative MethodYumeng Yan, Zeyu Wen, Di Zhang, Jiahua He, Shengyou Huang.School of Physics, Huazhong University of Science and Technology, Wuhan,China.RNA-RNA interactions play fundamental roles in gene and cell regulation.Therefore, accurate computation of RNA-RNA interactions is critical todetermine the complex structures and understand the molecular mechanismof the interactions. Here, we have developed a statistical mechanics-baseddouble-iterative strategy to determine the effective potentials for RNA-RNAinteractions based on a training set of 97 diverse RNA-RNA complexes.The double-iterative strategy circumvented the reference state problem inknowledge-based scoring functions by updating the potentials through itera-tion and also solved the decoys-dependent limitation in normal iterativemethod by generating the decoys iteratively. The derived scoring function,which is referred to as DIScore/RR, was evaluated on an independent testset of 60 diverse RNA-RNA complex structures from our RNA-RNA dockingbenchmark (http://huanglab.phys.hust.edu.cn/RRDbenchmark/) and comparedwith three other scoring methods. It was shown that for bound docking, ourscoring function DIScore/RR obtained an excellent success rate of 90% and98.3% in binding mode predictions when the top 1 and 10 predictions wereconsidered, compared to 63.3% and 71.7% for van der Waals interactions,45.0% and 65.0% for our ITScorePP, and 11.7% and 26.7% for ZDOCK2.1, respectively. For unbound docking, DIScore/RR also achieved a high suc-cess rate of 53.3% and 71.7% in binding mode predictions when the top 1 and10 predictions were considered, compared to 13.3% and 28.3% for van derWaals interactions, 11.7% and 26.7% for our ITScorePP, and 3.3% and6.7% for ZDOCK 2.1, respectively. The present scoring function is beneficialfor the prediction and design of both RNA structures and RNA-RNAcomplexes.

2160-Pos Board B176RNA Structure Prediction Guided by Coevolutionary InformationMehari Bayou Zerihun1,2, Alexander Schug2,3.1Physics Department, Karlsruhe Institute of Technology, Karlsruhe,Germany, 2Steinbuch Centre for Computing, Karlsruhe Institute ofTechnology, Karlsruhe, Germany, 3John von Neumann Institute forComputing, J€ulich Supercomputer Centre, J€ulich, Germany.RNAs play a significant role in cellular activities such as coding, decoding,regulation and expression of genes. Understanding their three-dimensionalstructure is crucial for understanding their function. However, the vastmajority of sequenced RNAs lack corresponding structural information. Dueto the extremely flexible nature of these molecules, experimental techniquesto determine their structure are challenging. One complementary theoreticalapproach is to extract information about spatially adjacent residues from se-quences of related RNAs using statistical analysis. Direct coupling analysis(DCA) has revolutionized the field of protein structure prediction andrecently started to be applied for RNAs. Here, we use DCA in the specificsof RNAs to infer pairs of spatially adjacent residues from multiple sequencealignment. Inferred residue pairs that show strong coevolution and that are notinvolved in the two-dimensional structure, are putative contacts within thetertiary structure of the biomolecule. This a priori information about spatialadjacency is integrated with molecular modeling tools for RNA folding.A number of folded decoys are generated and ranked according to score.The accuracy of the predicted structures is evaluated by computing the rootmean square deviation (RMSD) for RNAs whose structures are alreadyknown.

2161-Pos Board B177Helix-Based RNA Two-Dimensional Structure PredictionFengfei Wang1, Xiaojun Xu2.1School of Mathematics and Physics, Jiangsu University of Technology,Changzhou, Jiangsu 213001, China, 2Institute of Bioinformatics and MedicalEngineer, Jiangsu University of Technology, Changzhou, Jiangsu 213001,China.The structure of an RNA is determined by the complex pattern of base-baseinteractions, including the base-paired secondary structures and tertiary con-tacts (crossing base pairs). Stabilized by the canonical base pairing and stack-ing interactions, RNA helical regions contribute to the major part of the freeenergy of an RNA system, and play an essential role on the RNA global

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conformation, dynamics, kinetics, and adaptation. In this study, we use thecombination of helices and the dynamic programming algorithm to selectivelysample RNA two-dimensional (2D) structures. By calculating the free en-ergies of each sampled structures with the Vfold-derived loop entropic param-eters for secondary and pseudoknotted structures, and an empirical energymodel for kissing motifs, we predict most stable and metastable structuresfrom sequences. We use the stability of helices to balance the computationalefficiency and prediction accuracy. Benchmark test shows that the model im-proves the prediction of RNA 2D structures with and without crossing basepairs.

2162-Pos Board B178Martini Coarse-Grained Force Field for RNAJaakko J. Uusitalo, Helgi I. Ingolfsson, Siewert J. Marrink, Ignacio Faustino.Groningen Biomolecular Sciences and Biotechnology Institute and ZernikeInstitute for Advanced Materials, University of Groningen, Groningen,Netherlands.Given the central role of RNA molecules in cell biology, there is significant in-terest in studying the structural and dynamic behavior of RNA in relation toother biomolecules. Coarse-grain molecular dynamics simulations are a keytool to that end. Here, we have extended the coarse-grain Martini force fieldto include RNA after our recent extension to DNA. In the same way DNAwas modeled, the tertiary structure of RNA is constrained using an elasticnetwork. This model, therefore, is not designed for applications involvingRNA folding but rather offers a stable RNA structure for studying RNA inter-actions with other (bio)molecules. The RNA model is compatible with all otherMartini models and opens the way to large-scale explicit-solvent molecular dy-namics simulations of complex systems involving RNA. This opens upnumerous possibilities for CG simulations. The ease of backmapping the Mar-tini model to atomistic resolution furthermore allows its use in various hybridmultiscale setups.

2163-Pos Board B179Automated Force-Field Parametrization Guided by MultisystemEnsemble AveragesAndrea Cesari1, Sandro Bottaro2, Giovanni Bussi1.1Statistical and Molecular Biophysics, SISSA, Trieste, Italy, 2BiomolecularSciences, University of Copenhagen, Copenhagen, Denmark.RNA structure and dynamics play a fundamental role in many cellular pro-cesses such as gene expression inhibition, splicing and catalysis. Molecular dy-namics is a computational tool that can be used to investigate RNA structureand dynamics at atomistic resolution. However, its capability to predict andexplain experimental data is limited by the accuracy of the employed potentialenergy functions, also known as force fields. Recent works have shown thatstate-of-the-art force fields could predict unphysical RNA conformations thatare not in agreement with experiments. The emerging strategy to overcomethese limitations is to complement molecular dynamics with experimentaldata included as restraints. In a recent work, we suggested a maximum-entropy based method to enforce solution experiments in molecular dynamicssimulations. Importantly, this approach reduces the risk of overfitting by simul-taneously adapting the force-field corrections to multiple systems. We herepush this idea further and develop a general scheme to fit arbitrary force-field parameters given a set of ensemble averages. Such quantities can rangefrom NMR data, such as 3J couplings or NOE, to native state populations.The key feature of our method is the possibility to concurrently combineensemble averages from multiple systems into a unique error function to beminimized, drastically enhancing corrections’ transferability. The method isapplied to the difficult case of GAGA and UUCG tetraloops for which weare able to maximize their native state population by refining torsional poten-tials alone.

2164-Pos Board B180Topological Constraints and Their Conformational Entropic Penalties onRNA FoldsEthan N.H. Phan, Chi H. Mak.Chemistry, University of Southern California, Los Angeles, CA, USA.Functional RNAs can fold into intricate structures using a number of differentsecondary and tertiary structural motifs. Many factors contribute to the overallfree energy of the target fold. In this study we quantified the entropic costs fromthe loss of conformational freedom when the sugar-phosphate backbone is sub-ject to constraints imposed by secondary and tertiary contacts. Motivated by in-sights from topology theory, we design a diagrammatic scheme to representdifferent types of RNA structures so that constraints associated with a folded

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structure may be segregated into mutually independent subsets, enabling the to-tal conformational entropy loss to be easily calculated as a sum of independentterms. We used high-throughput Monte Carlo simulations to simulate large en-sembles of single-stranded RNA sequences in solution, in order to validate thebasic assumptions behind the diagrammatic scheme, examining the entropiccosts for hairpin initiation and many multiway junctions. Our diagrammaticrepresentation aides in the factorization of secondary/tertiary constraints intodistinct topological classes and facilitates the discovery of interaction amongstmultiple constraints on new RNA folds. This novel perspective leads to usefulinsights into the inner workings of some riboswitch RNA sequences, demon-strating how they might operate by transforming the structure among differenttopological classes.

2165-Pos Board B181Exaggerated Swivel Motions of the Small Subunit Head Domain areRequired for tRNA Translocation through the Bacterial RibosomeWataru Nishima1,2, Scott C. Blanchard3, Karissa Y. Sanbonmatsu1,2.1Los Alamos National Laboratory, Los Alamos, NM, USA, 2New MexicoConsortium, Los Alamos, NM, USA, 3Weill Cornell Medical College, NewYork, NY, USA.Translocation of tRNA through the ribosome is essential for protein synthesisand for maintaining the open reading frame of mRNA translation. Transloca-tion is associated with two large-scale rearrangements within the ribosome:an intersubunit rotation of the small subunit relative to the large subunit (rota-tion); and an intrasubunit rotation of small subunit head relative to its body(head swivel). Recent single-molecule FRET studies (smFRET) suggestedthat exaggerated head swivel motions accompany the translocation reaction co-ordinate. Here, we integrate molecular dynamics simulations of the 70S ribo-some translocation complex with single molecule FRET studies to morefully characterize the conformational changes in the ribosome accompanyingtranslocation, and the transit of key intermediates that have recently been iden-tified by structural and smFRET investigations. By explicitly incorporatingFRET labels into the simulations, we obtain configurations of the ribosomehighly consistent with measured FRET efficiencies for each translocation inter-mediate. We find that a larger amplitude head swivel configuration (‘superswivel’) satisfies the constraints of FRET efficiency and conformationalchanges predicted to accompany translocation. In so doing, our study supportsa simplified translocation pathway consistent with extant structural data and theorder of events during and the geometric constraints predicted by recentsmFRET initiatives. Our simulations also characterize the influence of dye flex-ibility on the smFRET distance estimates.

2166-Pos Board B182A Multi-color Riboswitch-Based Platform for Imaging of MRNA andSmall Non-coding RNA in Live Mammalian CellsEsther Braselmann1, Aleksandra Wierzba2, Jacob T. Polaski3,Miko1aj Chromi�nski2, Dilara Batan1, Dorota Gryko2, Robert T. Batey3,Amy Palmer1.1BioFrontiers Institute, Boulder, CO, USA, 2Institute of Organic Chemistry,Polish Academy of Sciences, Warzaw, Poland, 3Department of Chemistryand Biochemistry, University of Colorado Boulder, Boulder, CO, USA.Spatiotemporal dynamics of coding and non-coding RNAs play central rolesfor many cellular processes in mammalian and bacterial cells. For example,mRNAs are transiently sequestered in granules upon different stressors inmammalian cells. Currently, visualization of RNA in cells is largely limitedto methods that require fixing and staining of cells, with the notable exceptionof the recently developed ‘‘Spinach/Broccoli’’ and ‘‘Mango’’ aptamers, and theMS2-GFP system.We developed a complementary platform to visualize RNA live in mamma-lian cells that combines small size, robust applicability and multi-color fluo-rescent tags. A small RNA aptamer is attached as a fusion to an RNA ofinterest and addition of an organic probe induces fluorescence. This platformis based on the bacterial cobalamin riboswitch where the riboswitch RNAfunctions as the RNA aptamer tag. We synthesized variants of cobalaminthat are covalently attached to a series of organic fluorophores. Cobalamin it-self functions as a fluorescence quencher and we show that the fluorescencesignal increases upon binding of the cobalamin-fluorophore probes to the ri-boswitch RNA. Importantly, the riboswitch binds cobalamin, but not the flu-orophore, allowing us to alter fluorophores easily without compromisingRNA-probe affinity.We have demonstrated that mRNA recruitment to stress granules can be visu-alized by the riboswitch-based RNA tag in live cells using two differentprobes with different colors. Furthermore, we have tagged U1 snRNA using

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our riboswitch tag and demonstrated recruitment to cytosolic granules (U-bodies) in live cells. Together, we have developed a small and versatileRNA-tag that can be attached to diverse RNAs of interest while only mini-mally disrupting function.

2167-Pos Board B183Subnanometer Cryo-EM Structure of T-Box and tRNA ComplexZhaoming Su.Bioengineering, Stanford, Stanford, CA, USA.Single particle electron cryo-microscopy (cryo-EM) of RNA alone has not beenstudied at high resolution as in the case of RNA-protein complexes, because ofthe intrinsic conformational variability that presents great challenges in particleclassification for high resolution structure determination. T-box riboswitchesinteract with specific tRNAs and sense the corresponding amino acid levelsin Gram-positive bacteria. The complete structure of the T-box/tRNA (80kDa) complex remains unknown. We here report a subnanometer cryo-EMmap of this RNA complex, in which major and minor grooves are unambigu-ously resolved. This result differs from the two small angle X-ray scattering(SAXS) models reported recently, and illustrates the interaction interface be-tween tRNA and T-box riboswitch.

2168-Pos Board B184Probing Mechanical Properties of Biomolecules using NanoporesPrasad Bandarkar, Huan Yang, Robert Henley, Pradeep Waduge,Meni Wanunu, Paul C. Whitford.Northeastern University, Boston, MA, USA.Translocation of biomolecules through nanopores is a promising label-freesingle molecule technique to distinguish between molecules on the basis oftheir structural or dynamic properties. The confined nature of the nanopore af-fects the allowed conformations of the molecules, and in some cases, forcesthem to undergo large conformational changes to translocate through thenanopore. These effects are reflected in the observed current traces and thushelps us in measuring the flexibility of these biomolecules. Recently, weapplied molecular dynamics simulations using a structure-based model toobserve a correlation between the maximum RMSF of the protein and thewidth of the experimental current blockade distribution. This suggests thatprotein translocation can be utilized as a high-throughput method to distin-guish between functional conformers in proteins. Applying this technique totranslocation of tRNA offers an interesting challenge since the tRNA is ex-pected to undergo a conformational change due to the constricted size ofthe nanopore. To interpret the structural aspects of the conformation rear-rangements associated with nanopore translocation, we apply MD simulationsusing a simplified structure-based energetics model of tRNA. In our forcefield,interactions between the nanopore and the molecule represent the steric effectof the pore. Using energy landscape techniques, we have calculated the meanfirst passage time (MFPT) for crossing the rate-limiting free-energy barrier formultiple tRNA species. We find agreement between the MFPT values and theexperimental translocation times. Further, these calculations suggest that theexperiments specifically observe transient partial unfolding of tRNA. Theseresults provide a structural/energetic interpretation of current nanopore exper-iments, which can help guide the refinement and interpretation of single-molecule measurements, while advancing our understanding of biomoleculardynamics.

2169-Pos Board B185Ornithine Decarboxylase Antizyme Pseudoknot RNA Binding to SpermineRegulates Gene ExpressionJuliane Strauss-Soukup1,2, Jodi Monahan2, Katie Del Vecchio1,Molly McDevitt1, Zachariah Holmes1, Samantha Stoupa1, Garrett Soukup2.1Chemistry, Creighton University, Omaha, NE, USA, 2Biomedical Sciences,Creighton University, Omaha, NE, USA.Riboswitches are elements within messenger RNAs that directly bind tocellular metabolites and modulate gene expression through feedback regula-tion. Although riboswitches are widespread among bacteria, only one classfurther resides in fungi and plants, and no riboswitches have been found in an-imals. Here we propose the riboswitch function of a translational frame-shiftstimulatory pseudoknot RNA (pkRNA) that is highly conserved among verte-brate ornithine decarboxylase antizyme (OAZ) genes involved in the regulationof polyamine biosynthesis.Utilizing in-line probing and equilibrium dialysis, apparent binding affinity andspecificity for polyamines was determined. Mouse Oaz1-pkRNA binds to sper-mine with greater affinity than other polyamines. However, spermine binding toOaz1-pkRNA specifically elicits conformational change, a fundamental prop-

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erty of riboswitch RNAs. Closely related spermine analogs (with identical orgreater overall positive charge) have lesser affinity and specificity for theOaz1-pkRNA. Moreover, Oaz1-pkRNA specifically facilitates spermine-dependent reporter protein expression compared to another spermine-bindingframe-shifting pkRNA in identical context. Although spermine binding is notan exclusive property of Oaz1-pkRNA, results suggest that translationalframe-shifting in OAZ expression has evolved in context of the pkRNA forspermine-dependent regulation to avail of the binding event. Function of thepkRNA as a spermine sensor and mammalian riboswitch indicates a substan-tially broader distribution of riboswitches among eukaryotic organisms and of-fers a novel target for affecting a key metabolic process relevant to cancer andother diseases.

2170-Pos Board B186Selected Polycationic Surfactants as Sirna Carriers for Gene TherapyWeronika J. Andrzejewska1, Michalina Wilkowska1, Barbara Pepli�nska2,Maciej Kozak1.1Macromolecular Physics, Adam Mickiewicz University in Poznan, Poland,Poznan, Poland, 2NanoBioMedical Centre, Poznan, Poland.So far many drug delivery systems for transfer of nucleic acids in gene therapyhave been proposed but there is no universal system for transfer of genetic ma-terial into cells. The largest problem in the design of new nucleic acids deliveryvehicles is finding a carrier which fulfill such criteria as: stability in complexeswith nucleic acids, non-toxicity, non-immunogenicity, ability to safe degrada-tion and easy preparation protocol.A certain group of polycationic surfactants are able to meet these requirements.Their amphiphilic nature, structure flexibility and the ability to bind to siRNAor DNA oligomers are just some of the advantages. Our past research onselected dicationic compounds as DNA or siRNA delivery systems, indicatedthat an increase in the number of surfactant subunits is a promised directionto develop new efficient carriers of short nucleic acids.In this work we present results of the studies of the complexes based on selectednovel polycationic surfactants (eg. oligomeric imidazolium derivatives) with amodel system - double stranded 21-bp siRNA oligomer. By the use of biophys-ical methods (electrophoresis, circular dichroism, small angle scattering of syn-chtorton radiation and cryo-transmission electron microscopy) we examinedtheir complexation capacity, nanostructure of lipoplexes, conformationalchanges in bound siRNA oligomers and micromorphology of complexes.Our studies have shown the formation of stable systems with promising trans-fection properties. These systems exhibit various morphologies in frozen state.Acknowledgments: The study was supported by research grant ‘‘PREL-UDIUM’’ from National Science Center (Poland) - UMO-2016/23/N/ST4/01637.

2171-Pos Board B187Abiotic Fabrication of Sugar Phosphates and Ribonucleosides in WaterMicrodropletsInho Nam1, Jae Kyoo Lee1, Hong Gil Nam2, Richard N. Zare1.1Chemistry, Stanford University, Stanford, CA, USA, 2Institute for BasicScience, Daegu, Republic of Korea.As a ribonucleic acid (RNA) world hypothesis, RNA would exist on Earthbefore the evolution of deoxyribonucleic acid (DNA) and protein, becauseRNA has both genetic information and catalytic activity for biochemicalreactions in primitive cells. However, the feasibility of the prebiotic fabrica-tion of ribonucleosides which are the building blocks of RNA has been uncer-tain, because a condensation reaction between ribose and nucleobases isthermodynamically unfavorable in solution. Here, we discovered an abioticsynthetic path for ribonucleosides, both adenosine and cytidine in water mi-crodroplets, following a salvage reaction pathway in cells. The abiotic reac-tion path includes a condensation reaction between ribose and phosphate togive Rib-1-P and an exchange reaction between nucleobase and phosphatein the Rib-1-P. The reaction steps were driven by water microdroplets thatcan change the thermodynamic property of condensation reaction andacceler-ated by divalent magnesium ion, Mg2þ, as a catalyst. The yield for Rib-1-Pwas 6% and the values for adenosine and cytidine reached over 2% and0.5% under our conditions. At bulk, the condensation reaction for Rib-1-Phas the change of Gibb’s free energy, DG = þ5.4 kcal/mol. However, DGchanges to the value of �1.02 kcal/mol in microdroplets, which makes theabiotic salvage pathway spontaneous. As a consequence, both types of ribonu-cleosides, purine and pyrimidine, could have arisen through the same chemi-cal environment, which means that simultaneous production of randomribonuleosides, might be possible for generating chains of RNA in microdro-plets.

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Posters: Protein-Nucleic Acid Interactions III

2172-Pos Board B188Sensitized dsDNA-Peptide Complex and its Physicochemical PropertiesPawe1 Wityk, Janusz Rak.University of Gda�nsk, Gda�nsk, Poland.Nowadays, one of the leading causes of death is cancer and its common way oftreatment is photo- or radiotherapy. To increase the efficiency of these modal-ities one usually uses sensitizers that make cancer cell more sensitive to UV orionizing radiation. Modified nucleosides with high electron affinity (EA), proneto dissociative electron attachment are the examples of such compounds. Irra-diation of DNA modified with the radiosensitizing nucleosides with increasedEA leads to the production of radicals. These radicals are believed to inducefurther DNA damage after hydrogen abstraction from the sugar moiety. Whilethe first step of damage to the modified DNA - a nucleobase radical production -seems to be understood sufficiently well, the following degradation steps - i.e.hydrogen abstraction and further reactions resulting in a ‘‘fixed’’ damagerequire further studies. However, in order to test new radiosensitizers or under-stand the DNA degradation pathways models mimicking the native DNA envi-ronment are necessary. Indeed, in the cell nucleus DNA - a molecular target ofradio- or phototherapy - interacts with a number of proteins. Thus, access tospecific DNA-protein complexes, seem to be necessary to fully comprehendthe sensitization processes. Here, we will present how a complex of theBrdG/BrdA/BrdC or BrdU-labeled dsDNA with a covalently linked peptide(dsDNA-PEP) can mimic the sensitized DNA in its native environment andserve as a tool for the development of radio- or photosensitizers. LabeleddsDNA-PEP radiolytes and photolytes were fully characterized using theLC-MSmethod. Moreover, the electron affinities (AEA) and circular dichroism(CD) spectra of the labeled oligonucleotides were calculated and measured. Insummary, the obtained theoretical and experimental data allowed us to betterunderstand the processes involved in the photo- and radio-damage of DNAin a well-defined model system.

2173-Pos Board B189A General SAXS-Based Screening Protocol Validated in RNA-ProteinInteractionsPo-chia Chen1, Pawel Masiewicz1, Vladimir Rybin1, Dmitri Svergun2,Janosch Hennig1.1Structural and Computational Biology, EMBL Heidelberg, Heidelberg,Germany, 2EMBL Hamburg, Hamburg, Germany.RNA-protein interactions can be characterized by both sequence-specific andnon-specific interactions, due to the ubiquitous presence of charged contactsassociated with nucleic acid backbones. This multiplicity of binding modescomplicates screening efforts to identify strong-affinity RNA sequences (alsoknown as cognate motifs,) required to apply structural biology techniques. Inaddition, the cost of production of specific RNA sequences hinder current ef-forts using techniques such as ITC and NMR to validate motifs enrichedfrom affinity screens.We report a screening method based on small-angle X-ray scattering(SAXScreen) that summarises a ligand’s efficacy at eliciting signal changesas SD, a structural analogue to affinity KD that also captures receptor-ligandscattering and conformational changes. Since no structure-specific informationis required, the protocol can be generalised towards various biomolecular inter-actions and processes that are detectable via changes in scattering patterns. Uti-lising synchtron sources and automated sample-changer setups, we validatedthe approach by conducting sparse-search strategies covering �35 oligoneu-cleotide sequences around the UGUx motif of Sex-lethal protein, yieldingboth ligand ranking and differentiation between binding mechanisms that arefurther confirmed in ITC and NMR titrations.

2174-Pos Board B190Allosteric Control of Human CGAS Dimerization Underpins its Context-Dependent Response to Cytoplasmic DNARichard Hooy, Jungsan Sohn.Biophysics and Biophysical Chemistry, The Johns Hopkins UniversitySchool of Medicine, Baltimore, MD, USA.Cyclic GMP-AMP synthase (cGAS), a template-independent nucleotidyl trans-ferase, detects cytosolic double-stranded (ds)DNA and induces type 1 inter-ferons as part of the innate immune response to infection and cellular stress.Structural and biochemical studies have defined cGAS as a simple switchthat uses dsDNA as a regulator of its enzymatic activity. dsDNA inducescGAS dimerization and unlocks the substrate binding site allowing for catalysisand production of the unique secondary messenger, cyclic [2’-5’, 3’-5’] GMP-AMP (cGAMP). cGAMP triggers activation of the signaling adaptor, STING,

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which then propagates the signal. A hallmark of the cGAS pathway is that thecGAS sensor dynamically tunes the overall signaling output depending onvarious cellular contexts (e.g. dsDNA length, dsDNA concentration, cGASexpression level). Recent studies reported that dsDNA length dictates cGAS ac-tivity, and the ability of cGAS to form stable, ladder-like complexes on longerdsDNA is thought to underpin this phenomenon. Nevertheless, the currentmodel does not explain how cGAS dynamically responds to cytoplasmicdsDNA at the molecular level. Here, we investigate the allosteric coupling ofdsDNA binding and catalysis of human cGAS through rigorous thermodynamicand kinetic measurements. We use our data to derive a statistical thermody-namic model capable of explaining cGAS-dsDNA-length-dependent activityunder various contexts. Our data highlight key differences between mouseand human orthologues, enumerate rate constants and (apparent) binding affin-ities, and define thermodynamic linkage between signal recognition (dsDNAbinding) and signal transduction (enzymatic activity). Our model postulatesthat cGAS couples its intrinsic monomer-dimer equilibrium to dsDNA bindingand substrate binding, consequently resulting in dynamic, yet efficient re-sponses to cytoplasmic dsDNA.

2175-Pos Board B191Vizualizing the Mechanism of H-NS Gene RegulationKathy R. Chaurasiya, Ramon van der Valk, Bram Henneman,Remus T. Dame.Leiden University, Leiden, Netherlands.Bacterial genomes are organized into subdomains by nucleoid-associated pro-teins (NAP). The principal NAP in E. coli is H-NS, which is essential forstructural and functional organization of the genome and is a key transcriptionregulator that selectively silences genes involved in the bacterial response toenvironmental conditions. For example, H-NS regulates the ProU operon, atransport system that is upregulated under high osmolarity conditions.Although the mechanism remains unclear, the dsDNA bridging propertiesof H-NS suggest that it forms a loop between the upstream and downstreamregulatory elements (URE and DRE, respectively) of proU, which excludesRNAP promotor binding. In addition, recent ensemble biochemical experi-ments indicate that H-NS bridging induces RNAP pausing, backtracking,and Rho-dependent termination. Taken together, this suggests a model inwhich H-NS regulates proU by bridging the URE and DRE to inhibit tran-scription. In high salt conditions, H-NS dissociates, allowing RNAP to pro-ceed. Here, we test this model using single molecule fluorescence toquantify H-NS-mediated bridging of the URE and DRE in physiologicalsalt conditions.

2176-Pos Board B192G Quadruplex and Stem Interactions in RGG Box Domain RecognitionKendy A. Pellegrene, Mihaela-Rita Mihailescu, Jeffrey D. Evanseck.Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, USA.The role RNA G-quadruplexes (GQ) in various cellular processes has becomean important question in neurodegenerative diseases. Of specific interest is theC9ORF72 hexanucleotide repeat expansion (GGGGCC) associated withdevelopment of inherited amyotrophic lateral sclerosis (ALS) and frontotem-poral dementia (FTD). These repeats have been postulated to fold into GQstructures; however, little is known regarding GQ interactions with proteins.Previously, a synthetically derived stem-capped RNA GQ bound to the Frag-ile X Mental Retardation Protein (FMRP) (PDBID:5de5) arginine-glycine-glycine (RGG) box, crystal structure was reported. FMRP was shown tobind at the junction between the GQ and stem structures. In contrast,preliminary experimental evidence has brought into question the necessityfor the stem contribution for FMRP binding to the GQ. Utilizing moleculardocking, we probe the importance of the stem structure in the binding ofthe published crystal structure and FMRP. We will then apply this techniqueto our system of interest, the C9ORF72 hexanucleotide repeat expansion andRGG box containing proteins to further elucidate the protein nucleic acid in-teractions necessary for binding. Our studies are precursor to more elaboratemolecular dynamics studies to uncover how the GQ RNA secondary structureand proteins interact and establish the role they play in neurodegenerativediseases.

2177-Pos Board B193A Comparison of Basic Side Chain Internal Motions for the Free andDNA-Bound States of the Antennapedia HomeodomainDan Nguyen, Zoe A. Hoffpauir, Junji Iwahara.University of Texas Medical Branch, Galveston, TX, USA.For protein-DNA interactions, basic side chains at the interfacial region play acrucial role in forming intermolecular ion pairs with DNA phosphates. To

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further understand the role of basic side chains, we used NMR spectroscopyto investigate the basic side chain dynamics of these cationic groups. In thisstudy, we characterized the dynamics of Arg and Lys sidechains of the fruitfly Antennapedia homeodomain in the free and DNA-bound states. 15N relax-ation for Arg and Lys sidechains was measured at two different magneticfields. From these measurements, the generalized order parameters for thesecationic groups were determined in order to evaluate the dynamics. We foundthat the mobility of the R5 side chain, that forms a hydrogen bond witha thymine base in the DNA minor groove, was greatly dampened. In theDNA-bound state, several of the Lys and Arg side chains that formintermolecular ion pairs with the DNA phosphates were found to retain ahigh mobility, with an order parameter of < 0.6. However, one interestingobservation was that some of the interfacial cationic groups in the complex dis-played a higher mobility than in the free protein. In the protein-DNA associa-tion, the retained or enhanced mobility of the DNA-bound state should beentropically favorable.

2178-Pos Board B194Mechanistic Insight into the Assembly of the HerA-NurA Helicase-Nuclease DNA End Resection Complex using Native Mass SpectrometryZainab Ahdash1, Andy M. Lau1, Robert Thomas Byrne2, Katja Lammens2,Paula J. Booth1, Eamonn Reading1, Karl-Peter Hopfner2, Argyris Politis1.1King’s College London, London, United Kingdom, 2Gene Center andDepartment of Biochemistry, Ludwig-Maximilians-Universit€at M€unchen,Feodor-Lynen-Strasse, Germany, M€unchen, Germany.DNA double-stranded breaks are one of the most deleterious forms of DNAdamage. The HerA-NurA helicase-nuclease complex cooperates with Mre11and Rad50 to coordinate the repair of double-stranded DNA breaks. However,little is known about the assembly mechanism and activation of HerA-NurAcomplex. To investigate the oligomeric formation of HerA and to understandthe mechanism of nucleotide binding to the HerA-NurA from thermophilicarchaea, we combined native mass spectrometry with electron microscopy,molecular dynamics simulations and biochemical analyses. We reveal thatATP-free HerA and HerA-DNA complexes predominantly exist in solutionas a heptamer, and act as a DNA loading intermediate. The hexamericHerA is stabilised by the binding of either NurA or ATP suggesting thatHerA-NurA is activated by substrates and complex assembly. To study therole of ATP in DNA translocation and processing, we investigated how nucle-otides interact with the HerA-NurA complex. We show that while the hex-americ HerA binds six nucleotides in an ‘‘all-or-none’’ fashion, HerA-NurAharbors a highly coordinated pairwise binding mechanism and permits thetranslocation and processing of double-stranded DNA. Using moleculardynamics simulations, we uncover novel inter-residue interactions betweenthe internal DNA binding sites and the external ATP. Overall, we proposea stepwise assembly mechanism which details the synergistic activation ofHerA-NurA by ATP, which allows efficient processing of double-strandedDNA. Our finding that the helicase HerA predominantly exists as heptamerprior to hexamer formation and NurA nuclease recruitment may serve as amodel for DNA end resection in eukaryotes (Accepted, Nucleic AcidsResearch).

2179-Pos Board B195Are Cajal Bodies Droplet Organelles?Edward M. Courchaine, Karla M. Neugebauer.Molecular Biophysics & Biochemistry, Yale University, New Haven, CT,USA.Cajal Bodies(CBs) are subnuclear organelles responsible for spliceosomalsnRNP maturation in higher eukaryotes. Vertebrates must maintain highsnRNP levels during development, and a loss of CBs results in embryonic ar-rest due to defects in splicing. Like nucleoli and other cellular bodies, CBs arenot delimited by lipid bilayers and are RNA-rich. Current models for the for-mation of such ‘‘droplet organelles’’ invoke phase transitions promoted byintrinsically disordered regions (IDRs) of key components. CBs are scaffoldedby the protein coilin, which is assumed to be largely disordered; however, coi-lin’s biochemical and biophysical properties have not been fully examined. Itis currently unclear which coilin regions are structured and whether coilin it-self mediates a phase transition. For example, a protein or RNA interactionpartner may do so. We have purified coilin and subjected it to circulardichroism spectroscopy, finding that at least portions of coilin can adopt anordered state. In vivo staining with amyloid sensitive fluorescent dyes indi-cates that this structure is not cross-beta-sheet. Protein folds predicted withRaptorX include: 1) a ubiquitin-like fold at the N-terminus followed by 2)an IDR and 3) a C-terminal tudor-like domain. Prior immunoprecipitationresults have shown that the N-terminal domain mediates direct or indirect coi-lin ‘‘self-interaction’’, and UV crosslinking revealed RNA binding properties.

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We have identified single amino acid mutations that disrupt CBs, suggestingspecific functional sites within coilin are currently employing a protein-protein crosslinker (DPS) to implicate interactors. The major species appearsto consist of coilin-coilin dimers. Thus far, our findings are consistent witha model wherein the IDR promotes a phase transition yet two structuredregions impose specificity: an N-terminal coilin-coilin interaction domainand a C-terminal domain that binds to SMN, snRNP proteins and possiblysnRNA.

2180-Pos Board B196Liquid-Liquid Phase Transitions at the Origins of Life?Helen G. Hansma.Physics, UCSB, San Mateo, CA, USA.Membranes are fragile and are likely to be either too permeable or tooimpermeable, without elaborate channels and other proteins. This is aproblem for the idea that membranes were involved at the earliest stages oflife’s origins. On the other hand, liquid-liquid phase transitions generate struc-tures that are not bounded by membranes. [1] Liquid-liquid phase transitionsoften compartmentalize RNA and peptides/proteins, the components of ribo-somes, which contain some of the most ancient proteins. Where mightliquid-liquid phase transitions have occurred at the origins of life? The spacesbetween mica sheets have many advantages as a site for the origins of life.[2, 3] Other mineral compartments, in porous rocks, are possible sites forlife’s origins, but mica has an added advantage: mechanical energy frommoving mica sheets, compressing and stretching the molecules between thesheets. Mechanical energy is, arguably, the fundamental form of energyused by enzymes that use conformational changes to convert substrates intoproducts. Two types of mica are the best candidates for the origins of life,because these mica’s sheets are held together by potassium ions, which arepresent at high concentrations in all living cells. These two types of micaare the translucent Muscovite mica and the black mica, biotite, that is richin Fe(II) and Fe(III). Perhaps the mechanical energy of protein motion andthe high potassium concentrations in living cells are artifacts of life’s originsbetween mica sheets.1. Brangwynne, C.P., Phase transitions and size scaling of membrane-less or-ganelles. The Journal of cell biology, 2013. 203:875-881.2. Hansma, H.G., Possible origin of life between mica sheets. Journal of Theo-retical Biology, 2010. 266:175-188.3. Hansma, H.G., Better than Membranes at the Origin of Life? Life, 2017.7:28.

2181-Pos Board B197Protein-Sensing RiboswitchesRoee Amit.Technion - Israel Institute of Technology, Haifa, Israel.We study translational regulation by a 5’ UTR sequence encoding the bindingsite of an RNA-binding protein (RBP), using a reporter assay and SHAPE-seq,in bacteria. We tested constructs containing a single hairpin, based on the bind-ing sites of the coat RBPs of bacteriophages MS2, PP7, GA, and Qb, positionedin the 5’ UTR. With specifically-bound RBP present, either weak repression orup-regulation is observed, depending on the binding site. SHAPE-seq data for arepresentative construct exhibiting up-regulation indicate a partially-foldedhairpin and a hypo-modified upstream flanking region, which we attribute toan intermediate structure that apparently blocks translation. RBP binding stabi-lizes the fully-folded hairpin state and thus facilitates translation. This indicatesthat the up-regulating constructs are RBP-sensing riboswitches. This finding isfurther supported by lengthening the binding-site stem, which in turn destabi-lizes the translationally-inactive state. Finally, the combination of two bindingsites, positioned in the 5’ UTR and N-terminus of the same transcript can yielda cooperative regulatory response. Together, we show that the interaction of anRBP with its RNA target facilitates structural changes in the RNA, which is re-flected by a controllable range of binding affinities and dose response behavior.This implies that RNA-RBP interactions can provide a platform for construct-ing gene regulatory networks that are based on translational, rather than tran-scriptional, regulation.

2182-Pos Board B198Widespread Increase in Transcription Factor-DNA Binding due toMismatch DamageAriel Afek, Raluca Gordan.Duke University, Duke University, Durham, NC, USA.DNA mismatches occur when two non-complementary bases are aligned onopposite strands of a DNA duplex. Mismatches are generated during DNAreplication, genetic recombination, or by chemical reactions. Unrepaired mis-matches can lead to mutations, and have been associated with numerouscancers.

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Mismatches alter the DNA-structure and the DNA-bases functional groups,which can alter TF-DNA interactions. Currently, very little is known aboutthe effects of mismatches on TF-binding.We present the first high-throughput assay for characterizing the effect of mis-matches on DNA interactions with transcription-factors (TFs). We developedan in vitro technique to perform ‘saturation mismatching’, i.e. to generateand test all possible 1-base mismatches, in TF binding-sites (TFBSs). Weapplied our assay to measure binding of 18 TFs to thousands of mismatchedDNA sequences.We found that DNA mismatches within TFBSs can significantly increaseTF binding levels compared to the wild-type sequences. The increase in bind-ing affinity occurs even for sequences that perfectly match the consensusbinding site. The magnitude of the observed effect is large, with some mis-matches leading to a >5-fold increase in the TF binding-signal measured inour assay. For several TFs we identified genomic regions with no putativeTFBSs that became bound with high-affinity when certain mismatches wereintroduced.Importantly, we show that the effects single-base mismatches are distinct fromthose of single-bp mutations in which both strands are mutated. We investi-gated potential mechanisms by which mismatches might lead to increasedTF binding, including promoting a DNA structure more favorable for TF bind-ing, and increasing the number of protein-DNA contacts.The increased affinity of TFs for sites with certain DNA mismatches has thepotential to influence gene-expression and DNA-repair processes. Furthermore,studying TF binding to mismatched DNA will help us better understand DNAshape readout mechanisms.

2183-Pos Board B199Large Domain Movements Upon UvrD Dimerization and Helicase Activa-tionBinh Nguyen, Yerdos Ordabayev, Joshua Sokoloski, Elizabeth Weiland,Timothy M. Lohman.Biochemistry & Molecular Biophysics, Washington University in St Louis,St Louis, MO, USA.E. coli UvrD is an SF1A DNA helicase that functions in several DNA repairprocesses. As a monomer, UvrD can translocate rapidly and processivelyalong ssDNA, however, the monomer is a poor helicase. In order to unwindduplex DNA in vitro, UvrD needs to be activated either by self-assembly toform a dimer or by interaction with an accessory protein. However, the mech-anism of activation is not understood. UvrD can exist in multiple conforma-tions associated with the rotational conformational state of its 2B sub-domain and its helicase activity has been correlated with a ‘‘closed’’ 2Bconformation. Using single molecule total internal reflection fluorescence mi-croscopy, we examined the rotational conformational states of the 2B sub-domain of fluorescently labeled UvrD and their rates of interconversion.We find that the 2B sub-domain of the UvrD monomer can rotate betweenan open and closed conformation as well as two highly populated intermediatestates. The binding of a DNA substrate shifts the 2B conformation of a labeledUvrD monomer to a more open state that shows no helicase activity. Thebinding of a second unlabeled UvrD shifts the 2B conformation of the labeledUvrD to a more closed state resulting in activation of helicase activity. Bind-ing of a monomer of the structurally similar E. coli Rep helicase does notelicit this effect. This indicates that the helicase activity of a UvrD dimer ispromoted via direct interactions between UvrD subunits that affect the rota-tional conformational state of its 2B sub-domain.

2184-Pos Board B200Allosteric Effect of E. coli SSB C-Terminal Tails on RecOR Binding toDNAMin Kyung Shinn1,2, Alexander G. Kozlov1, Timothy M. Lohman1.1Department of Biochemistry and Molecular Biophysics, School ofMedicine, Washington University in St. Louis, St. Louis, MO, USA,2Department of Physics, Washington University in St. Louis, St. Louis, MO,USA.The E. coli single-stranded DNA binding (SSB) protein interacts with at least15 different proteins, known as SSB-interacting proteins (SIP), during DNAreplication, repair, and recombination. However, the specificity by whichSSB differentiates and recruits each SIP for different roles is not understood.The E. coli RecO protein is a recombination mediator protein (RMP) involvedin the RecF pathway of homologous recombination. RecO forms complexeswith RecF and RecR, and is thought to interact with the last 9 amino acidsof the intrinsically disordered C-terminal tails of SSB (SSB-Ct). We are exam-ining the interaction of SSB with RecOR by monitoring the change in intrinsicTrp fluorescence of RecO. We observe an allosteric effect of the SSB-Ct onDNA binding by RecOR, such that the SSB-Ct peptide enhances RecOR bind-

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ing to ssDNA. Whereas structures of RecOR complexes from other organisms,such as D. Radiodurans and T. Tengcongensis, have been determined, theydiffer in stoichiometry. Furthermore, structures of the E. coli RecOR complexare not available. We therefore are investigating the functional oligomeric stateof RecO and RecR, and the stoichiometry of the RecOR complex using analyt-ical ultracentrifugation and isothermal titration calorimetry. E. coli RecR hadpreviously been reported to form a dimer, but we find that it is in a dimer-tetramer equilibrium and the RecR tetramer appears to be the form that bindsRecO (supported by NIH GM030498 to TML).

2185-Pos Board B201Templated Cross Catalysis by Oligopeptides and OligonucleotidesEun Ae Park.California State University, Long Beach, Long Beach, CA, USA.A fundamental question on the origin of life asks how random chemistry over-came the laws of entropy to attain functional biological significance. The Cen-tral Dogma Theory states that DNA is transcribed to RNA then translated toamino acids. The unanswered question to this theory is, how can one comebefore the other when proteins are used to create proteins themselves fromthe inheritable genetic material? Our project will contribute to resolvingtwo questions, what is the fundamental quantity of information needed forlife to have begun and what are the catalytic properties rudimentary to bio-polymers that are required for information storage and catalytic capabilities.We hypothesize that oligopeptides and oligonucleotides began to ‘‘interpret’’and ‘‘replicate’’ each other. Short combinational oligopeptide and RNAhairpin libraries will be used to investigate the binding between two polymers,the statistical patterns of association and the catalysis of ligation andextension.

2186-Pos Board B202Molecular Dynamics Simulations of Brg1 Bromodomain Interacting withDNA in Both Presence and Absence of Brg1 AT-HookStefania Evoli, Jeff Wereszczynski.Department of physics, Illinois Institute of Technology, Chicago, IL, USA.ATP-dependent chromatin-remodeling complexes are large proteins involvedin remodeling the compact chromatin structure. The switching/sucrose non-fermenting (SWI/SNF) remodeling class contains a bromodomain importantfor the assembly and activity of the multicomponent complexes that are respon-sible for regulating chromatin structure and function. Human brahma-relatedgene 1 (Brg1), a central ATPase protein in human SWI/SNF chromatin remod-eling complex, contains a bromodomain, which in association with acetylatednucleosomes is critical in DNA damage response, and a small AT-hook, whichenhances the DNA-binding activity.NMR spectroscopy studies demonstrated that bromodomains can bind DNAthrough an interaction patch including which includes the aZ helix and theAB and ZA loops.Understanding the dynamics of the possible complexes is of key importance tostudy disease etiology.In our study, we combine docking and molecular dynamics simulations toinvestigate the interaction between three different DNA sequences and theBrg1 bromodomain, both in presence and absence of the AT-hook bromodo-main. Different binding modes have been found in correspondence of minorand major DNA grooves and they have been classified based on their free en-ergy differences, which have been calculated by means of free energy calcula-tions. These findings provide information about the specificity of bindingbetween DNA and Brg1 bromodomain.

2187-Pos Board B203Dissecting the Electrostatics of Nucleic AcidsMagdalena Gebala, Benjamin E. Allred, Daniel Herschlag.Stanford University, Stanford, CA, USA.Electrostatics are a major force in biology, affecting the structure and functionof all charged macromolecules in a cell. Nevertheless, the contribution of elec-trostatics to fundamental aspects of cellular function, such as the mesoscale or-ganization of the cell in membrane-less organelles (1) or the dynamiccompaction of chromatin into functional higher-order structures (2), are onlypoorly understood. The primary barrier to progress is the lack of experimentalapproaches to quantitatively study interactions between macromolecules andthe ions required to mitigate their charge and to correlate these effects with theirenergetic consequences. Importantly, the clear majority of ions that play thisrole are not site-specifically bound, but rather are part of a highly mobile andintrinsically disordered cloud of ions, which is referred to as the ionatmosphere.We will present an experimental approach, buffer equilibration-mass spectros-copy (BE-MS), that ‘counts’ the number of ions thermodynamically associated

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with a macromolecule or complex, allows dissection of energetic properties ofthe ion atmosphere, and provides direct comparison to theoretical results (3).We will discuss how ion size and charge affect the ion atmosphere and its en-ergetics for well-defined DNA, RNA, and the nucleosome and its constituents.We will show an additional new experimental approach that provides a localelectrostatic meter, determining the electrostatics surface potential of DNAmolecule via protonation of A�C wobble pair (4) This approach can also beapplied to dissect and understand the energetics of nucleic acid/proteincomplexes.1. Nott, T. J. et al. Mol. Cell 57, 936-947 (2015)2. Sun, J. et al. PNAS 102, 8180-8185 (2004)3. Gebala, M. et al. J. Am. Chem. Soc.137, 14705 (2015)4. Allred, B. E. et al. J. Am. Chem. Soc.139, 7540-7548 (2017)

2188-Pos Board B204Translation Initiation Complex eIFiso4F Targets Pokeweed AntiviralProtein (PAP) to Selectively Depurinate Uncapped Tobacco Etch Virus(TEV) RNAArtem V. Domashevskiy, Shu-Yuan Cheng.Sciences, John Jay College of Criminal Justice, CUNY, New York, NY,USA.Pokeweed antiviral protein (PAP) is a ribosome inactivating protein (RIP) thatdepurinates the sarcin/ricin loop (SRL) of rRNA, inhibiting protein synthesis.PAP depurinates viral RNA, and in doing so, lowers the infectivity of manyplant viruses. The mechanism by which PAP accesses uncapped viral RNAis not known, impeding scientists from developing effective antiviral agentsfor the prevention of the diseases caused by uncapped RNA viruses. Kineticrates of PAP interacting with tobacco etch virus (TEV) RNA, in the presenceand absence of eIFiso4F, were examined, addressing how the eIF affects selec-tive PAP targeting and depurination of the uncapped viral RNA. PAP-eIFs co-purification assay and FRET demonstrate that PAP forms a ternary complexwith the eIFiso4G and eIFiso4E, directing the depurination of uncapped viralRNA. eIFiso4F selectively targets PAP to depurinate TEV RNA by increasingPAP’s specificity constant for uncapped viral RNA twelve fold, whencompared to the depurination of an oligonucleotide RNA that mimics theSRL of large rRNA, and cellular capped luciferase mRNA. This explainshow PAP is able to lower infectivity of pokeweed viruses, while preservingits own ribosomes and cellular RNA from depurination: PAP utilizes cellulareIFiso4F in a novel strategy to target uncapped viral RNA. It may be possibleto modulate and utilize these PAP-eIFs interactions for their public healthbenefit, by repurposing them to selectively target PAP to depurinate uncappedviral RNA, many plant and animal diseases caused by these viruses could bealleviated.

2189-Pos Board B205Mapping Interactions of Single-Stranded (SS) DNA with the SS-DNABinding Protein (GP32) of the T4 DNA Replication Complex at SpecificNucleotide Residue PositionsBenjamin Camel, Anson Dang, Katherine Meze, Davis Jose,Peter H. von Hippel.University of Oregon, Eugene, OR, USA.The single-stranded (ss)DNA binding protein (gp32) of bacteriophage T4 playsa central role in regulating the functions and integration of the helicase, poly-merase and primase components of the T4 DNA replication system. To under-stand how gp32 interacts with itself and with the other regulatory proteins T4replication complex, we must first understand the structural details of how thisprotein binds to ssDNA lattices, both as isolated monomer subunits and ascooperatively bound gp32 clusters. We have explored these issues by moni-toring differences in the fluorescence and circular dichroism (CD) spectra ofsite-specifically positioned monomers and dimer-pairs of 2-aminopurine (2-AP) probes located at various ssDNA positions within the binding site. By map-ping spectral changes on binding of gp32 to ssDNA lattices we have been ableto characterize interactions at defined positions within the gp32 binding-cleft.We have extended these studies using acrylamide quenching and permanganatefoot-printing assays to monitor degrees of base exposure at various lattice po-sitions. Our results show that gp32 binds randomly at low concentrations, andthen shifts toward preferential binding at the 5’-ends of the lattice as coopera-tively bound gp32 clusters form at higher gp32 concentrations. Bases locatednear the middle of a gp32 binding site display lower solvent accessibilitythan those near the ends of the site. These differences in base ‘shielding’may reflect deeper burial of the middle bases within the electropositive bindingcleft, while bases at the ends may be made more accessible by fluctuations ofthe C- and N-terminal regulatory sub-domains of the protein. Insights intogp32-ssDNA interactions involved in controlling the functions of the T4DNA replication complex that result from these studies will be discussed.

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2190-Pos Board B206Quantifying Protein-DNA Interactions by Kinetics Exclusion AssayElizabeth Leung, Troy Rohn, Daniel Fologea.Boise State University, Boise, ID, USA.Protein-DNA interactions are vital components of the regulatory cellularmachinery in health and disease. Quantifying these interactions is essentialfor understanding their origin, specificity, and the role they play in regulatingintricate cellular processes. Although numerous laboratory techniques havebeen developed to study the intricate interactions between proteins andDNA, each one of them has limited utility regarding applications. For betterquantification of these important interactions, our work focused on usingthe Kinetics Exclusion Assay (Kinexa) technology. Our strategies includedusing either DNA or protein for constructing the titrant-coated solid phase.The captured constant binding partner was identified by using intercalatingfluorescent probes binding DNA molecules or fluorescently-labeled probestargeting proteins. The proposed methodologies provide a unique way toobtain true solution-phase measurements since the molecules in solutionare unmodified and the measurement process itself has a minimal impact onequilibrium.

2191-Pos Board B207Cooperation of DNA Helicases during dsDNA End ResectionKristina Kasaciunaite1, Fergus Fettes1, Maryna Levikova2, Petr Cejka3,Ralf Seidel1.1Leipzig University, Leipzig, Germany, 2University of Zurich, Zurich,Switzerland, 3Universita della Svizzera Italiana, Bellinzona, Switzerland.Double strand DNA (dsDNA) break repair by homologous recombination ineukaryotes is a complex process that is not well understood at the molecularlevel. A main step of this process is the 5’-strand resection at the DNA break.In yeast, one resection mechanism involves the cooperation of two molecularmotors: the RecQ helicase Sgs1 and the nuclease-helicase Dna2. These proteinsunwind DNA in opposite directions while the produced single stranded DNA(ssDNA) is protected by the ssDNA binding protein RPA.To gain insight into the potentially synergistic cooperation between these mo-tors we utilized magnetic tweezers. This technique allows stretching singleDNA molecules and this way monitoring the conversion of dsDNA to ssDNAby a change of the DNA length in real-time. We first characterized nuclease-dead Dna2 and Sgs1 individually. Dna2 showed processive unidirectional un-winding of long fragments being strictly dependent on the presence of RPA. Incontrast, the Sgs1 alone displayed gradual DNA unwinding, terminated byfrequent spontaneous DNA rezipping events. In presence of RPA, the unwind-ing velocity of Sgs1 was markedly shifted towards lower velocities and thehelicase could also close the DNA by gradual rewinding rather than rezipping.When reconstituting the full DNA resection process in vitro using wt Dna2,Sgs1 and RPA, an overall long-range and slow unidirectional DNA unwinding(Dna2-like) was observed. On top of this process, shorter repetitive openingand closing events (Sgs1-like) appeared. Furthermore, the presence of Dna2 re-verted the RPA-induced velocity reduction of Sgs1. These data suggest the for-mation of a Sgs1-Dna2 complex in which Sgs1 unwinds the dsDNA and feedsthe resulting ssDNA for degradation into Dna2. While formation of such aresection complex is similar to the well-known prokaryotic counterpartRecBCD, the coupling between the eukaryotic enzymes appears to be consid-erably weaker.

2192-Pos Board B208Investigation of mRNATranslation Regulation by FMRP via theMicrornaPathwayJoshua A. Imperatore, Brett A. DeMarco, Mihaela Rita Mihailescu.Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, USA.Fragile X syndrome (FXS) is the most common form of inherited mentalimpairment. The disease is a result of a cytosine-guanine-guanine (CGG)repeat expansion in the 5’ untranslated region (UTR) of the fragile X mentalretardation-1 (FMR1) gene. Overexpansion of the repeat causes hypermethy-lation of the cytosine residues, leading to transcriptional silencing of theFMR1 gene and loss of function of the fragile X mental retardation protein(FMRP). FMRP functions as a transporter protein and translational regulatorfor specific neuronal messenger RNA (mRNA) targets. It has been proposedthat the translation regulator function of FMRP is mediated via interactionswith the miRNA pathway. miR-125a has been implicated in the translationregulation of PSD-95 mRNA and FMRP has been shown to be required inthis process. Moreover, preliminary research has shown that FMR1 knockoutsin mice lead to the down regulation of mature miR-125a. Here, we proposethat FMRP regulates the translation of PSD-95 mRNA both directly byinteracting with this mRNA and the mature miR-125a, as well as indirectly,being implicated in the maturation of miR-125a. FMRP interactions with

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both premature and mature microRNA-125a were analyzed by various bio-physical methods.

2193-Pos Board B209Different Membrane Insertion Potential of Gene Nanoparticles Studied byusing Phospholipid Monolayer and Bilayer ModelsNabil Abdulhafiz Alhakamy1, Cory J. Berkland2, Prajnaparamita Dhar3.1Pharmaceutics, King Abdulaziz University, Jeddah, Saudi Arabia,2Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA,3Chemical and Petroleum Engineering, University of Kansas, Lawrence, KS,USA.Cell-penetrating peptides (CPPs) have been broadly used to enhance the intra-cellular delivery and transfection efficiency of genetic material (e.g., siRNAand pDNA). It is important to study the ability of CPPs and nanoparticles(NPs) to penetrate the cell membranes when we design them for genetic mate-rial (e.g., pDNA and siRNA) delivery applications. Still, the interaction/inser-tion mechanisms of CPPs and gene NPs to intracellular locations as well astheir intracellular stability are not completely understood. Here, phospholipidmonolayers and bilayers are used as cell membrane models to study the mem-brane interaction/insertion potential of CPPs and NPs by using the Langmuirand Quartz crystal microbalance with dissipation monitoring (QCM-D) tech-niques. We used zwitterionic and anionic phospholipid monolayers and bila-yers as models that mimic the cell membrane composition of the plasmamembrane and intracellular vesicles. Furthermore, we studied the membraneinteraction/insertion potential of CPPs and NPs at neutral and acidic pHs thatmimic the intracellular environment. The focus of this paper is to examinethe interaction/insertion potential of R9 and RW9 CPPs and NPs with phospho-lipids in the plasma membrane and endocytic pathway of eukaryotic cells(early/late endosome and lysosome membranes). Interestingly, our results sug-gest that not only the pH of the medium (neutral vs. acidic) play a significantrole to determine the interaction/insertion potential of the CPPs and NPs butalso the type of the membrane models that used in the experiments (monolayersvs. bilayers).

2194-Pos Board B210Kinetic Pathways of Topology Simplification by Type-II Topoisomerasesin Knotted, Supercoiled DNAAndreas Hanke1, Riccardo Ziraldo2, Stephen D. Levene2.1Physics and Astronomy, University of Texas Rio Grande Valley,Brownsville, TX, USA, 2Bioengineering, University of Texas at Dallas,Dallas, TX, USA.The topological state of covalently closed, double-stranded DNA is defined bythe knot type, K, and the linking number difference from relaxed DNA, DLk.DNA topoisomerases are essential enzymes that regulate topological states ofDNA in vivo: type-I topoisomerases (topo-Is) change DLk, thereby regulatingthe torsional tension, whereas type-II topoisomerases (topo-IIs) change both(DLk, K) by passing one DNA helix through another. A critical biological func-tion of type-II enzymes is the elimination of knots in DNA because their pres-ence impedes transcription and replication. It has been a long-standing puzzlehow small type-II enzymes select passages that unknot large DNA molecules,since topology is a global property which cannot be determined by local DNA-enzyme interactions. Previous studies addressing this question have focused onthe equilibrium distribution P(DLk, K). Motivated by the fact that topo-IIsreduce the knotting level below equilibrium at the expense of ATP hydrolysis,we set out to study topoisomerase activity in the framework of non-equilibriumthermodynamics. We consider the dynamics of transitions in a network of to-pological states (DLk, K) induced by type-II and type-I action by solving themaster equation for the time-dependent probability distribution P(DLk, K; t).We fully characterize non-equilibrium steady states generated by injectingDNA molecules in a given topological state in terms of stationary probabilitydistributions and currents in the network. This allows us, for the first time, topredict detailed kinetic pathways of topoisomerase action as a function of ge-ometry of the enzyme. In particular, we find that unknotting activity of topo-IIis significantly enhanced in DNA molecules which maintain a supercoiled statewith constant torsional tension; this is relevant for bacterial cells in which thetorsional tension is maintained by a homeostatic mechanism using topo I andDNA gyrase.

2195-Pos Board B211Identification of Suramin as a Potent and Specific Inhibitor of theMamma-lian High Mobility Group Protein at-Hook 2 (HMGA2)-DNA InteractionsLinjia Su1,2, Steve Vasile3, Layton Smith3, Fenfei Leng1,2.1Department of Chemistry and Biochemistry, Florida InternationalUniversity, Miami, FL, USA, 2Biomolecular Sciences Institute, Florida

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International University, Miami, FL, USA, 3Sanford-Burnham Center forChemical Genomics, Sanford-Burnham Medical Research Institute, Orlando,FL, USA.The mammalian high mobility group protein AT-hook 2 (HMGA2) is a nuclearprotein associated with epithelial-mensenchymal transition (EMT) during celldevelopment and differentiation. This protein plays an important role in the for-mation of a variety of tumors including malignant tumors, such as melanoma,lung cancer and hepatocellular carcinoma. These results suggest that HMGA2is a potential therapeutic target of anticancer drugs. HMGA2 is a DNA minor-groove binding protein specifically recognizing the minor groove of AT-richDNA sequences. Previous results showed that HMGA2-DNA interactions area potential target for chemical intervention. In this study, we developed an Al-phaScreen HTS Assay to screen inhibitors targeting HMGA2-DNA interac-tions. We are particularly interested in identifying non DNA-bindingcompounds that inhibit HMGA2-DNA interactions due to the fact that DNA-binding compounds are highly cytotoxic. After the HTS campaign, severalnon DNA-binding compounds have been identified to potently inhibitHMGA2-DNA interactions. Among them is suramin, a negatively charged anti-parasitic drug. Suramin potently inhibits HMGA2-DNA interaction with anIC50 of 2.58 micro molar. We also found that suramin and analogues stronglybind to HMGA2, suggesting that the inhibition of HMGA2-DNA interactions isthrough suramin binding to HMGA2 and therefore blocking HMGA2’s DNAbinding capacity. Suramin is an anticancer agent that inhibits tumor growthand metastasis for certain cancers including pancreatic cancer, prostate cancer,melanoma, and etc. The anti-cancer mechanism of suramin is still illusive. Thediscovery of suramin as a potent inhibitor of HMGA2-DNA interactions sug-gests that the anti-cancer activities of suramin may stem from its inhibitionof HMGA2-DNA interactions in vivo and opens a door for future research insuramin as an anticancer agent.

2196-Pos Board B212Engineering a Tunable DNA Loop in E. coliNicole A. Becker, Tanya L. Schwab, Karl J. Clark, L. James Maher III.Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA.In prokaryotes, genes are typically considered available for expression unlessswitched off by the binding of a repressor protein at or near the transcriptionalstart site. The level of gene repression can be further enhanced by the formationof a tunable DNA loop enclosing the transcription start site. One such exampleof gene control by loop formation occurs within the well-studied lac operon. InE. coli, lac loop formation occurs when LacI, a tetrameric protein, simulta-neously binds two operators within close proximity resulting in a DNA loop,repressing transcription of the downstream operon genes. A small molecule (al-lolactose) causes unlooping. To be useful, engineered loops should be similarlyreversible. We have created an artificial dimeric fusion protein capable of bind-ing DNA with designed sequence specificity to form a tunable DNA repressionloop. The fusion protein utilizes a transcription activator-like effector (TALE)protein as the DNA binding region fused to FK506 binding protein (FKBP) asthe protein-dimerizing domain. We show that the dimeric TALE-FKBP fusionbinds DNA in a bidentate manner, similar to LacI, resulting in repression loopformation in living E. coli cells. We assess a TALE-FKBP protein fusion tar-geted to the Osym operator in the context of our established in vivo lac pro-moter looping model system. We demonstrate �7 fold enhancement of generepression using the engineered TALE-FKBP fusion when a DNA loop isformed. The dimeric TALE-FKBP fusion can be tuned by addition of the smallmolecule FK506, which dissociates the FKBP dimer and derepresses the testpromoter. We thus demonstrate engineered regulation of bacterial gene expres-sion by DNA looping in living cells.

2197-Pos Board B213Protamin-Induced DNA LoopingAshley R. Carter, Obinna A. Ukogu, Adam D. Smith, Luka M. Devenica,Ryan McMillan, Yuxing Ma, Hilary Bediako.Amherst College, Amherst, MA, USA.DNA is dramatically condensed in sperm cells by protamine proteins. Theseprotamines bind to the major groove of the DNA and loop the DNA into a seriesof toroids, creating folded DNA that is near crystalline packing levels. Onequestion of interest is how protamine is able to fold the DNA into a singleloop. To investigate this question, we performed in vitro tethered particle mo-tion (TPM) assays on short (200 nm) DNAmolecules. In these assays, the DNAis biochemically tethered at one end to a glass coverslip and at the other end to amicron-sized polystyrene particle, forming a tethered particle. When we addlow concentrations (0.1-0.3 mM) of protamine, the DNA folds, and the rangeof motion of the tethered particle decreases as the DNA loops. We followedup these dynamic measurements with static atomic force microscopy (AFM)

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images of DNA molecules in the presence of protamine. We expected ourmeasurements to show a single folding event as the loop forms, but both mea-surements showed the presence of intermediate states. These results suggestthat DNA looping by protamine is more complex than previously thought,with interesting applications for biomaterials research.

2198-Pos Board B214A Tale of Two Mechanisms: DNA Recognition by the ETS-Family Tran-scription FactorsGregory M.K. Poon.Chemistry, Georgia State University, Atlanta, GA, USA.Transcription factors from the same eukaryotic gene family typically show lit-tle functional redundancy despite sharing structurally conserved DNA-bindingdomains with overlapping DNA sequence preferences. The ETS transcriptionfactors, an ancient family harboring a highly conserved winged helix-loop-helix (termed ETS) domain, exemplify this conundrum. Through extensive ex-periments and molecular dynamics simulations, we have identified an evolu-tionary transition in the mechanism of DNA recognition by ETS proteins. Inprimordial ETS members such as Ets-1, DNA binding is coupled to an induc-tion of localized sidechain dynamics, which map to structural elements con-necting the DNA binding surface to the distal end of the ETS domain. Thesedynamics correspond closely to correlated motion independently identified inautoinhibition, a regulatory mechanism in which small structures adjacent tothe distal end of the ETS domain partially unfolds to attenuate the associationkinetics and binding affinity. In ETS relatives that separated early in evolution,such as PU.1, dynamic induction has been replaced by a mechanism of DNArecognition in which sequence selection is coupled to interfacial hydration.Correspondingly, PU.1 is not autoinhibited but is regulated instead by the nega-tively cooperative formation of a site-specifically bound dimer. While bothDNA-binding mechanisms generate indistinguishable affinities under standardphysiologic conditions for the transcriptionally active, structurally conservedETS/DNA complex, they differ markedly in binding kinetics, sensitivity toepigenetic DNA modifications, and sequence discrimination power. Currentexperimental and MD work on resolving how such mechanistic heterogeneityarises out of the utilization of primary sequence space during ETS evolutionwill be discussed.

2199-Pos Board B215Visualization of Distinct Epigenetic States at the Single Molecule LevelLuke Strauskulage1, Olga Cisne-Thompson2, Jessica Hurst3,Barbara Panning1, Sy Redding1.1Biochemistry & Biophysics, UCSF, San Francisco, CA, USA, 2City Collegeof San Francisco, San Francisco, CA, USA, 3Molecular & Cell Biology, UCBerkeley, Berkeley, CA, USA.Covalent modification of DNA bases creates an additional node of regulationinfluencing gene expression and chromatin organization. These modificationscoordinate dynamic and differential cellular outputs from the same genome.In mammals, DNA methylation of cytosine is the most widespread and well-studied DNA modification. Additionally, methylated cytosines can be succes-sively oxidized to create unique DNA modifications, and we are still learningabout the distribution and the functional consequence of these marks. I amdeveloping a novel microfluidic assay based on DNA Curtains technology tostudy these epigenetic DNA modifications at the single-molecule level. By em-ploying fluorescently labeled naturally occurring ‘‘reader’’ domains, I can visu-alize the distribution of these DNA modifications over kilobase pairs of DNA,leveraging the throughput of our approach to achieve nucleotide resolution. Us-ing this highly amenable system I can study the dynamics of these marks inreal-time across a variety of unique DNA sequences to better understandhow these fundamental marks are distributed across our genome.

2200-Pos Board B216Regulation of UvrD Helicase Activity by MutLYerdos Ordabayev, Binh Nguyen, Anita Niedziela-Majka,Timothy Lohman.Biochemistry and Molecular Biophysics, Washington University, St Louis,MO, USA.Escherichia coli UvrD is a 3’ to 5’ superfamily 1 helicase/translocase involvedin multiple DNA metabolic processes, including methyl-directed mismatchrepair. Although a UvrD monomer can translocate along single-strandedDNA, a UvrD dimer is needed for processive helicase activity in vitro. Previousstudies have shown that E. coliMutL, a regulatory protein involved in methyl-directed mismatch repair, stimulates the helicase activity of UvrD, however, themechanism of stimulation is not well understood. Using single molecule andensemble fluorescence approaches, we show that MutL can activate latentUvrD monomer helicase activity. We also find that MutL binding to a UvrD

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monomer-DNA complex shifts the 2B sub-domain conformation of UvrD toa more closed state, which correlates with unwinding activation. We find thatMutL also stimulates the activity and DNA unwinding processivity of UvrD di-mers. This suggests that MutL acts as a processivity factor and moves withUvrD during DNA unwinding rather than facilitating loading of multipleUvrD molecules onto the DNA. Our work provides insight into how UvrDmight function as a part of larger molecular complexes in vivo.

Posters: Chromatin and the Nucleoid II

2201-Pos Board B217Probing the Liquid-Like Nature of Human Nucleoli and their Interactionwith the Interphase ChromatinChristina M. Caragine, Shannon C. Haley, Alexandra Zidovska.New York University, New York, NY, USA.The nucleolus is a membraneless organelle embedded in chromatin solution in-side the cell nucleus. It has recently been reported that nucleoli in frog oocytesand C. elegans embryos form by liquid-liquid phase separation [1,2]. Whilefusion of nucleoli in human cells has been observed, it is unclear if they areliquid-like or solid-like in nature [3,4]. Moreover, the role of the chromatinpolymer in the facilitation of nucleolar fusion is completely unknown. In thiswork, we investigate the kinetics of the nucleolar fusion in order to determineits physical nature, i.e. aggregation of solid-like particles vs. coalescence ofliquid-like droplets. We monitor and analyze the nucleolar shape during fusionevents and find it consistent with a coalescence of liquid droplets. Further, weanalyzed nucleolar fusion events and found that after fusion, the eccentricity ofthe new nucleolus decreases, indicating the nucleolus undergoes a structuralchange and behaves as a liquid-like structure. Historically, the nucleolar assem-bly in mammalian cells was believed to complete within two hours aftermitosis, when the final number of nucleoli forms via de novo assembly aswell as fusion events [3]. However, we found that fusion events can also occurlater in the cell cycle. To explore the interaction of nucleoli with chromatin, wealtered chromatin state using biochemical perturbations. We found that chro-matin is indeed involved in the positioning, shape and alignment of nucleoli in-side the cell nucleus.1. Brangwynne, CP et al., Proc. Natl. Acad. Sci., 4434, 20112. Berry, J, et. al. Proc. Natl. Acad. Sci., E5237, 20153. Savino, TM, et al. J. Cell Biol.,1097, 20014. Farley, KI, et al. Chromosoma, 323, 2015

2202-Pos Board B218Active Hydrodynamics of Interphase Chromatin: Coarse-GrainedModeling and SimulationsDavid Saintillan1, Alexandra Zidovska2, Michael J. Shelley3.1Mechanical and Aerospace Engineering, UCSD, La Jolla, CA, USA,2Physics, NYU, New York, NY, USA, 3Mathematics, NYU, New York, NY,USA.The three-dimensional spatiotemporal organization of genetic material insidethe cell nucleus remains an open question in cellular biology. During the timebetween two cell divisions, the functional form of DNA in cells, known aschromatin, fills the cell nucleus in its uncondensed polymeric form, which al-lows the transcriptional machinery to access DNA. Recent in vivo imagingexperiments have cast light on the existence of coherent chromatin motionsinside the nucleus, in the form of large-scale correlated displacements onthe scale of microns and lasting for seconds. To elucidate the mechanismsfor such motions, we have developed a coarse-grained active polymer modelwhere chromatin is represented as a confined flexible chain acted upon byactive molecular motors, which perform work and thus exert dipolar forceson the system. Numerical simulations of this model that account for stericand hydrodynamic interactions as well as internal chain mechanics demon-strate the emergence of coherent motions in systems involving extensile di-poles, which are accompanied by large-scale chain reconfigurations andlocal nematic ordering. Comparisons with experiments show good qualitativeagreement and support the hypothesis that long-ranged hydrodynamic cou-plings between chromatin-associated active motors are responsible for theobserved coherent dynamics.

2203-Pos Board B219A First-Principles Approach to Large-Scale Nuclear ArchitectureAnkit Agrawal1, Nirmalendu Ganai2, Surajit Sengupta3, Gautam I. Menon1.1Computational Biology, The Institute of Mathematical Sciences, Chennai,India, 2Physics, Forschungszentrum Julich, Julich, Germany, 3Physics, TIFRCentre for Interdisciplinary Sciences, Hyderabad, India.A few general organizing principles underlie nuclear architecture in metazoans.Chromosomes are structured into largely non-overlapping territories across

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multiple cell types, with gene-rich euchromatin more centrally disposed ofthan gene-poor heterochromatin. Such territories are not randomly positionedand both gene-density-based and size-based radial positioning schemeshave been proposed to describe the data. We propose that any realistic modelof these and other large-scale features of nuclear architecture mustaccount for ATP-fueled non-equilibrium activity, associated with transcrip-tional processes that are inhomogeneous within and across chromosomes.We describe a biophysical model for human cell nuclei which incorporatessuch activity.The model predicts the statistics of the shapes, positioning, and contact maps ofindividual chromosomes, with the differential positioning of the inactive andactive X chromosomes in female (XX), cells emerging as a natural conse-quence, and our results compare favorably to a broad spectrum of experimentaldata. We argue that the consequences, in mechanical terms, of the distributionof transcriptional activity across chromosomes should be the primary determi-nant of a chromosome positioning code.

2204-Pos Board B220Phase Separation Drives Heterochromatin Domain FormationAmy R. Strom1, Alexander V. Emelyanov2, Mustafa R. Mir3,Dmitry V. Fyodorov2, Xavier R. Darzacq3, Gary H. Karpen1.1Molecular and Cellular Biology, Biological Systems and Engineering, UCBerkeley, LBNL, Berkeley, CA, USA, 2Cell Biology, Albert Einstein Collegeof Medicine, New York, NY, USA, 3Molecular and Cellular Biology, UCBerkeley, Berkeley, CA, USA.Constitutive heterochromatin is made of repetitive sequences and is epigenet-ically identified by methylation of H3K9 and binding of HeterochromatinProtein 1a (HP1a). In cells, repetitive sequences from multiple chromosomesare organized into spherical domains important for maintaining transcriptionalsilencing and preventing aberrant recombination. Canonically, these functionsof heterochromatin are attributed to tight compaction of nucleosomes andconsequent exclusion of large protein complexes like polymerases andrecombinases. However, proteins within the domain are mobile, and size isnot the only factor that defines whether a protein can enter heterochromatin.We investigated whether the heterochromatin domain is similar to othermembraneless cellular compartments like nucleoli in that it is formed viaphase separation. We purified recombinant Drosophila HP1a and found thatin vitro, this protein is able to demix from aqueous solution to form dropletsthat fuse and flow like a liquid. In vivo, we observe similar droplet formationand fusion during the initial establishment of heterochromatin in the earlyDrosophila embryo. We used Fluorescence Correlation Spectroscopy-derived imaging methods to observe bulk movement of HP1a at the hetero-euchromatin interface, and found that proteins exhibit specific dynamicproperties associated with phase interfaces, indicating they are held in theheterochromatin domain by surface tension. Additionally, an inert probe(three tandem YFPs) is excluded from the domain in a similar manner,indicating that physicochemical properties of the heterochromatin domaindefine access to these sequences. The phase separation model we proposehere is consistent with historical data about heterochromatin domains,including the tendency for distal regions of heterochromatin to loop backand contact the main domain. We believe this work represents a shift inperspective of how we should view the nucleus; as a set of membraneless do-mains, each with its own chemical and physical properties that define theircomponents.

2205-Pos Board B221On the Origin of Shape Fluctuations of the Cell NucleusFang-Yi Chu, Shannon C. Haley, Alexandra Zidovska.Center for Soft Matter Research, Department of Physics, New YorkUniversity, New York, NY, USA.The nuclear envelope (NE) presents a physical boundary between thecytoplasm and the nucleoplasm, sandwiched in between two highly active sys-tems inside the cell: cytoskeleton and chromatin. NE defines the shape andsize of the cell nucleus, which increases during the cell cycle, accommodatingfor chromosome decondensation followed by genome duplication. In thiswork, we study nuclear shape fluctuations at short time scales of seconds inhuman cells. Using spinning disk confocal microscopy, we observe fast fluc-tuations of the NE, visualized by fluorescently labeled lamin A, and of thechromatin globule surface (CGS) underneath the NE, visualized by fluores-cently labeled histone H2B. Our findings reveal that fluctuation amplitudesof both CGS and NE monotonously decrease during the cell cycle, servingas a reliable cell cycle stage indicator [1]. Remarkably, we find that, whileCGS and NE typically fluctuate in phase, they do exhibit localized regions

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of out-of-phase motion, which lead to separation of NE and CGS. To explorethe mechanism behind these shape fluctuations, we use biochemicalperturbations. We find the shape fluctuations of CGS and NE to be both ther-mally and actively driven, the latter caused by forces from chromatin andcytoskeleton [1]. Such undulations might affect gene regulation as well ascontribute to the anomalously high rates of nuclear transport by, e.g., stirringof molecules next to NE, or increasing flux of molecules through the nuclearpores.[1] Chu F-Y, Haley SC, Zidovska A, Proc. Natl. Acad. Sci. USA, 114 (39),10338-10343, 2017

2206-Pos Board B222Phase Separation of Mitochondrial DNA in the Premature Aging DiseaseHutchinson-Gilford Progeria SyndromeMarina Mahynski, Tom Misteli.Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.Increased mutations and damage to mitochondrial DNA (mtDNA) have beenlinked to aging. Within the mitochondria, mtDNA is coated by proteins form-ing nucleoprotein complexes or nucleoids, which serve as the sites of mtDNAreplication and transcription. Mitochondrial nucleoids are tightly regulated asthey contain only 1-2 molecules of mtDNA and maintain a fixed size ofapproximately 100 nm. However, the biophysical properties of mtDNA orga-nization and their contributions to aging are unknown. Here, we probe thephase separation of mtDNA nucleoids in a model of premature aging. We hy-pothesize that mtDNA nucleoids represent phase separated nucleoproteincomplexes and that age-associated oxidative stress further drives nucleoidcondensation in the premature aging disease Hutchinson-Gilford ProgeriaSyndrome (HGPS). Using several microscopy techniques, we find that mito-chondria in HGPS cells are swollen and damaged, exhibiting remarkablyenlarged, spherical nucleoids, with diameters greater than 300 microns. More-over, in response to photoinduced ROS generation, mitochondria begin toswell, while the nucleoids undergo rapid homotypic fusion events with neigh-boring nucleoids. Nucleoid coalescence and coarsening are consistent with thephase behavior of liquid-liquid phase separation. Additionally, we performseveral assays to understand the functional consequences of nucleoid phaseseparation, including next generation sequencing of the entire mtDNAgenome as well as mitochondrial functional markers for membrane potentialand oxidative stress. We postulate that increased oxidative damage in HGPScells alters the phase behavior of mitochondrial nucleoids contributing tomitochondrial dysfunction. These results shed light on how the physical prop-erties and organization of mitochondrial nucleoids are influenced by oxidativestress, with potential implications for normal aging.

2207-Pos Board B223The EZH2 SANT1 Domain is a Histone Reader Providing Sensitivity to theModification State of the H4 TailTyler M. Weaver1, Jiachen Liu1, Katelyn E. Connelly2, Chris Coble1,Katayoun Varzavand1, Emily C. Dykhuizen2, Catherine A. Musselman1.1Department of Biochemistry, University of Iowa, Iowa City, IA, USA,2Department of Medicinal Chemistry and Molecular Pharmacology, PurdueUniversity, West Lafayette, IN, USA.Histone post-translational modifications (PTMs) are key determinants of thelocal chromatin landscape and critical for regulation of eukaryotic geneexpression. These histone marks are deposited by a vast number of histonemodifying enzymes and preferentially recognized by specific associatedreader domains. The SANT/Myb family of domains is one of the least char-acterized histone reader domains. These domains commonly occur in tran-scription factors, nuclear co-repressors and chromatin modifying complexes,and are known to have DNA and unmodified histone tail binding activity.However, no structural information currently exists for SANT domain interac-tion with unmodified histone tails. There are two SANT domains of unknownfunction in Enhancer of Zeste 2 (EZH2), the catalytic subunit of the PolycombRepressive Complex 2 (PRC2) histone methyltransferase. Here, we show thatthe first SANT domain (SANT1) of EZH2 is a histone binding domain withspecificity for the unmodified histone H4 N-terminal tail. Using NMR spec-troscopy and molecular modeling we structurally characterize the SANT1domain and determine the molecular mechanism of binding to the H4 tail pep-tide as well as in the context of the nucleosome. In addition, we find that acet-ylation of H4K16 (H4K16ac) or acetylation or methylation of H4K20(H4K20ac and H4K20me3) abrogate SANT1 binding, which is consistentwith these modifications being anti-correlated with H3K27me3 in-vivo. Ourresults provide the first characterization of the molecular mechanism of his-tone binding by SANT domains.

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2208-Pos Board B224Dynamics of Eukaryotic Histone Exchange with Single MoleculeResolutionMohamed Ghoneim1,2, Chia-Liang Lin3, Elizabeth A. McCormack3,Dale B. Wigley3, David Rueda1,2.1Single-Molecule Imaging Group, MRC London Institute of MedicalSciences, London, United Kingdom, 2Molecular Virology, Department ofMedicine, Imperial College London, London, United Kingdom, 3StructuralBiology, Department of Medicine, Imperial College London, London, UnitedKingdom.Remodelling chromatin structure is important for regulating gene expression,DNA replication and repair and other fundamental nuclear processes. The basicchromatin unit is the nucleosome. SWR1 is a multi-subunit complex, whosechromatin remodelling activity is associated with regulation of gene expressionin heterochromatin regions of chromosomes in plants and mammals, and withthe cellular response to DNA damage. In yeast, the simplest eukaryotic organ-ism, the SWR1 complex is responsible for the ATP-dependent nucleosome re-modelling by exchanging its canonical H2A histone with Htz1 variant (alsoknown as H2A.Z in mammalian cells). In spite of a large number of genetic,biochemical and structural studies on SWR1, its detailed histone exchangemechanism remains largely unknown. To investigate the mechanism of histoneexchange by SWR1, we have developed a single-molecule FRET assay, whichmonitors the interaction between individual nucleosomes and yeast SWR1complexes in real time. The data show distinct dynamic behaviours in the pres-ence or absence of ATP, or in the presence of non-hydrolysable ATP analogue.We hypothesize that the observed dynamics are important for the removal ofcanonical H2A histones or deposition of the histone variant Htz1. We anticipatethat our data will help elucidate the molecular mechanism of histone exchangeby SWR1.

2209-Pos Board B225GenomeWide Measurements of the Sequence Dependence of NucleosomalDNA FlexibilityAakash Basu1, Michael T. Morgan2, Basilio C. Huaman2,Tunc Kayikcioglu2, Thuy Ngo3, Qiucen Zhang3, Cynthia Wolberger4,Taekjip Ha4.1Biophysics and Biophysical Chemistry, Johns Hopkins University,Baltimore, MD, USA, 2Johns Hopkins University School of Medicine,Baltimore, MD, USA, 3Biophysics and Biophysical Chemistry, University ofIllinois Urbana Champaign, Urbana, IL, USA, 4Johns Hopkins University,Baltimore, MD, USA.The physical properties of DNA such as bending flexibility are sequence depen-dent. However, how DNA flexibility varies across the genome, how sequencemutations affect downstream biological processes by changing DNA flexi-bility, and how DNA flexibility may have contributed to the evolutionary selec-tion of DNA sequences, are questions that have rarely been explored. Bycombining single molecule techniques with next generation sequencing, wehave developed an assay to create a genome wide map of DNA flexibility.An input library of tens of thousands of short DNA fragment is end modifiedto permit cyclization. A selection condition is imposed, whereby stiffer mole-cules that fail to cyclize are preferentially digested. The surviving moleculesare sequenced and ranked in flexibility according to their survival probability.We have applied this technique to study the flexibility of DNA at ten thousandnucleosomal locations in Saccharomyces cerevisiae. We find that nucleosomaloccupancy correlates with local DNA flexibility, suggesting that DNA flexi-bility has played a role in the evolution of nucleosomal sequences. Correlationsbetween DNA flexibility asymmetry and histone occupancy asymmetry withinnucleosomes suggest that in vivo, nucleosomes exist in partially unraveledstates, and DNA is under tension. Finally, our data shows that DNA flexibilitypatterns on the first few nucleosomes downstream of transcription start sitesmay have evolved to facilitate initiation of transcription, and is a predictor ofgene expression levels. A detailed understanding of how DNA sequences,DNA chemical modifications, and DNA:protein interactions affect downstreambiological processes by altering local DNA flexibility, will allow us to appre-ciate how physical forces have shaped the evolution of genomes.

2210-Pos Board B226Exposing Chromosome Architecture andMechanics Using Optical Manip-ulation and Fluorescence MicroscopyAnna E.C. Meijering1, Kata Sarlos2, Anna H. Bizard2, Seyda Acar1,Andres B. Venegas2, Rahul Bhowmick2, Ying Liu2, Iddo Heller1,Ian Hickson2, Erwin J.G. Peterman1, Gijs J.L. Wuite1.1Physics and Astronomy, Vrije Universiteit Amsterdam, Amsterdam,Netherlands, 2Centre for Crhomosome Stability, University of Copenhagen,Copenhagen, Denmark.

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Chromosomes are highly flexible structures that undergo dramatic structuralchanges during the cell cycle. The highly condensed characteristic X-shapethat chromosomes adopt during mitosis suggest that shape and mechanicalproperties are essential for correct segregation. However, how chromosomesegregation is regulated and how chromosome structure supports correct segre-gation is still poorly understood. Moreover, many questions concerning theexact composition and structure of the mitotic chromosome and about howthey are formed are still open. To answer these questions, we have developeda technique that allows for the in vitro manipulation and imaging of individualhuman chromosomes. This technique relies on the attachment of chromosomes- at their telomeres or centromeres - to microspheres that we can manipulatewith an optical tweezers setup. We show that we can apply and measure forcesup to 1 nN either from the attachment site of microtubules at the kinetochore orfrom telomeres at the ends of the chromosome arms. At the same time, oursetup allows for quick buffer exchange and fluorescent imaging. This enablesus to measure not only the mechanics of chromosomes but also image specificproteins within or interacting with the chromosomes. We provide a proof ofprinciple of this new approach as well as data on the effect of external agentson chromosome condensation and structure.

2211-Pos Board B227Folding, Bridging, and Compaction of DNA by Nucleoid Associated Pro-tein HfqJohan R.C. van der Maarel1, Antoine Malabirade2, Veronique Arluison2.1National University of Singapore, Singapore, Singapore, 2Universite ParisSaclay, Paris, France.Nucleoid Associated Proteins (NAPs) play a key role in the folding, compac-tion and expression of the prokaryotic genome. We have investigated an exem-plary NAP, that is Escherichia coli Hfq. Hfq takes the form of a hexameric, N-terminal torus with six protruding C-terminal arms. It is a bacterial pleiotropicregulator that mediates several aspects of nucleic acids metabolism. It notablyinfluences translation and turnover of RNAs, but it also known to associate withdouble stranded DNA in vivo and in vitro. Here, we explore the role of Hfq inthe compaction of double stranded DNA. Various experimental methodologies,including fluorescence microscopy imaging of single DNA molecules confinedinside nanofluidic channels, atomic force microscopy, isothermal titrationmicrocalorimetry, small angle neutron scattering, and electrophoretic mobilityassays have been used to follow the assembly of full length Hfq as well as its C-and N-terminal regions on DNA. Results highlight the role of Hfq’s C-terminalarms in DNA binding, change in mechanical properties of the double helix andcompaction of DNA into a condensed form. The propensity for bridging andcompaction of DNA by the C-terminal domain might be related to aggregationof bound protein and may have implications for protein binding related generegulation.

2212-Pos Board B228Structure and Function of Archaeal HistonesBram Henneman1, Clara Van Emmerik1, Thomas Brouwer2,Ramon A. Van der Valk1, Nancy Kirolos1, Hugo Van Ingen1,John Van Noort2, Remus T. Dame1.1Leiden Institute of Chemistry, Leiden University, Leiden, Netherlands,2Leiden Institute of Physics, Leiden University, Leiden, Netherlands.The genomes of all organisms throughout the tree of life are compacted andorganized in chromatin by association of chromatin proteins. Eukaryotic ge-nomes encode histones, which are assembled on the genome into octamers,yielding nucleosomes. Like eukaryotes, most Archaea express histones, whichare believed to be involved in the compaction and organization of their ge-nomes. Instead of discrete multimers, in vivo data suggest assembly of ‘nucle-osomes’ of variable size, consisting of multiples of dimers, which are able toinduce repression of transcription. Based on these data and a recent modelderived from X-ray crystallography with the histone HMfB from Methano-thermus fervidus on artificial high-affinity DNA, it was recently proposedthat archaeal histones assemble on DNA into ‘endless’ hypernucleosomes.However, hypernucleosomes have to date not been shown to exist in solution,or on natural DNA. Also, the crystal structure does not reveal which interac-tions hold the hypernucleosome together. We use Tethered Particle Motion(TPM) and Magnetic Tweezers (MT) to show that HMfB cooperatively bindsand compacts naturally occurring DNA and forms a hypernucleosome insolution. Also, we show that stacking interactions between the layers of thehypernucleosome allow for stability of the hypernucleosome. Using thehistone HMfA, a natural mutant of HMfB, we find that the absence ofstacking interactions leads to loss of cooperativity and less dramatic DNAcompaction. Based on our findings, we propose structural models for therole of archaeal histones in repression of transcription and modulation ofrepressive activity.

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Posters: Membrane Physical Chemistry III

2213-Pos Board B229The Physical Characterization of Microvesicles Secreted From Thermoa-cidophilic Archaea and Liposomes Reconstituted fromMicrovesicle LipidsAlexander P. Bonanno, Parkson L.-G. Chong.Biochemistry, Temple University, Philadelphia, PA, USA.While microvesicles (MVs) from eukaryotes have been extensively studied,little is known about the biophysical properties of MVs secreted by thermoa-cidophilic archaea. In this study, we have isolated MVs from the crenarchaeonSulfolobus acidocaldarius (growth conditions: 75 oC and pH 2-3) and engi-neered liposomes comprised of the MV lipids, which have been reported tobe the tetraether lipids with the GTGT and GDGT core structure. TheseMVs are unique, as they are able to withstand the high temperature andlow pH environment, making them a very durable biomaterial for applicationssuch as carriers for therapeutics. In this study, we used dynamic light scat-tering, fluorescence spectroscopy, and dielectric measurements to characterizethe native MVs as well as reconstituted liposomes and painted planar mem-branes over pinholes. Both native MVs and reconstituted liposomes are stableover several months at ambient conditions. Laurdan fluorescence obtainedfrom both native MVs and reconstituted liposomes exhibit a much higherREES (red-edge excitation shift) value than that obtained from liposomesmade from diester lipids (e.g., POPC and DPPC), suggesting tight membranepacking in archaeal MVs and the liposomes reconstituted from MV lipids.Laurdan’s GP value exhibits a monotonic decrease from 0.15 to �0.08 overthe temperature range of 25-80 �C with no clear evidence of a phase transi-tion. Differential polarized phase-modulation fluorometry of DPH has alsobeen employed to characterize the archaeal MVs and the reconstituted lipo-somes. Planar membranes made from tetraether lipids extracted from S. acid-ocaldarius MVs exhibited a stable capacitance for over 8 hours compared tomembranes made from POPC, which lasted only for 2 hours (supported byNSF).

2214-Pos Board B230Biophysical Analysis of Extracellular VesiclesPietro Parisse.Elettra Sincrotrone Trieste, Trieste, Italy.Extracellular vesicles (EVs) are small vesicles ensuring transport of moleculesbetween cells and throughout the body. EVs contain cell-type specific signa-tures and have been proposed as biomarkers in a variety of diseases. Their smallsize and biological and physical functions make them optimal candidates fortherapeutic agents in immune therapy, vaccination, regenerative medicine,and drug delivery. Indeed there is no objective set of criteria available fordesigning synthetic EVs for a specific task in biomedicine. It is therefore urgentand critical to address these issues for EVs-based medicine to fulfill its promise.Here, we try to assess the phenotypic properties of EVs, through a multi-technique characterization based on Single Particle Tracking techniques,FTIR spectroscopy, Atomic Force Microscopy (AFM) and Small AngleX-Ray Scattering (SAXS). This detailed analysis allows us to model, visualizeand quantify EVs’ physical and chemical properties up to single vesicle leveland it serves as a basis for the correlation of phenotypic parameters of EVswith their functional activity. The proposed work could allow for the designof innovative strategies for their sorting and detection, and for the personalizednanomedicine in general.

2215-Pos Board B231Non-lameller Lipid Liquid Crystalline Phases - Controlling the FormedStructure using Lipolytic Enzymes with Different SpecificityMaria Wadsater1, Justas Barauskas2,3, Fredrik Tiberg1,2,Tommy Nylander1,4.1Physical Chemistry, Lund University, Lund, Sweden, 2Camurus AB, Lund,Sweden, 3Biomedical Science, Malmo University, Malmo, Sweden,4NanoLund, Lund University, Lund, Sweden.Lipids in living organisms do not always confined in bilayer structures, butthey can also assembly into intriguing 3D structures. Well-defined model sys-tem will help us understand the biological implication as well as develop newapplications, for e.g. biomedical devices and targeted delivery. Such differentstructures can be generated or evolved with the help of specific lipolytic en-zymes. Here we will demonstrate that the lipolysis-induced evolution of aparticular structure from reverse lipid phases formed by mixtures of lipids,which invoke different curvature, is indeed controlled by the type of lipolyticenzyme. For this purpose we used highly structured cubic micellar (Fd3m)nanoparticles of 50/50 (wt%/wt%) soy phosphatidyl choline (SPC)/glycerol

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dioleate (GDO). The two types of lipolytic enzymes used were phospholipaseA2 (PLA2) that catalyses degradation of the phospholipid component, SPC,and porcine pancreatic triacylglycerol lipase (TGL) that facilitate the hydroly-sis of the diglyceride, GDO. Phospholipase A2, which promotes the hydroly-sis of the lamellar forming component, SPC, induces a reversed micellarphase. However triacylglycerol lipase, which hydrolysis the reverse phaseforming compound, GDO, induces a lamellar phase. The lipid particleswere found to retain their integrity throughout the whole time of reactionstudied.

2216-Pos Board B232Depth-Dependent Physical Properties of Model Biological Lipid BilayersGanesh Shahane.Queen Mary University of London, London, United Kingdom.Lipid bilayers are thin, polar structures made up of two layers of lipid mole-cules. They are one of the constituents of the cellular plasma membrane, whichforms a curved and continuous barrier around the cells and are responsible forexchange of molecules in and out of the cell. The type and composition of lipidsthat make up the many different types of bilayers can alter its various physicalproperties that change significantly with depth, such as the highly fluctuatinglateral pressure profile. Studying the various depth-dependent properties cangive important insights into membrane functionality and the way they modulatemembrane proteins, which could be hard to investigate by experimental means.In this study, we use atomistic molecular dynamics simulations to quantify anumber of bilayer properties in model bacterial, mammalian and cancer mem-branes that have heterogeneous lipid compositions. We examine some basicproperties such as lipid area and volume, bilayer thickness and bending rigidity,as well as transmembrane profiles for electron density, lateral pressure anddipole potential. Our results show how individual lipids modulate physicalproperties of these heterogeneous bilayers, with potential implications on mem-brane function.

2217-Pos Board B233Biophysical Characterization of Lipid Membranes: Effect of Lipid HeadGroups and Tails on Synthetic and Natural Lipid MembranesYoung Hun Kim, Joon Lee, Ratnash Lal, Jerry Yang.University of California, San Diego, La Jolla, CA, USA.Lipid membranes play important roles in cell biology. They serve as a structurethat protects cellular organelles and maintains unique cell shapes and also as aregulator that controls cellular functions. Thus, understanding the biophysicalcharacteristics lipid membranes, e.g., fluidity and elasticity, is essential, asmembrane proteins in cells often modulate these characteristics to properlycontrol cellular activities. Here, we investigate various lipid head groups andtails by utilizing three different experimental techniques: 1) fluorescence recov-ery after photobleaching (FRAP), 2) black lipid membrane (BLM) electrophys-iology recordings, and 3) atomic force microscopy (AFM). Our results showlipid membranes composed of either negatively charged headgroups or lactocy-lated headgroups have slower lateral diffusion coefficients and reduced elastic-ity compared to lipids with neutrally charged headgroups or zwitterionicheadgroups. Addition of methyl groups in the acyl chain of the lipids decreaseslateral diffusion but does not change elasticity of the membrane. Further, westudy the biophysical characteristics of two natural lipid membranes extractedfrom E. coli and P. furiosus, which have known lipid compositions are distinctfrom each other, and compare our findings from synthetic lipids to understandthe biophysical characteristics of natural membranes. We observed that P. fur-iosus lipid membranes exhibit a slower lateral diffusion and less elasticitycompared with E. coli lipid membranes. From correlation among lateral diffu-sion, gA lifetime (local bending energy), and elasticity, we found that lipidmembranes with slower lateral diffusion shows bigger local bending energyand membrane elastic modulus.

2218-Pos Board B234Quantifying Asymmetry in Detergent-Membrane InteractionsHelen Y. Fan1, Ndjali Quarta2, Heiko Heerklotz2.1University of Toronto, Toronto, ON, Canada, 2University of Freiburg,Freiburg, Germany.Detergent-membrane interactions are important considerations in membraneprotein studies and in the formulation of pharmaceutics and pesticides.Symmetric interactions between detergents and model membranes arewell-described by the three-stage model, which requires fast flip-flop ofthe detergent between the two leaflets of the bilayer. Asymmetric interac-tions arise from slow flip-flop; the membrane may respond by sudden equil-ibration (cracking in), exovesiculation (budding off), or coexisting withalmost pure detergent micelles (staying out). These interaction mechanismsare influenced by membrane composition, ionic strength, and detergent

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structure. We use isothermal titration calorimetry, dynamic light scattering,and asymmetric flow field flow fractionation to quantify these interactionsand determine the conditions required for asymmetry.

2219-Pos Board B235Cardiolipin Partitioning in Mixed Membrane SystemsMargaret M. Elmer-Dixon, Bruce E. Bowler.University of Montana, Missoula, MT, USA.Cardiolipin (CL) is an unusual phospholipid found exclusively in the innermitochondrial membrane (IMM) of eukaryotes. During regular cell function,CL tethers Cytochrome c (Cytc) to the IMM. During the initial stages ofApoptosis, Cytc oxidizes CL facilitating the release of Cytc into the cyto-plasm where Cytc forms part of the apoptosome. With four mono-unsaturated acyl chains and a headgroup comprised of two phosphates,CL’s unique structure gives rise to preferential localization on concave sur-faces. CL constitutes 20% of the mitochondrial membrane and is often com-bined with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to model theIMM in vitro. Unlike CL, DOPC shows no preferential localization to curvedsurfaces. Studies investigating Cytc-CL binding using CL/DOPC mixed lipidsystems often assume unbiased lipid mixing throughout the model membranesystems. Using the recently synthesized fluorophore, 1,1,2,2,-tetrakis[4-(2-tri-methylamminoioethoxy)phenyl]ethene (TTAPE-Me), CL concentration wasevaluated on the inner and outer leaflets of 100 nm large unilamellar vesicles(LUV) at varying ratios of CL and DOPC. By exposing TTAPE-Me selec-tively to the inner versus the outer surface of the LUV, CL leaflet partitioningwas detected via fluorescence. Interestingly, CL partitioning occurred tovarying degrees at different lipid concentration ratios suggesting a mixingthreshold for the CL/DOPC system. These findings highlight the importanceof accounting for the bias in CL partitioning in evaluating Cytc-CL bindingdata. Cytc-CL binding was investigated under solution conditions fosteringelectrostatic Cytc-CL interactions for a mixed system of 20% CL/80%DOPC and for pure DOPC. Under these conditions, no Cytc binding was de-tected to either liposome system further demonstrating the preferential parti-tioning of CL to the inaccessible inner leaflet of the LUV membrane.

2220-Pos Board B236Structural Analogs of Palmitoyl Ceramide and their Functions inMembranesAnna Mouts1, Elina Vattulainen1, Takaaki Matsufuji2, Masanao Kinoshita2,Nobuaki Matsumori2, J. Peter Slotte1.1Faculty of Science and Engineering, Abo Akademi University, Turku,Finland, 2Department of Chemistry, Kyushu University, Fukuoka, Japan.Ceramides are precursor molecules in the sphingolipid family and consist of asphingosine backbone and an acyl chain. Normally the amount of ceramide isvery low in membranes, but can rise due to different stress stimuli or cell deathsignals. As ceramides form ordered domains in otherwise fluid membrane en-vironments, they can affect both lateral and transmembrane movement oflipids and membrane proteins. This is partly due to the hydrogen bonding ca-pabilities of the sphingolipid family. The small headgroup (hydroxyl) of cer-amide also allows for close interactions of ceramides with each other but alsowith other lipids in their environment. Both these factors promote phase-separation. In our study, we have investigated how modification of the1-OH group affects the ceramide behaviour in both binary and complex mem-brane systems. Ceramides with modifications at this position have not beenencountered in living cells as this functional group is important in biosyntheticpathways. Ceramides modified in the 1-OH position are thus also protectedfrom oxidation in cells. To elucidate the differences between these lipids wehave used fluorescence lifetime measurements and determined the thermosta-bility of the ordered domains by fluorescence anisotropy. From our results itseems that even if the 1-OH position is modified the ceramides maintain prop-erties similar to natural ceramide. This opens possibilities of developing inhib-itors of ceramide related enzymes.

2221-Pos Board B237Complex Effects of 24:1 Sphingolipids in Membranes ContainingDioleoylphosphatidylcholine and CholesterolAritz B. Garcia-Arribas, Emilio J. Gonzalez-Ramirez, Jesus Sot, Itziar Areso,Alicia Alonso, Felix M. Goni.Instituto Biofisika (CSIC-UPV/EHU), Leioa, Spain.The effects of C24:1 sphingolipids have been tested in phospholipid bilayerscontaining cholesterol. Confocal microscopy, differential scanning calorim-etry, and atomic force microscopy imaging and force curves have beenused. More precisely, the effects of C24:1 ceramide (nervonoyl ceramide,nCer) were evaluated and compared to those of C16:0 ceramide (palmitoylceramide, pCer) in bilayers composed basically of dioleoylphosphatidylcho-

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line, sphingomyelin (either C24:1, nSM or C16:0, pSM) and cholesterol.Combination of equimolecular amounts of C24:1 and C16:0 sphingolipidswere also studied under the same conditions. Results show that both pCerand nCer are capable of forming segregated gel domains. Force spectroscopydata point to nCer having a lower stiffening effect than pCer, while the pres-ence of nSM reduces the stiffness. DSC reveals Tm reduction by nSM inevery case. Furthermore, pSM seems to better accommodate both ceramidesin a single phase of intermediate properties, while nSM partial accommoda-tion of ceramides generates different gel phases with higher stiffnessescaused by interceramide cooperation. If both pSM and nSM are present, aclear preference of both ceramides toward pSM is observed. These findingsshow the sharp increase in complexity when membranes exhibit differentsphingolipids of varying N-acyl chains, which should be a common issuein an actual cell membrane environment.

2222-Pos Board B238Regional Cooperativity in Thermotropic Lipid Phase Transitions - aComment on the Fine Structure of the Main Transition PeakBeate Klosgen, Olesya P. Jensen, Brian B. Jensen, Chen Shen.Dept. for Physics, Chemistry, and Pharmacy, University of SouthernDenmark, Odense M, Denmark.Functional biomembranes are reactive platforms of life functions.Formally, they are complex bilayer systems established by a matrix of li-poids that carry further molecules, e.g. proteins. Model systems are cho-sen for studying physico-chemical aspects under well-defined constraints,for example in order to address material properties in reduced systems offew components only, and by a continuum physics approach. Systemsmay thus be characterized in terms of phase behaviour and miscibility,elasticity and sound propagation, heat and electric conductivity, andrelated structures and their dynamics. As for purely lipid membranes,model systems of phosphatidylcholines were established, e.g. di-myristoyl-phosphatidyl-choline and di-palmitoyl-phosphatidyl-choline(DMPC, DPPC). Their properties were extensively studied, and used forbenchmarking upon interaction with other biorelevant molecules ascholesterol or antibiotic peptides. Still, even these apparently simple sys-tems are left to be fully understood. One finding is another isothermalphase transition in DPPC monolayers that we recently reported, withpossible relevance for the understanding of lung surfactant. Anotherobservation is the distinct substructure in the thermotropic phase transi-tion of saturated phospholipids that still is under discussion. Here wereport more details on the phase transition of phosphatidylcholines, bothsaturated (DMPC) and non-saturated (palmitoyl-oleoyl-phosphatidylcho-line, POPC), and tracked by quasi-equilibrium differential scanning calo-rimetry (DSC, 0.05K/min) on aqueous suspensions of multilamellarvesicles (MLVs) or small unilamellar vesicles (SUVs, F=100nm). ForSUVs of DMPC, the main transition was found to comprise three contri-butions. Our new results indicate a regional cooperativity in the phasetransition. It is probably triggered by the chain melting and superim-posed by a contribution from the headgroups. The results further indicatean excess heat that may be attributed by bending contribution in freeenergy.

2223-Pos Board B239Lipid Interactions: Comparison of Experiment, Theory, and SimulationPaulo F. Almeida.Chemistry, Univ North Carolina, Wilmington, NC, USA.The measurement of interactions between lipids in a bilayer is a difficult exper-imental problem. Indirect measurements can be obtained from the experimentalheat capacity function Cp(T) of lipid phase transitions by comparing Cp(T) inexperiment with theory or simulation, and determining which interactionparameters lead to agreement between the two. These estimates have been ob-tained from Monte Carlo simulations by comparison with DSC data for a num-ber of lipids. The method works about a phase transition that absorbs or releasesheat. In lipid mixtures, the method is less sensitive, but it is possible to find setsof parameters that result in acceptable fits to Cp(T). The best experimentalapproach is probably the nearest neighbor recognition method developed byRegen and coworkers. This method uses lipids modified with a thiol (SH) inthe lipid headgroup, which allows monitoring lipid dimerization and quantifi-cation of nearest neighbor pairs. The method thus measures the equilibrium dis-tributions of homologous (AA, BB) and heterologous (AB) nearest neighbors inbinary lipid mixtures. The results yield an equilibrium constant given by KAB=[AB]2/([AA][BB]). The differential Gibbs energy of interaction between A andB is given by uAB=εAB �½(εAAþεBB), where the ε are, in general, Gibbs en-ergies. In the quasi-chemical approximation, the two quantities are related by

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uAB = �½ RT ln(KAB/4), where the factor of 4 is necessary to correct for thepurely statistical advantage of forming heterodimers over homodimers in arandom mixture. The question addressed here is, how good is the quasi-chemical approximation? In this work, KAB is calculated from Monte Carlosimulations using defined values of uAB and compared with the equation ofthe quasi-chemical approximation, uAB = �½ RT ln(KAB/4), and with KAB

available from experiment, for a number of lipid mixtures. (Supported byNSF grant CHE-1464769.)

2224-Pos Board B240Experimental Measurement of the Gibb’s Free Energy of Mixing forHydroxycholesterol-Phospholipid MonolayersJoan C. Kunz1, Blair Stewig2, Vision B. Bagonza2, Benjamin L. Stottrup3.1Chemistry, Augsburg University, Minneapolis, MN, USA, 2AugsburgUniversity, Minneapolis, MN, USA, 3Physics, Augsburg University,Minneapolis, MN, USA.Hydroxycholesterols are implicated in the management cell membrane choles-terol levels as well as various pathologies. These oxidized versions of choles-terol include a second hydroxyl functional group. We have previously usedfluorescence microscopy and traditional Langmuir trough techniques to mea-sure the unique phase behavior of multicomponent hydroxycholesterol-phospholipid monolayers (7ß-OH, 20-OH, 22(R)-OH, 22(S)-OH, 25-OH and27-OH ). Here we have extended that analysis to utilize pressure-area isothermsto measure area-condensation and expansion as well as the Gibbs free energy ofmixing. The unique area expansion exhibited by these model monolayer sys-tems at low compressions (large molecular area) and novel miscibility phasebehavior provide a potential insights into more commonly studied systems ofphospholipids and cholesterol.

2225-Pos Board B241iSCAT Microscopy of Phase Separated Lipid MembranesMatthew C. Blosser1,2, Helena L.E. Coker1,2, Mark I. Wallace2.1Chemistry, University of Oxford, Oxford, United Kingdom, 2Chemistry,King’s College London, London, United Kingdom.Characterizing the lateral organization of lipid membranes is important for un-derstanding the function of cell membranes. The most common way to deter-mine miscibility phase behavior is using fluorescence microscopy, where thepresence of separate phases is imaged by the preferential partitioning of alabeled lipid. However, fluorescence microscopy requires adding an additionallipid component, and can also lead to photooxidation. Interference scatteringmicroscopy (iSCAT) is a label-free imaging technique that images the differ-ences in light scattering from different regions of a sample. iSCAT is capableof imaging coexisting domains. We quantify the sensitivity of iSCAT to phys-ical parameters including thickness mismatch, topography, and the presence ofdefects.

2226-Pos Board B242Analyzing Simulations of Lipid Mixtures: Phase Boundaries, Tie-Linesand Critical PointsCl�ement Arnarez1, Siewert J. Marrink2, Manuel N. Melo1.1Instituto de Tecnologia Quımica e Biologica Antonio Xavier, UniversidadeNova de Lisboa, Lisbon, Portugal, 2Groningen Biomolecular Sciences andBiotechnology Institute, University of Groningen, Groningen, Netherlands.Formation of membrane domains, or phases, in different lipid mixtures is awell-known occurrence. It remains unclear how biologically relevant this phe-nomenon can be, but in spite of that — or perhaps because of it — membraneseparation has been extensively characterized.A range of experimental techniques has been employed for characterizing thephase separation of lipid mixtures, ultimately producing phase diagrams. How-ever, for the construction of these diagrams the definition of phase boundariesand the determination of tie-lines is a laborious and costly endeavor, requiringthe estimation of lipid compositions of the different domains that phase-separate.In recent years coarse-grained molecular dynamics became able to overcomethe time- and lengthscales needed to observe domain formation. This tech-nique positioned itself as a promising alternative that can yield unparalleledstructural information, while avoiding the costs associated with studyingincreasingly exotic lipids. Analyses of simulation results of phase-separationdata has, however, been rather qualitative and disconnected from thecompleteness aimed at in experimental studies. This is somewhat of aparadox, since simulations can provide a degree of detail that experimentscannot.

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In this work we automate the simulation analysis of 51 ternary mixtures of asaturated lipid (di-palmitoyl phosphatidylcholine), a polyunsaturated one (di-linoleyl phosphatidylcholine), and cholesterol. Local neighboring compositionfor each lipid and simulation frame is measured and plotted as an individualdata point in a ternary diagram. This clarifies the visualization of phase homo-geneity or separation. Further processing of the data allows us to define phaseboundaries and tie lines. Thus, by making a deeper use of the informationavailable in simulations our method can yield more complete phase diagrams,comparable to the ones produced experimentally, without any modification tosimulation methods.

2227-Pos Board B243Measuring Partition Coefficient between Liquid-Disordered (LD) andLiquid-Ordered Phases. Why are Phase Diagrams Important to Know?Thais A. Enoki, Gerald W. Feigenson.Department of Molecular Biology & Genetics, Cornell University, Ithaca,NY, USA.We study and exploit methods to measure the partition coefficient of fluores-cent molecules between liquid-disordered (Ld) and liquid-ordered (Lo)phases in 3 or 4-components lipid mixtures. A trajectory is created of 60-70 different lipid composition samples along a thermodynamic tieline, andthe fluorescence signal of a single probe is measured for each sample. Todeterminate the partition coefficient, the single dye fluorescence is fitted bya hyperbolic curve based on the fraction of Ld or Lo phase. Because self-quenching could disturb the fluorescence profile along the tieline, we developa self-quenching correction and formulate an equation to account for this ef-fect. Briefly, we first studied how the fluorescence self-quenching behaves forincreased dye concentration. Then we applied this correction along the tie-line, since the dye concentration and the self-quenching effects vary withthe fraction of Ld and Lo phases. Without this correction for fluorescenceself-quenching, significant partition coefficient errors can occur. We showexperiments using different dye concentrations where our corrected equationconveys the same partition coefficient measurement. We tested dyes thatfavor Ld phase, TopFluorPC and C12:0DiI, and TopFluor Ceramide that fa-vors the Lo phase. We also compare our results to partition coefficient mea-surements using giant unilamellar vesicles (GUVs). We call attention to howwell-resolved phase diagrams can be crucial for any measurement of thepartition coefficient.

2228-Pos Board B244Detection of Pure Cholesterol Bilayer Domains in Biological MembranesOverloaded with Cholesterol: Methodology Development and its Applica-tion to Porcine Lens Membrane StudiesLaxman Mainali1, William J. O’Brien2, James S. Hyde1,Witold K. Subczynski1.1Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA,2Ophthalmology, Medical College of Wisconsin, Milwaukee, WI, USA.In the eye lens’ fiber cell membranes, which are loaded with cholesteroland dense with integral membrane proteins, four purported lipid domainsare expected: bulk, boundary, and trapped lipids, as well as pure choles-terol bilayer domain (CBD). Three of these domains, namely, bulk,boundary, and trapped lipids, have been identified [1]. However, showingthat the CBD is present in these intact membranes, even though it wasclearly detected in lens lipid membranes prepared from total lipid extractsfrom fiber cell plasma membranes [2], was difficult. Here, this problemwas solved using an advanced saturation recovery (SR) electron paramag-netic resonance (EPR) spin-labeling approach (modified discriminationby the oxygen transport method), making it possible to detect CBDs inintact fiber cell membranes. CBDs were detected only in those intactmembranes in which CBDs were also detected in the lens lipid mem-branes. These intact membranes include those from porcine lens nucleibut not porcine lens cortices. Lens lipid membranes prepared from porcinelens cortices of two-year-old animals were CBDs free [3], while thoseprepared from porcine lens nuclei contained CBDs [3]. Results obtainedfor intact membranes, when combined with those obtained for lens lipidmembranes, contribute to the understanding of the organization of lipidsin fiber cell plasma membranes of the eye lens. Acknowledgements: Sup-ported by NIH grants R01EY015526, R01EB002052, and P41EB001980.Literature: [1] M. Raguz, L. Mainali, W.J. O’Brien, W.K. Subczynski,Exp. Eye Res. (2015), 132:78-90. [2] L. Mainali , M. Raguz, W.J.O’Brien, W.K. Subczynski, Curr. Eye Res. (2017), 42:721-731. [3] L.Mainali , M. Raguz, W.J. O’Brien, W.K. Subczynski, Exp. Eye Res.(2012), 97:117-129.

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2229-Pos Board B245Pure Cholesterol Bilayer Domains are Formed at Cholesterol ContentsSignificantly Lower than Cholesterol Solubility Thresholds inPhospholipid Membranes: EPR and DSC StudiesLaxman Mainali, Witold K. Subczynski.Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA.Recently, using spin-labeled cholesterol analogs and saturation-recovery EPRapproaches along with DSC measurements, we were able to show that forma-tion of cholesterol bilayer domains (CBDs) precedes formation of cholesterolcrystals in dimyristoylphosphatidylcholine membranes [1]. Here, we demon-strated that this is also true for other phospholipid membranes. Because ourresearch interests focused on fiber-cell plasma membranes that build humaneye lenses, we investigated membranes made of phosphatidylserine (PS),phosphatidylethanolamine (PE), and sphingomyelin (SM). These phospho-lipids, together with phosphatidylcholine (PC), are major components offiber-cell plasma membranes. Our recent and previous results showed thatCBDs begin to form at �33, �46, �48, and �50 mol% cholesterol for PE,SM, PS, and PC membranes, respectively. It was confirmed by DSC thatthe cholesterol solubility threshold is detected at �50, �66, �70, and �66mol% cholesterol for PE, SM, PS, and PC membranes, respectively. To pre-serve compositional homogeneity throughout the membrane suspension, lipidmultilamellar dispersions were prepared using a rapid solvent exchangemethod. Phospholipids contained both saturated acyl chains, or one acyl chainwas mono-unsaturated. These findings help us better understand the lateral or-ganization of membranes overloaded with cholesterol (such as the fiber-cellplasma membranes of the eye lens) and form new conclusions about the func-tion of cholesterol in these membranes. Acknowledgements: Supported byNIH grants R01EY015526, R01EB002052, and P41EB001980. Literature:[1] L. Mainali, M. Raguz, W.K. Subczynski, J. Phys. Chem. B. (2013),117:8994-9003.

2230-Pos Board B246Chemical Potential of Plasma Membrane Cholesterol Is RegulatedIndependently of Cell Cholesterol ContentArtem G. Ayuyan, Fredric S. Cohen.Physiology and Biophysics, Rush University Medical Center, Chicago, IL,USA.We have developed a method to measure the chemical potential of cholesterolin cell plasma membranes. We have used it to measure the cholesterol chemicalpotential in plasma membranes for a variety of cell types (e.g., erythrocytes,primary fibroblasts, cancer cell lines) under a variety of physiological condi-tions. We find that for erythrocytes, the chemical potential is z � 1 kBT;for primary cells in culture, z �2.2 kBT; for metastatic cancer cells, z� 1.3 kBT. We use crystalline cholesterol as our reference state, setat 0 kBT, so all our chemical potentials have negative values. The cholesterolchemical potential is directly dependent on cholesterol concentration in eryth-rocytes. However for nucleated cells, signaling molecules, such as cytokines,can cause chemical potentials to increase by z 1 kBT without a change inthe cells’ cholesterol content. We have also developed a means to set andhold (i.e., clamp) the cholesterol chemical potential of plasma membranes.Varying this clamped chemical potential over a wide range, from �2.1 kBTto �0.6 kBT, also does not significantly alter cell cholesterol content. Thisinvariance in cholesterol content occurs because when the cholesterol chemicalpotential of the bathing medium is adjusted, plasma membrane cholesterolquickly reaches equilibrium with the medium, achieving the same cholesterolchemical potential. We conclude that cells are able to actively regulate thechemical potentials of their membrane cholesterol, and do so, not by alteringthe amount of cholesterol, but by modifying interactions of cholesterol withother membrane components.

2231-Pos Board B247Molecular Dynamics Simulations Reveal the Impact of CompositionalAsymmetry in Phase-Separated Lipid Membranes on PhospholipidPhysical PropertiesMichael D. Weiner1, Gerald W. Feigenson2.1Field of Physics, Cornell University, Ithaca, NY, USA, 2Field of Biophysics,Cornell University, Ithaca, NY, USA.The plasma membrane of eukaryotic cells is compositionally asymmetric,meaning that different lipids are found in the cytosolic and exoplasmic leaf-lets. Since compositionally asymmetric bilayers are difficult to form in vitro,studies in silico play an especially important role. Model membranes embodythe behavior of the exoplasmic leaflet with coexisting liquid-ordered andliquid-disordered phases. We use all-atom Molecular Dynamics simulations

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conducted on the Anton2 supercomputer to study the effects of compositionalasymmetry and to examine the interactions between the two leaflets. In theouter leaflet, a phase-separated mixture of phosphatidylcholines and choles-terol is chosen, while the inner leaflet contains phosphatidylethanolamines,phosphatidylserines, and cholesterol. We consider the impact of asymmetryon order, clustering, and packing. Partitioning and clustering across from or-dered and disordered phases are studied. The effect of polyunsaturated acylchains is explored.

2232-Pos Board B248Asymmetry in Lipid Bilayers Supported on GlassAurelia R. Honerkamp-Smith.Physics, Lehigh University, Bethlehem, PA, USA.Supported lipid bilayers (SLBs) are frequently used as model systems tostudy the properties of lipids and membrane proteins. Important differencesbetween SLBs and GUVs have been observed, in particular, in the mobilityof lipids. Diffusion constants are reduced by the presence of a glass substrate,and liquid domains become immobile. In addition, some researchers find thatthe miscibility transition temperature is altered in supported bilayers contain-ing liquid-liquid phase coexistence. One explanation for this transition tem-perature shift is lipid asymmetry in the bilayer which may be induced by thepresence of the substrate or by the mechanism of SLB formation. Giant uni-lamellar vesicles formed from ternary mixtures of lipids can phase separate,forming micron-scale liquid domains below their miscibility transition tem-perature. When these GUVs are converted into SLBs by allowing them toburst onto a glass surface, the liquid domains are preserved and are visibleby fluorescence microscopy. Here I directly compare miscibility transitiontemperatures in GUVs with those of SLBs formed by rupturing GUVs ontoglass. I use fluorescence microscopy to determine transition temperaturesand systematically vary the lipid composition in order to identify asymmetriclipid components.

2233-Pos Board B249Manipulation of Length Scales in a Modulated Phase in Cell-DerivedGPMVs and Synthetic Model GUVsCaitlin E. Cornell1, Allison D. Skinkle2, Ilya Levental2,Kandice R. Levental2, Sarah L. Keller1.1Chemistry, University of Washington, Seattle, WA, USA, 2IntegrativeBiology and Pharmacology, McGovern Medical School at the University ofTexas Medical Center, Houston, TX, USA.A strikingly diverse set of physical and chemical systems exhibit the high-ly organized macroscopic patterns of a modulated phase. Lipid membraneswith modulated phases exhibit a spatially periodic pattern whose domainsize can be experimentally tuned. Here we investigate modulated phasesin cell-derived giant plasma membrane vesicles (GPMVs) and in syntheticgiant unilamellar vesicles (GUVs). Using fluorescence microscopy, weimage stripes and hexagonally-packed circular domains within ternaryGUV membranes containing excess area in the outer leaflet and within un-treated GPMV membranes. We tune the length scale of modulated patternsin these GUV and GPMV membranes through manipulation of tempera-ture, lipid composition, and osmotic pressure. Our results show qaulitativeagreement with theoretical predictions for a modulated phase in bilayermembranes.

2234-Pos Board B250Lipid Domain Size Distribution and Line Tension in LangmuirMonolayersBenjamin L. Stottrup1, Vision B. Bagonza2, Juan Tigre3,Joseph A. Zasadzinski4, Joan C. Kunz5.1Physics, Augsburg University, Minneapolis, MN, USA, 2AugsburgUniversity, Minneapolis, MN, USA, 3Physics, University of Minnesota,Minneapolis, MN, USA, 4Chemical Engineering & Materials Science,University of Minnesota, Minneapolis, MN, USA, 5Chemistry, AugsburgUniversity, Minneapolis, MN, USA.Phase coexistence between liquid phases in lipid membranes provides a idealmodel system to investigatemixing in twodimensions. In thisposterwewill extendour previous comparison of passive observational techniques for line tensionmea-surements in lipid monolayers to more complex systems. Using fluorescencemicroscopy and Langmuir troughs we will illustrate the role that experimentalparameters (composition, transition kinetics, etc.) play in determining the size dis-tribution for several lipid compositions. We also will utilize the novel two phaseliquid-liquid coexistence regions of hydroxycholesterol-phospholipid lipid mono-layers to test size distribution measurements of line tension.

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2235-Pos Board B251Reversible Separation of Living, Unperturbed Cell Membranes intoLiquid PhasesGlennis E. Rayermann, Scott P. Rayermann, Caitlin E. Cornell,Alexey J. Merz, Sarah L. Keller.Chemistry, University of Washington, Seattle, WA, USA.A contentious question is whether living, unperturbed cells employ spon-taneous lateral demixing of membranes into coexisting liquid phases toorganize proteins and lipids on micron scales. Since the 1960s, researchershave observed domains within yeast vacuolar membranes that are consis-tent with, but not conclusive of, liquid-liquid phase separation. Therefore,we imaged in vivo yeast vacuoles labeled with a Vph1-GFP fusion proteinin order to test whether vacuole membranes exhibited two defining indica-tors of phase separation: (1) domain coalescence and (2) the existence of amiscibility temperature, Tmix. We observed domains with rounded, smoothshapes that coalesce in seconds and subsequently change shape within tensof seconds, characteristic of fluid phases. In both in vivo and cell-free vac-uoles, we found a distinct Tmix above which domains disappear and belowwhich they reappear over multiple temperature cycles. Our results showthat large-scale membrane organization in living cells under physiologi-cally relevant conditions can be controlled by tuning a single thermody-namic parameter. Cells may access this same phase transition throughinternal or external molecular cues. Indeed, recent work by AlexandreToulmay, William Prinz, Ted Powers, and Jodi Nunnari (Journal of CellBiology, 2017) shows that membrane domains control a growth signalingpathway that is conserved across the broad evolutionary diversity ofeukaryotes.

2236-Pos Board B252How HIV-1 Takes Advantage of PI(4,5)P2 Clusters during Viral AssemblyYi Wen, Volker M. Vogt, Gerald W. Feigenson.Molecular Biology & Genetics, Cornell University, Ithaca, NY, USA.Located at the inner leaflet of the plasma membrane (PM), phosphatidyli-nositol 4,5-bisphosphate [PI(4,5)P2, here PIP2] has been proposed to existin distinct pools or microdomains. The origin of such spatially separatedpools and the contribution of this distribution to PIP2 function is unknown.PIP2 is critically involved not only in many cellular reactions, but also inHIV-1 particle assembly and release. For example, in vitro at a physiolog-ical level of 2 mol%, PIP2 significantly enhances membrane binding of theviral structural protein Gag. And the viral membrane is enriched in PIP2compared with the PM. Here, we exploited model membranes with PM in-ner leaflet lipid compositions to characterize PIP2 clustering and to under-stand how HIV-1 takes advantage of these clusters. Using acyl-chainfluorescently labeled TopFluor-PIP2 in fluorometric assays, we demon-strated that PIP2 begins to form clusters at approximately 0.05 mol%,far below physiological levels. This clustering is dependent on multivalentmetal ions. PIP2 clustering behavior is headgroup-specific and independentof acyl chain type. In cells PIP2 is likely to exist in two forms, either asindividual PIP2 molecules or else clustered with multivalent metal ionsand other PIP2 molecules. Manipulating PIP2 to either form clusters orstay as individual molecules in membranes enabled us to study whetherproteins recognize PIP2 clusters, and whether protein binding reorganizesPIP2 distributions. We found that in microscopic-based giant unilamellarvesicle (GUVs) assays, naturally myristoylated HIV-1 MA, the membranebinding domain of Gag, strongly prefers binding to PIP2 in clusterscompared with individual PIP2 molecules, whereas charge sensor proteinshardly sense any difference in between individual or clustered PIP2. Weare currently examining whether the PIP2 lateral distribution is influencedupon MA binding. We hypothesize that HIV-1 exploits PIP2 clusters assites of viral assembly.

Posters: Membrane Active Peptides and Toxins II

2237-Pos Board B253Membrane Cholesterol Reduces Polymyxin B Nephrotoxicity in RenalMembrane AnaloguesAdree Khondker1, Richard J. Alsop1, Alexander K. Dhaliwal1,Sokunthearath Saem2, Jose Moran-Mirabal2, Maikel C. Rheinstadter1.1Physics and Astronomy, McMaster University, Hamilton, ON, Canada,2Chemistry and Chemical Biology, McMaster University, Hamilton, ON,Canada.At the dawn of the post-antibiotic era, the use of ‘last-line’ antibioticscontinue to rise due to the emergence of antimicrobial resistant bacteria.

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Polymyxin B (PmB) is such a last-line antibiotic scarcely used due to itsnephrotoxicity. However, the molecular basis for antibiotic nephorotoxicityis not clearly understood. We prepared kidney membrane analogues ofdetergent-susceptible membranes, depleted of cholesterol, and cholesterolenriched, resistant membranes. In both analogues, PmB led to membranedamage. By combining X-ray diffraction, Molecular Dynamics (MD) simu-lations, and electrochemistry, we present evidence for two populations ofPmB molecules: peptides that lie flat on the membrane surface, and an in-serted state. In cholesterol depleted membranes, PmB forms aggregates onthe membranes leading to an indentation of the bilayers and an increase inwater permeation. The inserted peptides formed aggregates in the membranecore leading to further structural instabilities. While cholesterol did notinhibit peptide insertion, it minimized peptide clustering and water intakethrough stabilization of the bilayer structure, and suppression of lipid andpeptide mobility.[1] Khondker, A., Alsop, R. J., Dhaliwal, A., Saem, S., Moran-Mirabal, J.M.,Rheinst€adter, M. C., 2017, Membrane Cholesterol Reduces Polymyxin BNephrotoxicity in Renal Membrane Analogues, Biophysical Journal, Articlereference: BPJ_2017BIOPHYSJ307883, in press.

2238-Pos Board B254Selective Interaction of Colistin with Lipid Model MembranesFernando G. Dupuy1, Isabella Pagano2, Kathryn Andenoro2,Maria F. Peralta3, Yasmene Elhady2, Frank Heinrich2,Stephanie Tristram-Nagle2.1Biochemistry Institute, National University of Tucuman, San Miguel deTucuman, Argentina, 2Physics Dept., Carnegie Mellon University,Pittsburgh, PA, USA, 3Ferreyra Institute of Medical Research, NationalUniversity of Cordoba, Cordoba, Argentina.Although colistin’s clinical use is limited due to nephrotoxicity, colistin isconsidered an antibiotic of last resort since it is used to treat patients in-fected with multi-drug resistant bacteria. In an effort to provide moleculardetails about colistin’s ability to kill Gram-negative (G(-)) but not Gram-positive (G(þ)) bacteria, we investigated the biophysics of interaction be-tween colistin and lipid mixtures mimicking the cytoplasmic membrane ofG(þ), G(-) bacteria as well as eukaryotic cells. Two different modelsof G(-) outer membrane (OM) were assayed: lipid A with two deoxy-manno-octulosonyl sugar residues (KDO2) and Escherichia coli lipopoly-saccharide (LPS) mixed with dilaurylphosphatidylglycerol. We usedcircular dichroism and X-ray diffuse scattering at low and wide angle instacked multilayered samples, and neutron reflectivity (NR) of single, teth-ered bilayers mixed with colistin. We found no differences in secondarystructure when colistin was bound to G(-) vs G(þ) membrane mimics,ruling out a protein conformational change as the cause of this difference.However, bending modulus KC perturbation was quite irregular for G(-)IM, where colistin produced a softening of membranes at intermediate lip-id:peptide molar ratio but stiffening at lower and higher peptide concentra-tions, while in G(þ) and eukaryotic mimics there was only a slightsoftening. Acyl chain order in G(-) was perturbed similarly to KC. InG(þ) there was only a slight softening and disordering effect, while inOM mimics, there was a slight stiffening and ordering of both membraneswith increasing colistin. X-ray and NR structural results reveal colistin par-titions deepest into the hydrocarbon interior in G(-) membranes, but re-mains in the headgroup region in G(þ), OM and eukaryotic mimics. Itis possible that domain formation is responsible for the erratic responseof G(-) inner membranes to colistin and for its deeper penetration, whichcould increase membrane permeability.

2239-Pos Board B255Adsorption of Polyene Antibiotics on Supported Lipid Bilayers of DifferentLipid CompositionsArturo Galvan-Hernandez, Ivan Ortega-Blake.Universidad Nacional Autonoma de M�exico, Cuernavaca, Mexico.Amphotericin B (AmB) is still the gold standard for systemic fungal infection,in spite the collateral toxicity it presents and years of research aimed to find anew and less toxic drug[1]. The molecular mode of action is still under debateand several proposed models exist. The most accepted model is the pore-forming model [2] in which monomers of AmB adsorb to the lipid membraneand aggregate into pores that cause cell death by loss of Kþ. Following thisidea Huang et al. [3] demonstrated that AmB indeed forms channels onPOPC-Erg bilayers in monomeric form. However for POPC-Chol bilayersan aggregated form needs to be reached and for pure POPC bilayers higherorder aggregates are needed to observe ion channels. This particular behaviorcould account for the selective action of AmB that allows for its clinical use.

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We present Atomic Force Microscopy images of supported lipid bilayers ofPOPC:eSM with and without sterol and how AmB adsorbs to the surface ofthe bilayers at different concentrations. Additionally we test a new derivative,A21, obtained by our laboratory in collaboration with other laboratories andinstitutions [4]. A21 presents possible therapeutically advantages both in vitroand in vivo. In this work we compare the threshold concentrations at whichaggregates appear on the bilayers as well as the sites of adsorption andform of the aggregates for both AmB and A21. Funding: DGAPA-PAPIIT-IG100416. [1] Kami�nski, D. M. (2014). European biophysics journal,43(10-11), 453-467. [2] Finkelstein, A. & Holz, R. Membranes 2, 377-408(1973). [3] Huang, W., Zhang, Z., Han, X., Tang, J., Wang, J., Dong, S., &Wang, E. (2002). Biophysical journal, 83(6), 3245-3255. [4] Antillon, A.,de Vries, et al. (2016). PloS one, 11(9), e0162171.

2240-Pos Board B256An Exact Model of Daptomycin Binding to Lipid Bilayers: An UpdateAntje Pokorny, Tala O. Khatib.Chemistry and Biochemistry, Univ. North Carolina Wilmington,Wilmington, NC, USA.Daptomycin is a cyclic lipopeptide of clinical importance in the treatment ofmulti-drug resistant infections, including those caused by methicillin-resistant S. aureus (MRSA) strains. Similar to other antimicrobial peptides,daptomycin binds with preference to the anionic cytoplasmic membranes typi-cally found in prokaryotes. However, in contrast to most linear, alpha-helicalpeptides, daptomycin binds to lipid bilayers only in the presence of calciumions and its activity is absolutely calcium-dependent. We measured the interac-tion of daptomycin with anionic lipid membranes using kinetic binding exper-iments and equilibrium titrations. The data were analyzed using an exact modeldescribing the interactions of daptomycin with the lipid bilayer that includessolution and membrane-bound states, and the influence of calcium ions ondaptomycin-lipid interactions. A preliminary version of the model was pre-sented at the Biophysical Society Meeting in 2017. Here, we present a morecomplete analysis.

2241-Pos Board B257How does the Membrane-Active Antibiotic Daptomycin Work?Huey W. Huang1, Nicholas E. Charron1, Ming-Tao Lee2,Meng-Hsuan Hsieh3, Yu-Yung Chang2.1Physics and Astronomy, Rice University, Houston, TX, USA, 2NationalSynchrotron Radiation Research Center, Hsinchu, Taiwan, 3Institute ofBiotechnology, National Taiwan University, Taipei, Taiwan.Daptomycin (Dap) is one of a few membrane-active antibiotics approved byFDA so far. Despite years of research, its molecular mechanism remainsmysterious. Accumulated evidence supports the mode of action discoveredlong ago that its main target is the bacteria’s cytoplasmic membranes, whereDap causes lion leakage leading to loss of membrane potential and cell death.It is also well established that its antibacterial activity is Ca2þ-dependent andcorrelates with the target membrane’s content of phosphatidylglycerol (PG).Many have suggested that Dap forms ion channels but there is neither support-ing evidence nor a convincing molecular model. A recent study shows thatDap has only two molecular states: state-A was detected in the absence ofeither Ca2þ or PG and state-B in the presence of both. When Dap was intro-duced to a DOPC/DOPG giant vesicle (GUV) with Ca2þ, Dap initially boundto GUV and expanded its membrane area; subsequently the GUV areadecreased and simultaneously small lipid-Dap aggregates appeared. This phe-nomenon was described as a lipid extracting effect. One obstacle for Dapstudies is the difficulty of preparing uniformly mixed multilayers of lipids/Dap/Ca2þ. Having solved this problem, we now present X-ray results showingthat in the A-state Dap thins DOPC/PG bilayer implying it is imbedded in themembrane. In the B-state Dap does not thin the bilayer implying it is outsidethe lipid bilayer. Altogether, Dap initially binds to the membrane in the A-state; then the reaction with PG and Ca2þ turns it into the B state. The kineticprocess of A-state transforming to B-state involves lipid extraction, similar tomembrane-mediated transformation of penetratin from helices to beta-sheets.Ion leakage occurs only during the kinetic transition from the A-state to the B-state. Both Dap and penetrain kill bacteria by this so-far undiscovered molec-ular mechanism.

2242-Pos Board B258Membrane Selectivity of an Antimicrobial Lipopeptide using MolecularDynamics SimulationsSreyoshi Sur, Alan Grossfield.Chemistry, University of Rochester, Rochester, NY, USA.

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Fengycin is an antimicrobial cyclic lipopeptide synthesized by the bacterialfamily Bacillus as an immune response against fungus. Fengycins works bybinding to fungal cell membranes and disrupting them. Previous experimentaland simulation work from our group and others suggests that fengycin’s aggre-gation state is crucial to its ability to selectively damage fungal but not bacterialmembranes. Fengycin has minimal activity against mammalian cells, althoughthey are far more similar to fungi than to bacteria. It has been hypotehsized thatthe presence of cholesterol in mammalian cell membranes is the key protectiveagent. To test this idea, we use coarse-grained molecular dynamics simulationscoupled with the weighted ensemble enhanced sampling technique to directlymeasure the free energy change for fengycin to both bind and aggregate as afunction of membrane composition. Preliminary results suggest that cholesterolsignificantly increases the barrier to entry, and may alter the tendency to aggre-gate once bound. Using weighted ensemble techniques to assess the free energyassociated with lateral ordering is a general technique that will have applica-bility beyond fengycin to other phenomena involving membrane lateralordering or demixing.

2243-Pos Board B259Synergistic Action of Fungicidal Lipopeptides as a Mechanism of TargetMembrane SelectivityLisa Dietel1, Quang Huynh2,3, Sebastian Fiedler4, Heiko Heerklotz1,4.1Pharmaceutical Sciences, University of Freiburg, Freiburg, Germany,2Molecular Structure and Function, Hospital for Sick Children, Toronto, ON,Canada, 3Biochemistry, University of Toronto, Toronto, ON, Canada, 4LeslieDan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada.Bacterial lipopeptides of the surfactin, fengycin, and iturin families are usedas biological fungicides for crop protection. Their activity involves the perme-abilization of the cellular membrane of the target pathogen. Depending on thenature of the target membrane, they have been shown to synergize or antag-onize with each other, with other fungicides, or with simple detergents. For adetailed, mechanistic understanding of these interactions, we have studied thepermeabilizing effect of combinations of these agents against liposomes ofdifferent lipid compositions. The fluorescence lifetime based leakage assaybased on time correlated single photon counting experiments was utilized tocharacterize in detail the efflux of calcein from these liposomes. The typesof concerted action are discussed on the basis of the specific modes of actionof the individual agents. First, this opens a new avenue to improving the ac-tivity of membrane-active, antimicrobial agents for crop protection and,possibly, medical use. Second, target dependent, mutual enhancement or inhi-bition is discussed as a new mechanism governing target selectivity of antimi-crobial agents. It is easy to kill cells in a petri dish (you recall: so does ahandgun), so being able to fine tune selectivity is the key to manyapplications.

2244-Pos Board B260Molecular Mechanism of Synergy between the Antimicrobial PeptidesPGLa and Magainin 2 in MembranesErik Strandberg1, Jonathan Zerweck2, Parvesh Wadhwani1,Johannes Reichert1, Jochen B€urck1, Anne S. Ulrich2.1Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology,Karlsruhe, Germany, 2Institute of Organic Chemistry, Karlsruhe Institute ofTechnology, Karlsruhe, Germany.The two antimicrobial peptides PGLa and magainin 2 (MAG2), both found inthe skin of the African frog Xenopus laevis, are known to exhibit strong syn-ergistic effects in bacterial killing, lipid vesicle leakage, and other membrane-related activities [1]. Here, we studied the synergy of the two peptides in anattempt to find the molecular mechanism behind their synergistic effect [2]. Alarge number of mutants of the two peptides were investigated, and the syn-ergy tested was using three complementary methods: (i) a checkerboard assayagainst two bacteria; (ii) a vesicle leakage assay; and (iii) 15N-NMR, showingthe insertion of PGLa into a transmembrane orientation (suggesting the forma-tion of a pore) in the presence of MAG2. Several mutations were identifiedthat reduced or inhibited the synergy of the peptides, and a single mutationon PGLa from Gly to Ala was enough to completely abolish synergy. Atthe same time, the designer-made and related but completely non-synergistic peptide MSI-103 could be modified by two point mutations tobecome strongly synergistic with MAG2. Also in this case Gly residueswere involved, indicating that a GxxxG motif on PGLa is critical and neces-sary for synergy. Based on these results, a 3D model could be developed ofthe PGLa-MAG2 complex in the membrane that is synergistically active.An oligomeric pore is likely formed from antiparallel dimers of transmem-brane PGLa peptides, wherein each PGLa molecule makes contact to aMAG2 peptide that is aligned on the membrane surface.

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References: [1] E Strandberg, P Tremouilhac, P Wadhwani, AS Ulrich (2009).Biochim Biophys Acta 1788, 1667. [2] J Zerweck, E Strandberg, O Kukharenko,J Reichert, J B€urck, P Wadhwani, AS Ulrich (2017) Sci Rep, DOI:10.1038/s41598-017-12599-7.

2245-Pos Board B261Systematic Analysis of Hybrid Antimicrobial PeptidesHeidi M. Wade1, Louise E.O. Darling2, Donald E. Elmore1.1Department of Chemistry and Biochemistry Program, Wellesley College,Wellesley, MA, USA, 2Department of Biological Sciences and BiochemistryProgram, Wellesley College, Wellesley, MA, USA.Antimicrobial peptides (AMPs) are part of the immune response of all clas-ses of life and have gained attention as promising alternative treatments forinfectious bacteria resistant to conventional antibiotics. AMPs kill bacteriathrough two known mechanisms of action. Some AMPs, such as parasinand magainin II, kill bacteria by inducing membrane permeabilization.Other AMPs, such as buforin II (BF2) and DesHDAP1, readily translocateacross the membrane and interact with intracellular components includingnucleic acids. In recent years, there has been increased interest in devel-oping hybrid AMPs that combine two distinct AMPs into a single peptide.These hybrid AMPs have been shown to be more potent than their individ-ual AMP components. To date, few studied hybrid have combined AMPsthat follow different mechanisms. Here, we focus on using a variety ofcellular assays and confocal imaging to characterize the activity and mech-anisms of action of hybrid AMPs that combine one permeabilizing AMP(parasin or magainin II) with one translocating AMP (BF2 or DesHDAP1)in different orientations and with different linkers. We show that thesehybrid AMPs are generally more potent than their individual AMP compo-nents and that the permeabilizing peptide (parasin or magainin II) domi-nates the mechanism of action when combined with the translocatingpeptide (BF2 or DesHDAP1). These observations of 16 hybrid peptideshave elucidated trends that will promote the rational design of AMPswith enhanced activity.

2246-Pos Board B262Aggregation vs. Fusion of Negatively Charged Lipid Bilayers Induced byBactenecin and Magainin DerivativesMojtaba Bagheri.Institute of Biochemistry and Biophycics, University of Tehran, Tehran,Islamic Republic of Iran.Antimicrobial peptides (AMPs) have been studied for their future promisingperspectives to overcome the arising health-treating issues of bacterial super-bugs, alone or in synergy with other antibiotics. Although AMPs clinical ap-plications are still obscured owing to their formulation challenges, saltsensitivity and stability issues, their mechanisms of action upon encounteringthe bacterial cell membranes is a matter of debate and require understating ofboth the intrinsic physicochemical properties of the membrane and crucialrole of the peptide primary structure. In this study, we wanted to investigatethe membrane bindings of highly selective antimicrobial bactenecin and mag-ainin 2 variants to the negatively charged mixed POPC/POPG and POPE/POPG liposomes with the lower and higher spontaneous negative curvatures,respectively. Unlike magainin 2 derivatives, increasing the molar ratio of bac-tenecins to both lipid systems followed by sample turbidity and decrease inthe fluorescence emission spectra of tryptophan, particularly for POPE/POPG bilayer. The electron microscopy showed the formation of stable andfragile colloidal aggregates of about 1-15 micron for the mixed liposomecomposed of POPE and POPG, respectively. Whereas these preliminary re-sults were further supported by the FRET analysis using POPE/POPG systemmixed with Rhod-DPPE and NBD-DOPE, liposome fusogenic properties weresuggested for the membrane-lytic magainin peptide. It seems, though, bothpeptide variants are membrane-active and antimicrobials at the very low com-parable concentrations, different antimicrobial mode of actions are expectedfor them. The results here turn out to dictate the design of next generationof AMPs which are less vulnerable to the membrane-associated mechanismof bacterial resistance.

2247-Pos Board B263Conformations and Dynamic Transitions of a Melittin Derivative in LipidBilayerAnna E. Pittman1, Gavin M. King1,2.1Department of Physics and Astronomy, University of Missouri-Columbia,Columbia, MO, USA, 2Department of Biochemistry, University of Missouri-Columbia, Columbia, MO, USA.

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MelP5 is a lipophilic peptide with unique physical properties including the abil-ity to create large and stable pores at low concentrations. Self-assembly intomembrane spanning pores makes MelP5, systematically evolved from the pri-mary toxic component of bee venom, a promising candidate for future applica-tions in the pharmaceutical arena, as it offers a robust mechanism of drugdelivery. Despite significant importance, little is known about the mechanismby which MelP5 remodels the lipid bilayer upon binding. Here, we demonstrateby atomic force microscope imaging that MelP5 interacts with a lipid bilayer inone of two ways: surface bound, which causes a thinning of the bilayer, orinsertion into the membrane, which creates pores. Thinning of the bilayerwas measured to be �0.4 nm below the upper leaflet of the bilayer and wasa prerequisite to pore formation. Pores exhibited many stable sizes, some ofwhich were quite large. For example, approximately 20% of the pores exhibitedfootprint areas of 47 þ/- 19.8 nm2. Time lapse analysis demonstrated that thepeptides transitioned reversibly between the membrane thinned state and porestate, yielding upper and lower bounds (0.2 < t< 180 s) on the characteristictime scale of transitions between these states.

2248-Pos Board B264Dynamic Membrane Bound Structures of Melittin and Alamethicin asRevealed by Solid-State NMR and MD SimulationAkira Naito1, Takashi Nagao1, Kazushi Norisada1, Namsrai Javkhlantugs2,Daisuke Mishima1, Izuru Kawamura1, Kazuyoshi Ueda1.1Yokohama National University, Yokohama, Japan, 2National University ofMongolia, Ulaanbaatar, Mongolia.Melittin is a bee venom peptide that disrupts acidic dimyristoylphosphatidyl-glycerol (DMPG) bilayers as well as neutral dimyristoylphosphatidylcholine(DMPC) bilayers. 13C chemical shift anisotropy of [1-13C]-labeled melittinshowed oscillatory shifts with the index number of residues. Analysis of the13C chemical shift oscillation properties indicated that melittin bound to aDMPG membrane adopts a bent a-helical structure with tilt angles for theN- and C-terminal helices of �32 and þ30�, respectively. The transmembranemelittin in DMPG bilayers indicates that the peptide protrudes toward theC-terminal direction from the core region of the lipid bilayer to show a pseudo-transmembrane bent a-helical structure [1, 2].The structure topology and orientation of membrane-bound antibiotic alamethi-cin were studied using solid state NMR spectroscopy. 13C chemical shift interac-tion was observed for [1-13C]-labeled alamethicin. The chemical shift oscillationanalysiswas performedwith the assumption that the adjacent peptide planes forman angle of 100� or 120� when it forms a-helix or 310-helix, respectively [2, 3].These properties lead to an oscillation of the 13C chemical shift anisotropy withrespect to the phase angle of the peptide plane. The chemical shift oscillationcurves revealed that the N- and C-termini formed a-helix and 310-helix, andthe N- and C-termini were tilted 17� and 32� to the bilayer normal, respectively.References[1] K. Norisada, N Javkhlantugs, D. Mishima, I. Kawamura, H. Saito, K. Ueda,A. Naito, J. Phys. Chem.B. 2017, 121, 1802-1811.[2] A. Naito, N. Matsumori, A. Ramamoorthy, Biochim. Biophys. Acta, Gen-eral Subject. In press 2017. DOI: 10.1016/j.bbagen.2017.06.004.[3] T. Nagao, D. Mishima, N. Javkhlantugs, J. Wang, D. Ishioka, K. Yokota, K.Norisada, I. Kawamura, K. Ueda, A. Naito, Biochim. Biophys. Acta, Bio-membrane 2015, 1848, 2789-2798.

2249-Pos Board B265Impact of Metallation and Oxidized Lipids on the Structure andMembrane Disruptive Effects of Host Defense Peptides Piscidin 1 andPiscidin 3Myriam Cotten1, Ella Mihailescu2, Anna De Angelis3, Ratan Rai3,Dana Moore1, Yawei Xiong1, Alfredo Angeles-Boza4, Stanley Opella3.1The College of William and Mary, Williamsburg, VA, USA, 2University ofMaryland, Rockville, MD, USA, 3University of California San Diego, LaJolla, CA, USA, 4University of Connecticut, Storrs, CT, USA.This research investigates the membrane-interacting host defense metallopep-tides piscidin 1 (P1) and piscidin 3 (P3) that are active on drug resistant bacte-ria. Bacterial membranes are battlegrounds for these host defense peptides(HDPs), which can leverage multiple antimicrobial strategies (e.g. copper-associated oxidative stress; structural and chemical modifications of phospho-lipids) to sensitize cell membranes and achieve high antimicrobial potency.We previously showed that P1 and P3 achieve metal binding through theiramino terminal Cu and Ni (ATCUN) binding motif and that their metallationenhances their antimicrobial activity. Here, we employed biophysical tools toinvestigate the impact of metallation and oxidized lipids (OxPL) on peptideconformation and bilayer thickness. We used UV spectroscopy to demon-strate peptide metallation and membrane binding. For high resolution struc-

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tural studies, we employed both oriented sample and magic angle solid-stateNMR multi-dimensional experiments on 15N- and 13C-labeled P1 and P3bound to nickel or copper ions in the presence of 3:1 phosphatidylcholine/phosphatidylglycerol (PC/PG) as well as 2.6:1:0.4 PC/PG/OxPL. Using x-ray diffraction, we quantified bilayer thinning resulting from adding metal-lated P1 and P3 to 3:1 PC/PG.In parallel to these studies, we carried out biochemical assays to establish levelsof lipid peroxidation in membranes of live E. coli cells. We tested both metal-lated P1 and P3 at their minimum inhibitory concentration in the presence andabsence of a copper chelator to determine if the ATCUNmotif is directly impli-cated in lipid peroxidation and peptide potency.These comprehensive studies help us achieve a deeper understanding of howcopper-binding HDPs and other agents of the immune system responsecombine their antimicrobial effects at pathological membranes. This newknowledge could be useful to the rational design of novel anti-infectivetherapeutics.

2250-Pos Board B266Unraveling the Role of Peptidoglycan in the Interaction of AntimicrobialPeptides BP100 and MSI-78 with Bacterial Cell Envelopes through 2HNMR ExperimentsNury Paula Santisteban, Michael Morrow, Valerie Booth.Memorial University of Newfoundland, St. John’s, NL, Canada.Specific activity against many different pathogens is one of the reasons whyantimicrobial peptides (AMPs) are tantalizing candidates to address the cur-rent crisis of antibiotic-resistant infections. Nevertheless, many questionsabout the exact pathogenic mechanisms of AMPs remain unsolved. Bio-physical studies on AMPs have shown a destabilizing effect on lipidbilayers. In addition, studies performed in whole bacterial cells have shownthat some AMPs may have additional targets different from the lipid bilayer.Thus, it is possible that the destabilizing effect of at least some AMPs onmembranes may be a collateral effect of transiting through the membraneon the way to an intra-cellular target, or that there is more than one mech-anism by which AMPs harm target cells. Moreover, understanding the roleof non-lipid components of the bacterial envelope in the interaction AMPswith bacterial membranes could help us better understand how AMPs selec-tively target pathogenic bacteria. In this work, we study the role of the pepti-doglycan layer in the interaction AMPs with the bacterial membrane. To doso, we implemented 2H NMR in intact Bacillus subtilis cells. In the absenceof AMP, the 2H NMR spectrum of the labeled bacterial membrane is a su-perposition of doublets characteristic of fast axially symmetric chain reor-ientation. Prominent shoulders, at þ/-12 kHz, reflect an order parameterprofile having a plateau near the headgroup end of the chain. In the presenceof AMP, intensity shifts from the plateau spectral region to smaller quadru-pole splittings suggesting a peptide-induced disordering of the bacterialmembrane. Finally, we show that for MSI-78 and BP100, peptidoglycandisruption does not affect the AMPs’ ability to affect the lipid membraneof intact cells.

2251-Pos Board B267Gramicidin Subunits that Cross Membranes and form Ion ChannelsMatthew Brownd1,2, Matthew J. McKay1, Denise V. Greathouse1,Olaf S. Andersen3, Roger E. Koeppe1.1Dept of Chemistry & Biochemistry, University of Arkansas, Fayetteville,AR, USA, 2John Brown University, Siloam Springs, AR, USA, 3Dept ofPhysiology and Biophysics, Weill Medical College of Cornell University,New York, NY, USA.Gramicidin A (formyl-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp9-D-Leu-L-Trp11-D-Leu-L-Trp13-D-Leu-L-Trp15-ethanolamine) formstransmembrane channels that are selective for monovalent cations. Thechannels are dimers, consisting of two gramicidin (gA) subunits that areheld together by hydrogen bonding. Due to the affinity of Trp for the mem-brane/water interface, the subunits are anchored by the four Trp residues.The interfacial affinity prevents subunits from crossing the membrane andrequires that gA subunits be introduced to both sides of a lipid bilayer inorder to facilitate channel formation. Nevertheless, it has been observedthat analogues in which Trp residues 13 and 15 have been replaced withPhe (designated [Phe13,15]gA) are able to cross the membrane with relativeease, presumably in a double-stranded conformation, and engage in channelformation. Based on this finding, we have investigated a longer 17-residuepeptide endo-Gly-Gly-[Phe13,15]gA, which is likely to be an effectivechannel-former in thicker membranes. We report the synthesis and spectralproperties of [Phe13,15]gA and endo-Gly-Gly-[Phe13,15]gA, along with pre-liminary comparisons of the respective subunits in DMPC bilayer mem-

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branes. The investigation was conducted primarily using deuterium NMRspectroscopy, made possible because we incorporated deuterated alaninesat the 3rd and 5th positions of both peptides, together with circular dichroismspectroscopy and tryptophan fluorescence. Both analogues exhibit confor-mational heterogeneity. In each case, a major conformation closely resem-bles that of the single-stranded channel conformation of unmodified gA.The kinetics of channel formation, under conditions of one-sided addition,are under investigation.

2252-Pos Board B268Lipid Interdigitation Promotes Thermal Stabilization of Lipid Polymor-phisms Induced by Surfactant Peptide B1-25

Nhi T. Tran1, Justin Kurian2, Avni Bhatt2, Gail E. Fanucci1,Joanna R. Long2.1Chemistry, University of Florida, Gainesville, FL, USA, 2Biochemistry andMolecular Biology, University of Florida, Gainesville, FL, USA.Here we characterize dynamics and morphologies of major lipid constituentsin a pulmonary surfactant (PS) model system containing therapeutic levels ofsurfactant peptide B1-25 (SP-B1-25) using

2H and 31P NMR spectroscopy, dif-ferential scanning calorimetry (DSC) and transmission electron microscopy(TEM). We observe peptide induced isotropic lipid phase behavior in hydrat-ed assemblies of 4:1 DPPC/POPG, reflecting non lamellar lipid morphologiesinvolved in lipid trafficking within the aqueous alveolar sub phase. 31P T2

relaxation times confirm the isotropic phase to be consistent with a lipid cubicphase, illustrating the role of SP-B1-25 in promoting an architectural frame-work for rapid lipid transit between lipid lamellae. The appearance and ther-mal stability of the lipid cubic phase observed with NMR is highly dependenton thermal cycling of peptide/lipid mixtures. Rapid heating of frozen samplesto room temperature lead to coexistence of a cubic and interdigitated lipidphase, isolated to DPPC lipids. This interdigitated lipid phase exhibits boththermal stability up to physiologic temperature (37 �C) and hysteretic phasebehavior, consistent with formation of the well characterized DPPC ripplephase. An interdigitated DPPC phase is also reported with surfactant proteinC (SP-C) at physiologic temperature (Roldan et al., 2017), suggesting a uni-versal mechanism in modulating lipid organization within the dynamic alve-olar environment. We propose a unique role for DPPC in stabilizingenergetics of SP-B1-25 induced lipid polymorphisms, given its preponderancein native mammalian PS. Our results highlight complexities between equili-bration temperature and thermal cycling of peptide/lipid mixtures in produc-ing both a dynamic and thermally stable lipid phase. This study presents anew perspective in evaluating the therapeutic success of current clinical prep-arations and applications of PS peptide/lipid mixtures, with broad impacts inmembrane biophysics, particularly on thermotropic phase behaviors of non-lamellar lipid polymorphisms.

2253-Pos Board B269Influence of Saturation and Hydrophobic Length of Lipid Bilayers onTwin-Arginine Containing Helical PeptidesKarli A. Lipinski, Ashley N. Martfeld, Denise V. Greathouse,Roger E. Koeppe II.University of Arkansas, Fayetteville, AR, USA.Membrane proteins are essential for many cell processes yet are understood lesswell than soluble proteins. Charged residues such as arginine often contributesignificantly to the function of membrane proteins. To characterize the effect ofthese residues on transmembrane proteins, it is useful to employ a modelpeptide system such as GWALP23 (acetyl-GGALW5LAL8ALALALAL16

ALW19LAGA-amide), a designed transmembrane peptide with interfacialtryptophan anchors. We have substituted R8 and R16 in place of L8 and L16in GWALP23, equidistant from the center, and incorporated specific2H-labeled alanine residues in the core of the helix for detection by means ofsolid-state 2H NMR. The pattern of 2H-Ala quadrupolar splitting magnitudesalong the helix indicates a significantly tilted, similar transmembrane orienta-tion in DLPC and DMPC bilayers, while multiple orientations are indicatedin DOPC (18:1 D9 cis) bilayers. Inclusion of 10-20 mol% cholesterol inDOPC bilayers causes the peptide to adopt a surface orientation with interfacialaccess for both charged arginine residues. One arginine residue has greater ac-cess than the other and further investigations will discern whether helix un-winding allows greater access to the second arginine residue. In bilayers of ashorter unsaturated lipid 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine(16:1 D9 cis), the peptide adopts a single transmembrane orientation, similarto orientations in DLPC and DMPC. The results suggest hydrophobic thicknessas a more important factor than lipid saturation for the twin arginine peptidedynamics.

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2254-Pos Board B270Lysine Position Affects Binding of Aib-Rich Model Antibiotics to LipidVesiclesJack M. Geiger, Adrienne Loh.Chemistry & Biochemistry, U. Wisconsin - La Crosse, La Crosse, WI, USA.Due to the increased development of drug resistance and decrease of antibioticdrug effectiveness, there is an immediate need for new antimicrobials. Apossible, promising solution is in the form of charged peptide-based molecules,which can function as antibiotics by interacting with and disturbing bacterialmembranes. We are exploring these interactions using model peptidescomposed of hydrophobic, branched amino acid Aib (a-aminoisobutyricacid), and large unilamellar vesicles (LUVs) composed of DMPG andDMPC. These LUVs model bacterial (negatively charged) and non-bacterial(neutral) cell membranes, respectively. Aib naturally occurs in antibioticsused by some bacteria and biases peptides to adopt a helical structure. We pre-sent results using two model peptides in which two positively charged lysinemolecules were placed in either adjacent positions of the helix (KK45), or ahelical turn apart (KK36). Interactions between the LUVs and peptides areinvestigated using Isothermal Titration Calorimetry (ITC), resulting in bindingenthalpies, entropies, and binding constants. Our initial results indicate thatboth KK45 and KK36 bind in a multi-stage interaction to the DMPG vesicles(bacterial models), while showing very little affinity when binding to DMPC(non-bacterial models). One stage appears to be enthalpy-driven and is consis-tent with electrostatic interactions between peptide sidechains and lipid head-groups, while the other stage appears to be entropy-driven and is consistentwith hydrophobic interactions. Overall KK45 binds more favorably toDMPG vesicles, which is interesting given that the KK45 helix is kinked, whilethe KK36 helix is not.

2255-Pos Board B271Understanding the Effect of Cationic Residue Identity on Lipid Interac-tions of Translocating Antimicrobial PeptidesShelby N. Kranc1, Donald E. Elmore2.1Department of Chemistry, Wellesley College, Wellesley, MA, USA,2Department of Chemistry and Biochemistry Program, Wellesley College,Wellesley, MA, USA.Histone-derived antimicrobial peptides (HDAPs) are an interesting family ofantibacterial agents that utilize both translocation and permeabilization-basedmechanisms of action. Previous work demonstrated that arginine-rich mutantsof two membrane translocating HDAPs, buforin II (BF2) and DesHDAP1,were more active than wild type or lysine-rich mutants. To provide a struc-tural explanation for these results, we have performed over 3 ms of moleculardynamics (MD) simulations to compare the lipid membrane interactions ofBF2 and DesHDAP1 variants with differing arginine compositions. Thesesimulations show increased lipid hydrogen bonding of arginine-rich peptidescompared to other forms, with most differences in hydrogen bonds arisingbetween the lipid and arginine residues themselves. Additionally, thearginine-rich HDAPs appear to reside closer to the lipid bilayer in simula-tions, showing their increased ability to begin potential translocation eventsin comparison to other variants of the peptides. The improved understandingof peptide-lipid interactions from these simulations of BF2 and DesHDAP1will aid in future work aimed at enhancing the activity of HDAPs and otherpeptides through altering the composition of cationic residues.

2256-Pos Board B272Hydrophobic Interactions Modulate Peptide Cell SpecificityKonstantin Andreev1, Michael W. Martynowycz1, Mia L. Huang2,Kent Kirshenbaum2, David Gidalevitz1.1Illinois Institute of Technology, Chicago, IL, USA, 2Department ofChemistry, New York University, New York, NY, USA.Hydrophobic interactions govern specificity for natural antimicrobial pep-tides. No such relationship has been established for synthetic peptoids thatmimic antimicrobial peptides. Peptoid macrocycles synthesized with fivedifferent aromatic groups are investigated by minimum inhibitory and hemo-lytic concentration assays, epifluorescence microscopy, atomic force micro-scopy, and X-ray reflectivity. Peptoid hydrophobicity is determined usinghigh performance liquid chromatography. Disruption of bacterial but not eu-karyotic lipid membranes is demonstrated on the solid supported lipid bila-yers and Langmuir monolayers. X-ray reflectivity studies demonstrate thatintercalation of peptoids with zwitterionic or negatively charged lipid mem-branes is found to be regulated by hydrophobicity. Critical levels of peptoidselectivity are demonstrated and found to be modulated by their hydrophobicgroups. It is suggested that peptoids may follow similar optimizationschemes as their natural analogues.

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2257-Pos Board B273Development of Novel Antimicrobial Peptides with Improved Hemocom-patibility via Combinatorial Library Screening and Rational EngineeringCharles G. Starr, William C. Wimley.Biochemistry, Tulane University, New Orleans, LA, USA.Development of antimicrobial peptides (AMPs) as next generation clinicalantibiotics has been a pursuit of the scientific community for severaldecades. AMPs are attractive drug candidates because of their potent anti-bacterial activity and a low propensity for eliciting resistant phenotypes.However, despite substantial efforts and a myriad of approaches, AMPshave yet to make inroads in the clinic due to toxicity concerns and activityloss in vivo. We hypothesized that eukaryotic cytotoxicity and antibacterialactivity loss are intricately related in that peptide-induced tissue or hostcell damage corresponds to depletion of free peptide available to targetbacterial cells. Using human red blood cells (RBCs) as a model eukaryoticcell, we have previously demonstrated that a cross-section of AMPs loseappreciable antibacterial activity when preincubated with 1x109 RBCs/mL and that this behavior can be explained by plasma membrane binding.To approach this problem in a unique manner, we synthesized a combina-torial peptide library based on the potent AMP, ARVA, and screened thelibrary for activity in the presence of concentrated RBCs. We isolated nineunique, but similar sequences from the screen. During the screening pro-gram, we discovered that RBC-peptide interactions lead to peptide degra-dation through the release of cytosolic proteases. We used this knowledgeto design a consensus sequence based on the nine peptides isolated fromthe library screen and synthesized it using only D-isomer amino acids.The novel peptide displays excellent antimicrobial activity against severalhuman pathogens in the presence and absence of concentrated RBCs, hasreduced toxicity towards eukaryotic cells, and is not susceptible to cleav-age by cellular proteases.

2258-Pos Board B274Population Dynamics of Antimicrobial Peptides and BacteriaPaul Talledo1, Mehdi Snoussi2, Nathan Del Rosario2, Bae-Yeun Ha3,Andrej Ko�smrlj4, Sattar Taheri-Araghi1.1Department of Physics and Astronomy, California State University,Northridge, Northridge, CA, USA, 2Department of Biology, California StateUniversity, Northridge, Northridge, CA, USA, 3Department of Physics andAstronomy, University of Waterloo, Ontario, Waterloo, ON, Canada,4Department of Mechanical and Aerospace Engineering, PrincetonUniversity, Princeton, NJ, USA.Antimicrobial peptides (AMPs) are broad-spectrum antibiotics that utilizeelectrostatics to target bacteria selectively. Like most antibiotics, AMPsneed a minimum concentration to inhibit the growth of a bacterial popu-lation. Despite our knowledge of the molecular structures and membraneinteractions of AMPs, we poorly understand AMP’s dynamics at thecellular and the population level. Here we demonstrate that the minimuminhibitory concentration (MIC) of AMPs is strongly dependent on the celldensity, even in dilute cultures where direct cell-to-cell interactions areminimal. We hypothesize that this dependence arises because individualcell absorbs a significant number of AMPs which considerably reducesthe effective concentration of AMPs in the culture. To investigate this hy-pothesis, we used a live single-cell imaging platform to track fluorescentlytagged AMPs and the time evolution of their translocation into bacteria.We also developed a chemical reaction-like model to elucidate the kineticsof interacting bacteria and AMPs. Our single-cell analysis shows that bac-teria not only absorb a significant fraction of AMPs from the culture butalso retain them even after cell death, which sequesters AMP’s availabilityfor attacking more cells.

2259-Pos Board B275First Example of Kinetic Modelling of Multi-state Membrane Binding,Exapnsion and Disruption for an Antimicrobial PeptideMibel Aguilar, Daniel Hirst, Tzong-Hsien Lee.Monash University, Clayton, Australia.Antimicrobial peptide disruption of the membrane bilayer involves a seriesof defined states during binding and insertion, and are all associated withsignificant changes in the bilayer structure. We recently identified a com-plex series of bilayer state changes for the binding of an analogue ofmagainin 2, (Ala-8,13,18)-Magainin 2-amide (MagA), to lipid bilayers. How-ever, the presence of multiple structural states of the membrane preventedthe determination of kinetic constants using currently available kineticmodels, but this information is necessary to allow the full definition ofthe mechanism of action. In this study the binding and dissociation of

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MagA to lipid bilayers with different charge and fluidity properties wasstudied with dual polarization interferometry, an optical technique capableof measuring real-time simultaneous changes in mass and birefringence (anoptical parameter representing bilayer order). The dependence of birefrin-gence vs. mass were categorised into several discrete mechanistic profilesand new multiple-state kinetic models were developed and fitted to theexperimental data, with a three-state model with lateral bilayer expansionproviding distinctly superior fits to simpler model types. Overall this isthe first study to quantitatively analyse complex peptide-membrane bindingdata in terms of specific states of membrane disruption by an antimicrobialpeptide.

2260-Pos Board B276Characterizing Changes in Antimicrobial Peptide Mechanism AgainstDifferent Bacterial StrainsKatrina P. Montales1, Heidi M. Wade2, Dania M. Figueroa3,Louise E.O. Darling4, Donald E. Elmore2.1Department of Chemistry, Wellesley College, Wellesley, MA, USA,2Department of Chemistry and Biochemistry Program, Wellesley College,Wellesley, MA, USA, 3Biochemistry Program, Wellesley College,Wellesley, MA, USA, 4Department of Biological Sciences and BiochemistryProgram, Wellesley College, Wellesley, MA, USA.Antimicrobial peptides (AMPs) are part of the innate immune response ofall classes of life and are effective in killing a broad range of bacteria,making them potential candidates for combating multidrug resistant organ-isms. AMPs can be classified into two categories based on their mechanismof action. One type of AMP operates by permeabilizing and disrupting thecell membrane, while others work by translocating across the membraneand disrupting internal cellular processes. Although the activity of AMPshas been observed to differ between bacterial strains relatively few studieshave considered whether peptide mechanisms of action are strain depen-dent. In this study, we have utilized confocal microscopy to compare themechanism of AMPs against gram-negative and gram-positive bacteria.Due to the small size and orientation of bacteria, cell wall deficient formsof bacteria were developed to produce higher quality images with moreclearly resolved membrane and internal spaces. Our initial imaging focusedon BF2, which is primarily translocating against gram-negative Escherichiacoli, and parasin, which is primarily membrane localizing in E. coli. Ourresults showed that both peptides exhibited altered mechanisms againstgram-positive Bacillus megaterium. Ongoing work is focused on expandingthese studies to a wider range of peptides and bacterial strains to morethoroughly investigate trends in mechanisms against gram-positive andnegative bacteria.

2261-Pos Board B277Exploring Conformations of the Teixobactin-Lipid II Complex inMembranePo-Chao Wen1, Juan M. Vanegas2, Susan B. Rempe3, Emad Tajkhorshid1.1Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL,USA, 2Physics, University of Vermont, Burlington, VT, USA, 3Nanobiology,Sandia National Laboratories, Albuquerque, NM, USA.Teixobactin is a recently discovered antibiotic that works against gram-positive bacteria without detectable resistance. The bactericidal mechanismof teixobactin is believed to be the inhibition of cell wall synthesis due tothe binding to lipid II and lipid III, which likely takes place at the pyro-phosphate moiety that shared by both targets. Despite numerous chemicalanalogs of teixobactin have been synthesized and functionally assessed,and the pharmacophore of one analog has been identified, detailedstructural information is still lacking for the conformations of teixobactinand lipid II in their complex. Here we use molecular modeling and multi-ple sets of microsecond-scale molecular dynamics simulations to createatomic models of teixobactin-lipid II complex at the membrane surface.Two major lipid II binding conformations of teixobactin have beencaptured, and both show pyrophosphate binding by the backbone amidesnear the C-terminal cyclic depsipeptide (D-Thr8—Ile11) ring, as well asby the side chains of Ser7 and the unique allo-enduracididine. Interest-ingly, the major difference between the two conformations is the swappingof two groups of hydrogen bond donors that coordinate one of the lipid IIphosphates, which results in opposite orientations of lipid II binding be-tween two conformations. In addition, regardless of the detailedphosphate-binding interactions, residues D-allo-Ile5 and Ile6 always func-tion as membrane anchors in both conformations. Based on the teixobactin-

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lipid II interactions captured in their complexes, as well as their partitiondepths at the membrane surface, we propose that the bactericidal mecha-nism of teixobactin is to arrest cell wall synthesis by selectively inhibitingthe transglycosylation of peptidoglycan, while the transpeptidation ispossibly unaffected. This pyrophosphate caging mechanism of lipid II in-hibition appears to be similar to some lantibiotics, but different fromthat of vancomycin or bacitracin.

2262-Pos Board B278Macromolecular Crowding Effects on Energetic Residue Contributions toPeptide-Nucleic Acid InteractionsCarla P. Perez1, Donald E. Elmore2, Mala L. Radhakrishnan2.1Department of Chemistry, Wellesley College, Wellesley, MA, USA,2Department of Chemistry and Biochemistry Program, Wellesley College,Wellesley, MA, USA.Antimicrobial peptides (AMPs) are small, cationic peptides that are natu-rally found in a variety of organisms. AMPs have therapeutic potentialas antibiotics due to their antimicrobial activity against a wide range ofbacteria and other pathogens. Some AMPs function via translocation,which involves the undisruptive crossing of bacterial cell membranes fol-lowed by the targeting of an intracellular component. For example, buforinII (BF2) is believed to act by targeting intracellular nucleic acids. In aneffort to better understand BF2 activity, previous studies have examinedBF2-nucleic acid binding under dilute buffer conditions. These studiesare expanded here to account for the macromolecular crowding environ-ment present under physiological cellular conditions. In particular, weare interested in trying to determine the most effective computationalmodels for considering these crowding effects. To this end, the bindingof BF2 to DNA under crowded cell conditions is studied using a varietyof computational crowding models. Crowding effects are accounted forthrough a representation of crowding as a lowered outer dielectric constant,as spherical crowders, or as explicit protein crowders. These models areapplied to snapshots extracted from molecular dynamics (MD) simulationson which continuum electrostatics calculations are performed to examineany crowding effects on particular residue contributions to BF2-DNA bind-ing. The use of MD enables better modeling of the dynamics and potentialarrangements of crowders in solution. Preliminary analyses show thatparticular cationic residues exhibit an increased contribution to binding in-dependent of the particular crowding model used. However, the magnitudeof this increased contribution to binding appears to be dependent on theparticular crowding model. These observations will help guide the modelsused by future work aimed at designing more potent peptides withenhanced nucleic acid binding.

2263-Pos Board B279Effects of Macromolecular Crowding on Antimicrobial Peptide ActivityHannah B. Schmidt1, Mala L. Radhakrishnan2, Donald E. Elmore2.1Department of Chemistry, Wellesley College, Wellesley, MA, USA,2Department of Chemistry and Biochemistry Program, Wellesley College,Wellesley, MA, USA.Due to the increasing public health concern of antibiotic resistant bacteria,antimicrobial peptides (AMPs) are a promising area of research for the devel-opment of new antibiotics. AMPs induce bacterial cell death in one of twoways: translocating into the cell and interfering with its nucleic acids or per-meabilizing the cell membrane and inducing cell lysis. While a large amountof previous research has focused on AMPs in dilute buffers, this does notaccurately reflect the physiological conditions where AMPs would be activeas drugs. Blood plasma and the extracellular matrix contain large concentra-tions of macromolecules that can affect the AMP’s ability to interact with thebacterial cell membrane. This study compares the activity of magainin, awell-characterized membrane-permeabilizing AMP, in dilute buffer to activ-ity when crowded with dextran, a large polysaccharide molecule without itsown antimicrobial properties. Propidium iodide uptake assays are commonlyused to measure the membrane permeabilization induced by antimicrobialpeptides. Our results have shown that magainin’s membrane permeabilizationmeasured by this assay is significantly decreased in the presence of crowdingmolecules. We have also employed circular dichroism spectrometry to give astructural interpretation of the crowding effects on magainin. These initialstudies emphasize the importance of developing appropriate experiments toconsider the potential effects of macromolecular crowding on antimicrobialpeptide activity.

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Posters: General Protein-Lipid Interactions II

2264-Pos Board B280SANS Observation of Precrystallization Intermediates of Bacteriorho-dopsin in the Lipidic Cubic PhaseThomas Edgar Cleveland, Paul Butler.Center for Neutron Research, National Institute of Standards andTechnology, Gaithersburg, MD, USA.Membrane proteins can be incorporated into the lipidic cubic phase (LCP)for crystal growth and structure determination. LCP crystallization hasbecome an important tool in the field of membrane protein crystallography(particularly, but not solely, with GPCRs and other small membraneproteins). However, many details of this process are not well understood.There is little direct experimental evidence for the localization of proteinand detergent after incorporation into LCP; the mechanisms of nucleationand crystal growth; and the details of how the cubic phase modifies theinteractions between protein molecules. We are using Small AngleNeutron and X-Ray Scattering (SANS/SAXS) to study each step of the cu-bic phase crystallization process using Bacteriorhodopsin (bR) as a modelsystem.Using SANS, it is possible to contrast-match the non-protein componentsof the system, i.e. detergents and lipids. This allows us to measure the pro-tein scattering directly and in isolation, greatly simplifying the data inter-pretation from these complex multicomponent systems. At high bRconcentrations, it is possible to measure structure factors, from which in-formation on protein-protein interactions can be obtained. We havemeasured the concentration-dependent scattering of bR: (1) in solution;(2) after incorporation into LCP; and (3) as a function of precipitantconcentration. Solution structure factor measurements at lower salt concen-trations are consistent with a charged sphere interaction model. In contrast-matched LCP at lower concentrations of bR and precipitant, scatteringfrom bR monomers could be observed, similarly to bR in solution. Athigher bR and precipitant concentrations, a series of higher-order structureswere observed by SANS, as well as protein-dependent Bragg reflections insamples in which macroscopic crystals were later observed.

2265-Pos Board B281Polymer Nanodiscs as New Platforms for Membrane ProteinsMariana C. Fiori1, Yunjiang Jiang1, Wan Zheng1, Miguel Anzaldua2,Mario J. Borgnia3, Guillermo A. Altenberg1, Hongjun Liang1,2.1Department of Cell Physiology and Molecular Biophysics, and Center forMembrane Protein Research, Texas Tech University Health Sciences Center,Lubbock, TX, USA, 2Department of Chemical Engineering, Texas TechUniversity, Lubbock, TX, USA, 3Laboratory of Cell Biology, Center forCancer Research, National Cancer Institute, National Institutes of Health,Bethesda, MD, USA.Lipid nanodiscs (LNDs) are discoidal nanostructures that consist of a lipidbilayer membrane patch encased within membrane scaffold proteins (MSPs)derived from apolipoprotein A1. LNDs are playing increasingly importantroles in studies of the structure and function of membrane proteins(MPs). A recent development is the use of styrene-maleic acid (SMA) co-polymers for solubilization and reconstitution of MPs into nanodiscs. Thesepolymer-encased nanodiscs (SMALPs, for SMA lipid particles) are prom-ising platforms for studies of MPs in a near-physiologic environmentwithout the use of detergents. However, current SMA copolymers displaysevere limitations in terms of buffer compatibility and ensued flexibilityfor various applications. In addition, the development of nanodiscs as aMP-supporting platform, or a drug targeting and delivery vehicle, is under-mined by the fluidic and labile nature of the lipid bilayer. Here, we intro-duce new approaches to address some of the drawbacks of SMALPs andLNDs by using a set of new block copolymers to replace the MSPs andanother set of block copolymers to replace the lipid bilayer. Our new familyof zwitterionic styrene-maleic acid-derivative copolymers (zSMAs) do notaggregate at low pH or in the presence of polyvalent cations (as commercialSMAs do), and can be used to solubilize MPs and produce nanodiscs ofcontrolled sizes. We also introduce polymer nanodiscs (PNDs), discoidalamphiphilic block copolymer membrane patches encased within MSPs.PNDs are novel two-dimensional nanomembranes that maintain the advan-tages of LNDs while addressing their stability weakness. We expect that thehigher mechanical and chemical stability of block copolymer membranesand their chemical versatility for adaptation will open new opportunitiesfor applications built upon diverse MP functions, or involved with drug tar-geting and delivery.

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This work was supported in part by NSF grants DMR-1623241 and CBET-1623240.

2266-Pos Board B282The Role of Angiomotin Coiled-Coil Homology Domain Arginine/LysineResidues in Vesicle Fusion ActivitySeth Sears1, Ann Kimble-Hill2.1Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA,2Biochemistry and Molecular Biology, Indiana University School ofMedicine, Indianapolis, IN, USA.Angiomotins (Amot) are a family of adaptor proteins that control cellularsignaling responsible for cellular differentiation and proliferation. Thesecellular events have been liked to regulation of invasive ductal carcinoma,the most common form of breast cancer. Their characteristic coiled-coilhomology (ACCH) domain is of particular interest because of its capabilityto selectively bind phosphatidylinositol lipids (PI). These binding events sub-sequently lead to lipid membrane deformation and juxtanuclear endosomalvesicle fusion to the apical membrane. Our library of arginine and lysine res-idue mutations in the ACCH domain were screened for a loss of vesiclefusion activity. The mutations at the following residues led to a diminishedfusion activity: R40T, K49E, K72E, K76E, R85T, R103G, K111E, K126E,K136E, K187E, R140S, R221Q, R224E, and R234G. Next, we endeavoredto characterize how each of these residues participated in vesicle fusion bydetermining the kinetic rate of vesicle fusion. In this study, fluorescence reso-nance energy transfer between probes from 2 different lipid populations as afunction of incubation time and protein concentration was utilized to deter-mine the rate of fusion. Careful analysis of this data will provide insightinto the ACCH domain structural elements that drive membrane fusionevents.

2267-Pos Board B283Membrane Solubilization by Styrene-Maleic Acid Copolymers: Impor-tance of Polymer Length and Comonomer SequenceAdrian H. Kopf1, Nelmari Harmzen2, Juan J. Dominguez1,Martijn C. Koorengevel1, Rueben Pfukwa2, Bert Klumperman2,Antoinette J. Killian1.1Membrane Biochemistry & Biophysics, Bijvoet Center and Institute ofBiomembranes, Utrecht University, Utrecht, Netherlands, 2Department ofChemistry and Polymer Science, Stellenbosch University, Matieland, SouthAfrica.Styrene-maleic acid (SMA) copolymers have emerged as a powerful alter-native to detergents for the extraction of membrane proteins from cellularmembranes. These polymers can solubilize membranes in the form ofnanodiscs that are stabilized by the polymer, allowing characterization ofmembrane proteins in their native environment. However, our understand-ing of the parameters that determine the efficiency of solubilization or theproperties of the resulting nanodiscs is still limited. This is partly due tothe heterogeneity of commercially available SMA copolymers, where thereare large variations in both the length distribution of the polymers and thecomonomer sequence distribution. Here we successfully synthesized a se-ries of copolymers by Reversible Addition-Fragmentation Chain Transfer(RAFT) mediated polymerization in conjunction with repetitive chainextension. These copolymers were found to have a well-defined length,composition and monomer sequence by size exclusion chromatography(SEC) and DEPT 13C NMR, among others. We investigated the solubiliz-ing efficiency of these polymers as well as the properties of the resultingnanodiscs using both lipid model membrane systems and E.coli mem-branes. Results of these experiments will be shown and the effects of thepolymers will be compared with those of commercially available polymers.

2268-Pos Board B284Characterizing the Lipid Annulus Surrounding Membrane Proteins withNative Mass Spectrometry of NanodiscsJames E. Keener, Deseree J. Reid, Dane Evan Zambrano, Ciara Zak,Michael T. Marty.Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA.It is challenging to characterize the transient and heterogeneous interac-tions between membrane proteins and their surrounding lipids. High reso-lution structural biology techniques can detect tightly bound structurallipids but generally cannot detect weakly bound annular lipids. Spectros-copy techniques can detect relative proximity of lipids but require labelsthat may disrupt their behavior. By using nondenaturing ionization to pre-serve noncovalent complexes for mass analysis, native mass spectrometry(MS) provides a label-free strategy for detecting and characterizing

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protein-lipid complexes. However, conventional native MS of membraneproteins uses detergent micelles to solubilize the membrane protein, whichlimits analysis to complexes with only a handful of lipids bound. To allowanalysis of a wide range of lipid interactions, we employ lipoprotein nano-discs to deliver membrane proteins for native MS. Using optimized ioniza-tion conditions, we can measure the mass of the intact nanodisc, whichallows direct measurement of the oligomeric state of the membrane proteinwithin the lipid bilayer of the nanodisc. Collisional activation of nanodiscsin the gas phase releases membrane proteins surrounded by a completeannulus of lipids, which can be further activated to dissociate the weaklybound lipids in the annulus. We are developing new approaches usingnative MS of nanodiscs with binary combinations of lipids to characterizethe composition and stoichiometry of this lipid annulus. Our ultimate goalis to develop a general method for characterizing protein-lipid interactionsof varying strengths and specificities.

2269-Pos Board B285Human ATG3 Binding to Lipid Bilayers. Role of Lipid Geometry andElectric ChargeJavier H. Hervas, Ane Landajuela, Zurine Anton, Anna Shnyrova,Felix M. Goni, Alicia Alonso.Instituto Biofisika (CSIC-UPV/EHU), Leioa, Spain.Specific protein-lipid interactions lead to a gradual recruitment ofAuTophaGy-related (ATG) proteins to the nascent membrane during auto-phagosome (AP) formation. ATG3, a key protein in the movement of LC3towards the isolation membrane, has been proposed to facilitate LC3/GA-BARAP lipidation in highly curved membranes. In this work we have per-formed a biophysical study of human ATG3 interaction with membranescontaining phosphatidylethanolamine, phosphatidylcholine and anionic phos-pholipids. We have found that ATG3 interacts more strongly with negatively-charged phospholipid vesicles or nanotubes than with electrically neutralmodel membranes, cone-shaped anionic phospholipids (cardiolipin and phos-phatidic acid) being particularly active in promoting binding. Moreover, anincrease in membrane curvature facilitates ATG3 recruitment to membranesalthough addition of anionic lipid molecules makes the curvature factor rela-tively less important. The predicted N-terminus amphipathic a-helix ofATG3 would be responsible for membrane curvature detection, the positiveresidues Lys 9 and 11 being essential in the recognition of phospholipidnegative moieties. We have also observed membrane aggregation inducedby ATG3 in vitro, which could point to a more complex function of this pro-tein in AP biogenesis. Moreover, in vitro GABARAP lipidation assays sug-gest that ATG3-membrane interaction could facilitate the lipidation of ATG8homologues.

2270-Pos Board B286Detection of Helix Fraying of a Transmembrane Peptide with TwoInterfacial Arginine ResiduesSara Sustich, Fahmida Afrose, Denise V. Greathouse, Roger E. Koeppe II.Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA.Transmembrane peptides are often flanked by interfacial aromatic residuesthat may play a role in anchoring the peptide to assist in the stabilizationof a tilted transmembrane orientation. The peptide GWALP23 (acetyl-GG2-AWLALALALALALALWLAG22A-amide) containing two interfacialTrp residues has been shown to have a stable transmembrane orientationin lipid-bilayers of varying thickness. GWALP23 has provided a usefulframework for additional investigations of the protein-lipid interactionsin biological membranes. Previously GWALP23 family peptides werecharacterized with a double mutation of G2 and G22 to arginine (J.Biol. Chem. 285, 13723-13730, 2010) and the properties of the transmem-brane peptide remain quite similar to the host GWALP23 peptide. Thegoal of this research is to investigate the partial unwinding of the endsof R2,22WALP23 and for this purpose we have incorporated 2H labelsat Ala3 and Ala21. These alanines are outside of the core helical regionof the peptide and could be sensitive to the helical integrity. Thisapproach reveals the influence of the interfacial residues on the extentof unwinding of the helix ends. Solid-state 2H NMR was performed onthe peptide in the lipid-bilayers of DOPC, DMPC, and DLPC. The deute-rium labeled alanines showed unwinding at both ends of the peptide ineach lipid membrane. Further research will investigate the extent of theunwinding in each bilayer membrane and whether the Trp residues at po-sitions 5 and 19 are unwound as they aid in anchoring the peptide to thelipid-bilayer.

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2271-Pos Board B287Ionization and Dynamic Properties of Single and Multiple HistidineResidues on a Transmembrane Helical BackboneFahmida Afrose, Denise V. Greathouse, Roger E. Koeppe II.Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA.Many membrane proteins contain functionally important His residues withintheir transmembrane domains. To address the ionization properties of Hisresidues at various locations within the lipid bilayer, we have employedGWALP23 (acetyl-GGALW5 (LA)6LW

19 LAGA-amide) as a host frame-work. Histidine may also substitute for tryptophan as a stabilizing interfacialresidue, as it has similar hydrogen bonding ability. We have incorporatedpairs of histidines in different positions on either side of the helix midpoint,such as (H5, H19); (H2, H22) and (H8, H16) of GWALP23 and tested theionization properties of these pairs over a pH range from 2-8. Solid-state2H NMR measurements were made possible by the inclusion of deuteratedalanine residues in the core helix and near the ends of peptide. The resultsvary with the His positions and hydrophobic depth within the bilayer.H2,22WALP shows little change from pH 2-8, yet the 2H quadrupolar splittingfor A9 does not fit the wave plot in DLPC. The GH5,19ALP helix in DLPC orDOPC bilayers also changes little between pH 2 and pH 8. In both cases thetransmembrane orientation and dynamics do not vary much compared to thehost peptide. When the His residues are more buried, GWALP23-H8,16 seemsto adopt a more defined orientation in DOPC as opposed to DLPC bilayers.Additional investigation is required to obtain a clear idea about the dynamicaspects of GWALP23-H8,16. Experiments are underway with single His res-idues at position 2, 5, 19 or 22 to help clarify the results. The unwinding ofhelix ends and their variation with pH is also a matter of interest.

2272-Pos Board B288HIV GP41 Envelope Protein Early and Late Membrane Fusion Stages areImpaired by a Sphinganine Based Lipo-PeptideYoel A. Klug1, Avraham Ashkenazi1, Mathias Viard2, Ziv Porat1,Robert Blumenthal2, Yechiel Shai1.1Weizmann Institute of Science, Rehovot, Israel, 2National Institutes ofHealth, Frederick, MD, USA.HIV infectivity can be blocked by lipid conjugated peptides, termed lipo-peptides, which modulate various biological systems including viral fusion.These compounds have advanced the understanding of membrane protein func-tions and the roles of lipids in the membrane milieu. However, a single functionhas been suggested for the lipid, which is binding to the membrane thuselevating the peptide’s local concentration at the target site. Utilizing biophys-ical and biochemical methods coupled with fully infectious virions we chal-lenged this argument. We investigated the antiviral mechanism of lipo-peptides comprised of an HIV envelope derived peptide and sphinganine, thelipid backbone of dihydrosphingomyelin enriched in the HIV and target cellmembrane. We uncovered a partnership that reduced CD4 diffusion andHIV-1 fusion peptide mediated lipid mixing. In addition, the lipo-peptideslocalized to the virus-cell and cell-cell contact sites and disrupted HIV-1 fusionprotein assembly and folding. Overall, the findings might help uncover a rolefor elevated dihydrosphingomyelin in HIV-1 and its target cell membranesand may be implicative to lipid-protein interactions in various biologicalsystems.

2273-Pos Board B289Molecular Simulations Detail the Thermal Expansion of thePre-endosomal Dengue VirusJan K. Marzinek1,2, Roland G. Huber1, Kamal K. Sharma2,Thorsten Wohland2, Chandra Verma1,2, Peter J. Bond1,2.1Bioinformatics Institute, A*STAR, Singapore, Singapore, 2Department ofBiological Sciences, National University of Singapore, Singapore, Singapore.The dengue virus (DENV) is a positive single-stranded RNA flavivirus thatinfects �400 million a year. At room temperature (�25�C), the mature flavi-viruses has a ‘‘smooth’’ envelope (E) with 90 dimers embedded in a lipidbilayer that exhibits significant curvature. In the endosome the lowered pHtriggers a major conformational change of the E proteins: a dimeric-to-trimeric transition. Prior to this conformational change, the E proteins areprimed for the transition by the change of temperature in the host organism(37�C). This primed state has been captured at low resolution bycryo-electron microscopy (cryo-EM), and shows the DENV-2 to possessan expanded protein shell, with the E protein ectodomains raised by0.15 nm above the, now, spherical membrane. Due to the low resolution of

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the cryo-EM data, the atomic details of this ‘‘bumpy’’ state are still elusive,and the positions of the E and membrane (M) stem/transmembrane (TM) re-gions remain unclear. In order to provide these details, we have performedmicrosecond time scale coarse-grained targeted molecular dynamics simula-tions of the entire DENV-2 envelope, complete with E/M proteins embeddedin a realistic lipid vesicle. During the transition from ‘‘smooth’’ to ‘‘bumpy’’,the diffusion of water was seen to aid in the dissociation of the E protein ec-todomain domains from the lipid bilayer. The final ‘‘bumpy’’ model also pro-vides the position of the E/M and TM regions that were not resolvable in thecryo-EM data. Additional simulations and experiments reveal the influence ofdivalent cations and that specific Fab antibodies bound to the 2-fold verticeshave the highest propensity to be dislodged, agreeing with cryo-EM observa-tions. We also propose that two mutations (E172 and S298C) can block thevirus opening in silico. These insights provide details of potential novel ther-apeutic targets.

2274-Pos Board B290Characterizing MA Interactions with Mimetic Membranes in the Presenceand Absence of tRNALYS3

Emre Tkacik, Christy Gaines, Michael Summers.UMBC, Baltimore, MD, USA.Human immunodeficiency virus-1’s (HIV-1) matrix domain (MA) of the Gagpolyprotein targets Gag to the plasma membrane through interactions betweenMA’s highly basic region (HBR) and phosphatidylinositol 4,5-bisphosphate[PI(4,5)P2] on the plasma membrane, allowing viral assembly to occur.CLIP studies have shown that MA also specifically binds to certain tRNAsin vivo, including tRNALys3, the primer for reverse transcription. This workand liposome flotation assays led to a proposed mechanism that tRNA bindsto MA preventing nonspecific binding to membranes other than the plasmamembrane, where PI(4,5)P2 outcompetes the tRNA for binding to MA. Thispromotes myristoyl exposure, allowing anchorage into the plasma membranefor budding to occur. Using isothermal titration calorimetry, we found that asexposure of MA’s N-terminal myristoyl group increases, MA’s affinity fortRNALys3 decreases. To test this competition between tRNA and PI(4,5)P2,we conducted 1H-1D NMR liposome binding assays to examine how MA in-teracts with liposomes both containing or lacking PI(4,5)P2, in the presenceand absence of tRNALys3. As expected, tRNALys3 outcompetes liposomesthat lack PI(4,5)P2, however, it also outcompetes liposomes that containPI(4,5)P2. There is evidence that Gag targets to lipid rafts, not just PI(4,5)P2. We repeated the experiment using liposomes high in cholesterol and nega-tively charged lipids, that were designed to mimic lipid rafts, the plasma mem-brane, and the HIV viral envelope, but again, tRNALys3 outcompeted theliposomes for binding to MA regardless of liposome composition. This sug-gests that the targeting mechanism is not yet complete. Liposome flotation as-says performed by other labs that saw PI(4,5)P2 outcompete tRNA for bindingused Gag rather than MA. Future work will determine the effect the other do-mains of Gag may have on tRNA-MA interactions and their role in membranetargeting.

2275-Pos Board B291Function, Property, and Interaction of Archaeal Lipids: A MolecularDynamics Simulation StudyShasha Feng1, Jeffery B. Klauda2, Wonpil Im3.1School of Life Sciences, Peking University, Beijing, China, 2Department ofChemical and Biomolecular Engineering and Biophysics Program,University of Maryland, College Park, MD, USA, 3Department of BiologicalSciences and Bioengineering, Lehigh University, Bethlehem, PA, USA.The archaea form a diverse kingdom, with many species existing in extremeenvironments like hot springs and salty lakes. To cope with such harsh environ-ments, archaea have developed their characteristic membrane lipids, such asether-linked lipids instead of typical ester-linked ones and single membrane-spanning lipids rather than bilayer-forming lipids. The tails of these lipids oftencontain repeated isoprene units. These characteristics render their membranesmore rigid and resistant to high temperature and ion permeability. In addition,proteins originating from archaea like archaerhodopsin-3 have inspired plentyof useful biological tools in past decades. In our study, we developed theCHARMM topologies and parameters of these unique lipids, simulate thearchaeal membranes, and analyze the simulation trajectories for membraneproperties as well as their interactions with archaeal archaerhodopsin-3.Coupled with experimental findings, our study provides insight into the func-tion and property of archaeal lipids, and also helps understand proteins ofarchaeal origin.

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2276-Pos Board B292Structure-Activity Correlations of Amphipathic Cationic AMPs: MDSimulations and Antibacterial MeasurementLoan K. Huynh, Roel Rabara, Jeanette Velasquez, Supratim Basu,Hau Nguyen, Goutam Gupta.Biology, New Mexicoconsortium, Los Alamos, NM, USA.Bacterial drug resistance is one of the largest issues that impede the develop-ment of new antibiotics. Since their discovery in multiple hosts, antimicrobialpeptides (AMPs) appear to be a viable alternative to antibiotics. Here, we reportstructure-activity correlations of an amphipathic cationic peptide P1, KKLIK-KILKIL, and its analogs, that target bacterial membrane unlike the antibioticsthat target intracellular machineries. We performed molecular dynamics simu-lations on P1 and its analogs to study their insertion into a model lipid bilayer.We also measured the antibacterial activities of P1 and its analogs on a wild-type E. coli and a resistant strain evolved against P1. Combining experimentand molecular dynamics simulations, we show that: (i) neutralization of theC-terminal L of P1 enhances both membrane insertion and antibacterial activ-ity; (ii) reduction of charge and hydrophobicity in P2, KKLAKEILKIL (withunderlined amino acid substitutions) drastically reduces the antibacterial activ-ity; (iii) P3 with C-terminal charge neutralization remains susceptible to resis-tance mechanism evolved against P1; (iv) however, increase in electrostaticinteraction with the lipid in P4, RRLIRRILRIL with K to R substitutions, ren-ders protection against bacterial resistance. In summary, combination of molec-ular dynamics simulations and antibacterial activity measurement provideinsight into the mechanism of membrane insertion of amphipathic cationicAMPs and offer guidelines to increase their activity and decrease their suscep-tibility to bacterial resistance.

2277-Pos Board B293Conformational Changes in Marburg Virus VP40 upon PlasmaMembrane AssociationNisha Bhattarai, Prem P. Chapagain, Bernard S. Gerstman.Physics, Florida International U, Miami, FL, USA.Infections with the Marburg and Ebola viruses from the Filoviridae familycause hemorrhagic fever that often results in high fatality rates in human.The Marburg virus is a lipid enveloped virus and its viral matrix is formedby the matrix protein VP40. Like in Ebola virus VP40, the crystal structureof Marburg VP40 (mVP40) also contains basic residues that form lipid bindingpatch but the basic patch of mVP40 is significantly broad and extended thanthat of Ebola VP40 (eVP40). This suggests the possible differences in theplasma membrane (PM) binding and phospholipid specificity between VP40dimers. Here, we investigate the roles of various residues and lipid types inPM association as wells as conformational changes of mVP40 dimer in bothlipid and lipid free environments using molecular dynamics simulations.Despite the significant structural differences in the crystal structure, mVP40dimer is found undergo conformational rearrangements of the protomers andadopt a configuration similar to eVP40 after associating with membrane.These conformational changes upon lipid binding allow mVP40 to localizeand stabilize at the membrane surface similar to the eVP40 dimer, but maygive subtle differences in its function due to the differences in the solutionconformations.

2278-Pos Board B294Orderphobic Effect of Proteins in Multicomponent MembranesClay H. Batton1, Shachi Katira2, Kranthi K. Mandadapu1.1Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, CA,USA, 2Chemistry, UC Berkeley, Berkeley, CA, USA.We examine the structure and dynamics of a multicomponent membrane sys-tem exhibiting a liquid-disordered and liquid-ordered phases along with modelproteins. It has been shown in previous work that proteins with hydrophobicthickness mismatch can exhibit pre-transition effects stabilizing an order—dis-order interface, termed the orderphobic effect [1]. This effect is mediated by afirst order phase transition between solid ordered and liquid disordered phasesor liquid-ordered (Lo) and liquid-disordered (Ld) phases. The orderphobic ef-fect provides a mechanism for assembly and mobility of proteins and phases inthe membrane. In this work, we examine the consequences of the orderphobiceffect with respect to Lo-Ld first-order phase transition. We perform simulationstudies probing the phase behavior of a model multicomponent membranesystem with proteins that induce a liquid-disordered phase within a bulkliquid-ordered system. The composite phase diagrams of proteins and multi-component lipid membranes are analyzed with respect to key membrane pa-rameters and adjusting the strength of the orderphobic effect. Theimplications of this behavior with respect to membrane budding and lipid rafts

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are discussed. In particular, we discuss the length and time scales of theemerging lipid rafts.

2279-Pos Board B295Cell-Space Confinement Effects on Min Protein Waves InsideMicrodropletsShunshi Kohyama.Graduate School of Fundamental Science and Technology, Keio University,Yokohama, Japan.Min system is the determinant mechanism of the position of division plane inbacterial cells. Reaction-diffusion coupling of Min system components (MinD,MinE, and ATP) emerges pole-to-pole oscillation of Min proteins (Min wave),and it inhibits assemblies of cell division initiator (Z ring) except at the centerof cell. Although Min wave has been successfully reconstituted on 2D planarmembrane and in fully confined systems, we found that physiological concen-tration of Min system components does not emerge Min wave in microdropletscovered with E. coli polar lipids. This finding is contrary to the results from pre-vious studies, and thus, we investigated the mechanism underlying this differ-ence.Consequently, we found ‘‘cell-space confinement effects’’ are the cause ofthe difference. Recent studies have shown that ‘‘cell-space confinement ef-fects’’ changes behaviors and dynamics of biological systems. In the case ofMin system, our biochemical investigations showed that the cell-space confine-ment changes physicochemical parameters of Min proteins, and hence, behav-iors of Min wave were altered by the confinement. Furthermore, we showed amacromolecular crowding reagent counteract the change of the physicochem-ical parameters of Min proteins, and ensure stable emergence of Min wave in-side microdroplets covered with E.coli polar lipids. We also found that artificiallipid composition of the previous study using microdroplets works similarly tothe macromolecular crowding reagent we used.Our results predict that unde-fined factors are critical for stable emergence of Min wave in living cells,and evoke that importance of cell-size confinement effect for understandingcoupling of reaction with diffusion in living systems.

2280-Pos Board B296New Continuum Approaches to Study how Arbitrary Shape ProteinsInduce Membrane DeformationsDavid Argudo, Michael Grabe, Neville Bethel, Frank Marcoline.Pharmaceutical Chemistry, University of California, San Francisco, SanFrancisco, CA, USA.The influence of the membrane on transmembrane proteins is central to variousbiological phenomena, notably the gating of stretch activated ion channels.Conversely, membrane proteins can influence the bilayer, leading to the stabi-lization of specific membrane shapes, topological changes that occur duringvesicle fission and fusion, and shape-dependent protein aggregation. Contin-uum elastic models of the membrane have been widely used to studyprotein-membrane interactions. These mathematical approaches produce phys-ically interpretable membrane shapes, energy estimates for the cost of deforma-tion, and a snapshot of the equilibrium configuration. Moreover, elastic modelsare much less computationally demanding than fully atomistic and coarse-grained simulation methodologies; however, it has been argued that continuummodels cannot reproduce the distortions observed in fully atomistic moleculardynamics simulations. We suggest that this failure can be overcome by usingchemically and geometrically accurate representations of the protein. We pre-sent a fast and reliable hybrid continuum-atomistic model that couples the pro-tein to the membrane. We show that the model is in excellent agreement withfully atomistic simulations of nhTMEM16 lipid scramblase and simulations ofthe ion channel gramicidin. Our continuum calculations not only reproduce themembrane distortions produced by the inserted protein but also accuratelydetermine the protein’s orientation.

2281-Pos Board B297Investigating the Influence of Transmembrane Proteins on the LocalMembrane EnvironmentGergo F€ulop.Applied Physics, TU Wien, Wien, Austria.It is well established that lipids and proteins are not just independent compo-nents of the plasma membrane of eukaryotic cells but that their arrangement,dynamics and function are interdependent.Besides direct lipid-protein interaction, transmembrane proteins are thought tobind a shell of annular lipids, which are more or less tightly associated with theproteins. Furthermore, highly ordered nanoscopic membrane domains havebeen proposed to act to compartmentalize proteins and their interactions, buthave thus far not been directly observed. In general, detailed information on

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lipid-protein interactions in living cells is largely missing. The reason forthis is that such interactions are inherently difficult to study and currentmethods hardly allow for quantitative characterization of the dynamic associa-tion of lipids and proteins under physiologically relevant conditions.In this study, we use protein micropatterning combined with single-moleculetracking to directly measure lipid-protein interactions in the plasma membraneof living cells: different fluorescently labelled transmembrane proteins of inter-est (POIs) were captured and enriched within well-defined areas in the plasmamembrane, leaving regions depleted of POI, which function as reference areas.From the distribution and diffusion behaviour of lipids and proteins withrespect to the POI patterns, we were able to conclude on the local membraneenvironment of the POI. We found that a palmitoylated protein based on thetransmembrane domain of hemagglutinin (HA-mGFP) influences its membraneenvironment well beyond the size of the transmembrane helix. The same effectwas observed for a palmitoylation-deficient mutant allowing us to rule out for-mation of a more ordered membrane domain around HA-mGFP as the cause forthis apparently increased protein size.

2282-Pos Board B298A Combined Computational and Experimental Study to Investigate theRole of COQ9 in Promoting COQ BiosynthesisDeniz Aydin1, Danielle C. Lohman2, David J. Pagliarini2, Matteo DalPeraro1.1Ecole Polytechnique F�ed�erale de Lausanne, Lausanne, Switzerland,2University of Wisconsin-Madison, Madison, WI, USA.Integral and peripheral membrane proteins account for one-third of the humanproteome, and they are estimated to represent the target for over 50% of modernmedicinal drugs. Despite their central role in medicine, the complex, heteroge-neous and dynamic nature of biological membranes complicates the investiga-tion of their mechanism of action by both experimental and computationaltechniques. Among the different membrane bound compartments in eukaryoticcells, mitochondria are highly complex in form and function, and they harbor aunique proteome that remains largely unexplored. A growing number of in-herited metabolic diseases are associated with mitochondrial dysfunction,which necessitates the structural and functional elucidation of mitochondrialproteins. In this work, we combine computational and experimental methodsto explore the lipid binding activity of COQ9, a member of the mitochondrialcoenzyme Q synthesis machinery that is structurally characterized but remainsfunctionally obscure. We elucidate the mechanistic details of its membranebinding process, which gives hints about possible interaction surfaces withother members of the CoQ synthesis machinery and has implications on howit mediates functional interactions with lipids. Together, our data suggest amodel wherein COQ9 has a lipid presentation role at the penultimate step ofCoQ biosynthesis that helps to overcome the hydrophobic barrier of the mem-brane. Collectively, this work is a good illustration of the interplay betweenexperiment and modeling in protein research and specifically in understandinghow proteins perform their action in direct synergy with membraneenvironments.

2283-Pos Board B299Identification of Cardiolipin Binding Sites on Membrane Proteins using anAccelerated Computational Membrane ModelAndres S. Arango1, Tao Jiang1, Ricky Cheng2, Merritt Maduke2,Emad Tajkhorshid1.1Center for Biophysics and Quantitative Biology, University of Illinois atUrbana-Champaign, Urbana, IL, USA, 2Molecular & Cellular Physiology,Stanford University, Stanford, CA, USA.Cardiolipin (CDL), a negatively-charged lipid found primarily in bacterial andmitochondrial membranes, is believed to play a major role in membranemorphology, some hereditary heart diseases, as well as acting as a proton reser-voir in the inner mitochondrial membrane. CDL has been shown to regulatemany integral membrane proteins involved in bioenergetics, often bindingwith nanomolar affinity. In this study we incorporate CDL into the Highly Mo-bile Membrane Mimetic (HMMM), a membrane model consisting of lipid headgroups partitioned by an organic solvent mimicking hydrophobic acyl tails,which allows for fast lipid diffusion, nanosecond spontaneous membrane inser-tion of proteins, and providing atomistic sampling of CDL-protein interactions,which gives rise to the identification of potential CDL binding residues. As anapplication, we chose CLC chloride transporter (CLC-ec1), an integral mem-brane protein involved in maintaining proton gradients, which initial experi-mental results show that CDL increases CLC-ec1 activity, likely due toCDLs ability to act as a proton reservoir. The increased lipid diffusion and sam-pling of CDL containing HMMM membranes aided in determining potential

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binding sites for ClC Chloride transporter. Incorporating CDL into the HMMMmodel involved applying harmonic restraints to each carbonyl carbon for eachtail to maintain membrane thickness, as well as applying RMSD based collec-tive variable restraints on ClC-ec1. Using ten one-hundred nanosecondHMMM simulations of CLC-ec1 embedded into a 80:20 POPE:CDL mem-brane, interactions between CDL and protein residues were measured. Theoverall microsecond simulation time, together with the enhanced dynamicsoffered by the HMMM membrane, provided a large sample size, which ledto narrowing down the potential CDL binding sites to two sites, each stabilizedby two basic side chains, thus expediting and guiding ClC-ec1 mutagenesisstudies.

2284-Pos Board B300Lipid-Dependent Alternating Access Mechanism of a Bacterial MultidrugABC Transporter: A Molecular Dynamics StudyJeevapani Hettige, Seyed Hamid Tabari, Mahmoud Moradi.University of Arkansas, Fayetteville, AR, USA.We have performed an extensive set of all-atom molecular dynamics (MD)simulations on a bacterial multidrug ABC transporter, Sav1866 in explicitPC and PE membrane environments. This study focuses on the energeticallydownhill, outward-facing (OF) to inward-facing (IF) conformational transitionof an ABC transporter. In the current study, we have characterized an interme-diate, IF occluded conformation of Sav1866 transporter embedded in a PE lipidenvironment, which is believed to appear during the OF to IF conformationaltransition.However, we have not been able to capture the IF conformation of the trans-porter during any of the microsecond level MD simulations. Nonetheless,such OF to IF occluded conformational transition was not observed in the trans-porter, when the transporter was in a PC lipid membrane environment. To ourknowledge, this observation directly coincides with the lipid dependentbehavior of the alternating access mechanism of the ABC exporter.

2285-Pos Board B301AT the Air-Water Interface, the Reversibility of the PhospholipidMonolayers in the Presence of Mini-B, Depends on the LipidHead-Group Charge of the MixturesAishik Chakraborty1, Saba Ghazvhini2, Alan J. Waring3,Prajnaparamita Dhar1.1Chemical & Petroleum Engineering, The University of Kansas, Lawrence,KS, USA, 2Biofrontiers Institute, University of Colorado Boulder, Boulder,CO, USA, 3Department of Medicine, Harbor-UCLA Medical Center,Torrance, CA, USA.Lung surfactants [LSs] are complex lipo-protein mixtures that cover the air-water interface of the alveoli. LSs help in reducing the surface tension of thewater, and this, in turn, conserves the work needed for expanding the lungs.Deficiency or dysfunction of the LSs is highly detrimental and can become fatalif left untreated. One fundamental property of the native LSs is their ability torespread at the interface and subsequently retain material. This property allowsthe LSs to go through multiple cycles of compressions and expansions avoidinga substantial loss. At the end of each compression cycle, the monolayer col-lapses from its 2-D state to a 3-D one, which appears as folds on the surface.In this study, we have focused on the impact of Mini-B, a synthetic derivativeof the native LS protein SP-B, on the reversibility of LS monolayers. To under-stand the mechanism of collapse, we have used fluorescence imaging alongwith active interfacial micro-rheology, where the re-orientation of Nickelnano-rods has allowed us to monitor the surface viscosity of differentphospholipid-Mini-B combinations. We have observed that Mini-B alongwith DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine):POPG (1-palmi-toyl-2-oleoyl-sn-glycero-3-phospho- (1’-rac-glycerol)) collapses with folds,whereas, the mixtures containing DPPC:POPC (1-palmitoyl-2-oleoyl-sn-glyc-ero-3-phosphocholine) with Mini-B doesn’t form similar structures. Further-more, the surface viscosities of the mixtures are also substantially modified

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in the presence of Mini-B, depending on the lipid head-group charge. There-fore, we can use this information to optimize the composition of the surfactantsfor treating the relevant diseases.

2286-Pos Board B302Giant Lipid Vesicles with Inner Compartments to Mimic Eukaryotic CellsNaresh Yandrapalli, Tabea Kirchhofer, Tom Robinson.Theory and Bio-Systems, Max Planck Institite of Colloids and Interfaces,Golm-Potsdam, Germany.Eukaryotic cells are highly complex with many processes being compartmen-talized within membrane bound organelles. Until now, synthetic lipid vesi-cles have provided the simplicity that is required to controllably model andunderstand some cellular processes. However, there are no existing platformsto study compartmentalized lipid membrane systems reliably. We aim tocreate multi-compartment giant unilamellar vesicles (GUVs) with a definedsize and number of independent inner lipid vesicles. Such a platform is alogical step in achieving the long-term goal of a minimum artificial cell,but we also use it to study protein-membrane interactions and to mimiccell polarization. In our work, we implemented the phase transfer methodand microfluidic technology to develop our multi-compartmental lipid vesi-cles. Both of these emulsion-based methods can be employed to encapsulatethe desired components (liposomes, large proteins, and DNA). The producedGUVs are characterized for their morphology and unilamellarity using analpha-hemolysin based leakage assay. By comparing the results from boththe methods, we found that the microfluidic approach can reliably produceinner compartments with respect to encapsulation efficiency, size and numberof intraluminal vesicles. In addition to the development of these compart-mentalized vesicles, we studied the interaction of the GUVs with their innercompartments via protein-protein adhesion. Such a controllable approach hasgiven us the opportunity to visualize the entire process of adhesion and theeffect of molecular crowding on lipid bilayers. Furthermore, we observedmembrane bending and symmetry breaking of the outer GUV upon interac-tions with the inner compartments. We aim to control this adhesion event byemploying light-switchable proteins in the membranes of both vesicles. Thiswork is part of the MaxSynBio consortium which is jointly funded by theFederal Ministry of Education and Research of Germany and the Max PlanckSociety.

2287-Pos Board B303Liposomes as Targets for ToxinsMarcelo Ayllon1, Andy Bogard2, Juliette Kay Tinker1, Daniel Fologea1.1Biomolecular Science Graduate Programs, Boise State University, Boise,ID, USA, 2Boise State University, Boise, ID, USA.Many toxins and viruses are known to target components of cell membranes,which raises the question if such membranes may constitute effective targetsfor their elimination from biological fluids. To demonstrate this hypothesis,we investigated removal of free cholera toxin subunit B (CTB) from solutionsby using unilamellar liposomes as preferred targets. Since CTB is known topreferentially bind to the ganglioside GM1, we prepared liposomes containingvarious amounts of GM1 and investigated the binding to CTB. Unilamellar li-posomes produced by extrusion and comprising various ratios of asolectin,cholesterol, and GM1 have been exposed to fluorescent CTB and the specificbinding was assessed by fluorescence microscopy. In addition, we reconstitutedgiant vesicles supported by hard core glass beads, which facilitated quantifica-tion of interactions in terms of Kd by both fluorescence spectroscopy and Ki-netics Exclusion Assay (Kinexa�). With the long-term goal of using suchliposomes in vivo, we investigated membrane-CTB binding by includingPEG-lipids in the membrane structure to suppress their premature uptake bymacrophages and attack by immune system. Our results suggest that liposomesmay constitute excellent targets for macro-molecules and bio-molecular assem-blies, which open novel avenues for applications in nanomedicine, diagnosisand therapy.

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Posters: Membrane Receptors and SignalTransduction II

2288-Pos Board B304Mechanism of TRK Receptor Dimerization and ActivationFozia Ahmed, Kalina Hristova.Materials Science and Engineering, Johns Hopkins University, Baltimore,MD, USA.We have previously hypothesized that all Receptor Tyrosine Kinases(RTKs) follow a single ‘‘unified’’ model of activation, characterized bythe (i) existence of RTK unliganded dimer, (ii) varying levels of unligandeddimer stabilities (and thus dimer abundance at physiological concentrations)and (iii) ligand-induced dimer structural changes that propagates to theintracellular domains. Depending on the exact value of the two-dimensional dissociation constant and the expression levels, receptors mayappear to be predominantly monomeric or predominantly dimeric at a spe-cific expression level. In support of this unified model of RTK activation,for several RTKs it has been shown that ligand-induced structural changesoccur in the dimer and that they involve rearrangement of the transmem-brane helices. However, it is not yet known if all RTK dimers undergo astructural change upon ligand binding. Here we investigate the mechanismof activation of the Tropomyosin Receptor Kinases (Trks) family of RTKs.The Trk receptors are expressed in neuronal tissues, and guide the develop-ment of the central and peripheral nervous systems. They have been impli-cated in the progression of several cancers, including colon and thyroidpapillary carcinomas, and breast cancer. We used FRET based technologyto determine mechanism of dimerization and activation for Trk receptors.Results of our studies show that the Trk receptors follow the hypothesizedmodel of activation.

2289-Pos Board B305Investigating the Association of Ephrin Type-B Receptor Tyrosine Kinasesin Live CellsTaylor Patrick Light, Kalina Hristova.Materials Science and Engineering, Johns Hopkins University, Baltimore,MD, USA.Ephrin (Eph) receptors represent the largest family of receptor tyrosine ki-nases (RTKs). RTKs are involved in signal transduction cascades that areactivated by receptor dimerization, a process that is modulated by ligandbinding. Eph receptors mediate cell-cell contacts for intercellular communi-cation through interaction with specific ephrin ligands on neighboring cells.Independent of ligand binding, Eph receptors are also known to regulatemultiple developmental processes and tissue homeostasis. The biophysicalbehavior of the fourteen Eph receptors is not well characterized. The broadgoals of this research are to determine the mechanism of Ephrin type-B re-ceptor homo- and hetero-association in the absence and presence of ephrinligands and to determine which domains are critical for mediating the inter-action. Here, two of the Ephrin type-B receptors, EphB2 and EphB6, areused toward these aims. The fully quantified spectral imaging FRET methodis used to probe the association of these receptors in the plasma membrane oflive cells. Our preliminary data suggest that both EphB2 and EphB6 self-interact in the membrane. Discovering a molecular basis of the homo- andhetero-interactions of these receptors is important for understanding thecomplex mechanisms that underlie cell-cell communication and Ephreceptor-related cancers.

2290-Pos Board B306Investigating the Hetero-interactions of Receptor Tyrosine Kinases in LiveCellsMichael D. Paul1, Kalina Hristova2.1Biophysics, Johns Hopkins University, Baltimore, MD, USA, 2MaterialScience and Engineering, Johns Hopkins University, Baltimore, MD, USA.Receptor tyrosine kinases (RTKs), the second largest class of membrane recep-tors, transduce signals across the plasma membrane via lateral association.They play an important role in development, regulating tissue growth, and con-trolling cell migration. Mutations in RTKs are associated with many cancers,and thus anti-RTK drugs are being actively developed and tested. Whilemany drugs have entered the market, they largely have only been moderatelysuccessful. One possible explanation for this is a lack of biophysical under-standing of how these receptors interact with each other. There is a growingbody of evidence that RTKs from different subfamilies (e.g., VEGFs, Ephs,FGFRs) can form hetero-species; however, the current picture of this interac-tome is rather incomplete. It is possible that presently unknown interactionsaffect the response of a given RTK to its ligands and inhibitors. Using a quan-

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titative FRET spectroscopy method known as Fully Quantified Spectral Imag-ing (FSI), we demonstrate the existence of specific interactions betweenmembers of different RTK subfamilies. Finally, we present thermodynamicmodels that show the relative population of heterodimers under differentconditions.

2291-Pos Board B307Investigating the Interactions between VEGFR2 and EGFRHana N. Grubb1, Michael D. Paul2, Kalina Hristova3.1Valencia College, Orlando, FL, USA, 2Department of Biophysics, JohnsHopkins University, Baltimore, MD, USA, 3Department of Materials Scienceand Engineering, Johns Hopkins University, Baltimore, MD, USA.Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) and EpidermalGrowth Factor Receptor (EGFR) are two receptor tyrosine kinases (RTKs)that play critical roles in the process of angiogenesis. Angiogenesis is theprocess by which new blood vessels can form from pre-existing vascula-ture. Angiogenesis is a process that tumors can take advantage of in orderto obtain needed nutrients. Currently, little is known regarding the interac-tions between these two receptors. Insight into these interactions willbroaden our knowledge of RTK interactions, and may possibly provideus with a better understanding of drug resistance in cancer treatments.Here, we describe the interactions between VEGFR2 and EGFR usingFRET.Fully Quantified Spectral Imaging Forster Resonance Energy Transfer (FSIFRET) was used to study the interactions between VEGFR2 and EGFR. Inbrief, HEK 293T cells were transiently transfected with VEGFR2 taggedwith MTurquoise and EGFR tagged with YFP. Cells were then imaged usinga two-photon microscope, and analyzed with custom written MATLAB soft-ware to determine FRET and the two-dimensional concentration of the recep-tors in the membrane.Our work indicates that VEGFR2 and EGFR interact with one another. Webelieve these results provide insight into how RTKs function, and furtherwork is needed to understand the implications for both normal biological pro-cesses and cancer.

2292-Pos Board B308EGF Activation of EGFR Drives Crosstalk with RON at the Plasma Mem-braneCarolina Franco Nitta1,2, Ellen W. Hatch1,2, Justine M. Keth1,2,Rachel M. Grattan1,2, Elton D. Jhamba1,2, Mara P. Steinkamp1,2,Bridget S. Wilson1,2, Diane S. Lidke1,2.1Pathology, University of New Mexico Health Sciences Center,Albuquerque, NM, USA, 2Comprehensive Cancer Center, University of NewMexico Health Sciences Center, Albuquerque, NM, USA.The Epidermal Growth Factor Receptor (EGFR) and Recepteur d’OrigineNantais (RON) are members of the Receptor Tyrosine Kinase (RTK) familythat play important roles in cell signaling and tumorigenesis. Previousbiochemical studies have shown that interactions between RON and EGFRcan alter cell signaling outcomes. However, the molecular mechanisms ofcrosstalk between these two seemingly disparate RTKs are unknown. Wehave integrated cellular imaging and biophysical techniques with biochem-ical assays to unravel the mechanisms and dynamics of EGFR-RON inter-play. Using cell lines stably expressing a range of EGFR:RON expressionratios, we first characterized the ligand-induced signaling of these two recep-tors by western blotting. We found that EGF activation of EGFR results inRON phosphorylation, while activation of RON by MSP (macrophage-stim-ulating protein) does not cause EGFR phosphorylation. Furthermore, resultswith EGFR- and RON-selective kinase inhibitors implicates EGFR as acontributor to RON phosphorylation during crosstalk. Interestingly, EGF-induced crosstalk is absent in cells expressing lower EGFR:RON ratios.These results suggest that crosstalk is dependent on EGF-stimulated activa-tion of EGFR only at high EGFR density. To understand whether protein-protein interactions facilitate EGFR/RON crosstalk, we examined EGFR/RON distributions using immunogold electron microscopy and found thatthey often co-localize on the plasma membrane. Single particle tracking ofquantum dot-tagged RON or EGFR revealed that EGF stimulation resultsin a diffusional slowdown of both EGFR and RON, consistent with the abil-ity of EGFR and RON to dimerize or engage in higher order oligomers.Despite this apparent interaction at the plasma membrane, confocal imagingstudies showed that EGF stimulation induces rapid EGFR endocytosis that isunaccompanied by RON. Together these results suggest that crosstalkbetween EGFR and RON occurs in a unidirectional manner, is density-dependent, and involves transient receptor interactions at the plasmamembrane.

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2293-Pos Board B309Activation of Preformed EGFR Dimers by Binding of Single EGF Mole-cules: Negative CooperativityEi-ichiro Saita, Ichiro N. Maruyama.Information Processing Biology Unit, Okinawa Institute of Science andTechnology, Okinawa, Japan.Epidermal growth factor receptor (EGFR) plays roles in cell proliferation, in-hibition of apoptosis and angiogenesis among others. It has long been thoughtthat binding of the ligand induces the dimer formation of monomeric EGFR(the ‘‘ligand-induced dimerization model’’). Upon ligand binding, tyrosine res-idues in the C-terminal tail of EGFR are phosphorylated, and recruit effectorproteins such as Shc1 and Grb2, which trigger activation of downstreamsignaling cascades. However, a pletora of recent studies indicate that EGFR ex-ists in dimeric form (a preformed dimer), which is incompatible with theligand-induced dimerization model.To understand the molecular mechanism of activation of EGFR, we directlyobserved interactions among EGFR, EGF labeled with two different fluorescentcompounds, and GFP-tagged Shc1 by multi-color TIRF microscopy. Within 30seconds of application of the dye-conjugated EGF to cultured cells expressingEGFR, three different fluorescent spots of EGF and Shc1 appeared on the cellsurface. EGFR mutant that have no kinase activity could bind EGF but not re-cruit Shc1. Two different fluorescent colors derived from EGF molecules donot co-localize, and binding of a single EGF molecule induces Shc1 recruit-ment to EGFR. These results suggest that binding of a single EGF moleculecan activate preformed EGFR dimer, consistently with negative cooperativityin EGF binding to EGFR.

2294-Pos Board B310Integrating Multiplex Single-Molecule Pull-Down (SiMPull) Data andComputational Modeling to Understand EGFR SignalingEmanuel Salazar-Cavazos1,2, Bridget S. Wilson1,2, Keith A. Lidke3,William S. Hlavacek4, Diane S. Lidke1,2.1Department of Pathology, University of New Mexico, Albuquerque, NM,USA, 2Cancer Research and Treatment Center, University of New MexicoSchool of Medicine, Albuquerque, NM, USA, 3Department of Physics andAstronomy, University of New Mexico, Albuquerque, NM, USA,4Theoretical Biology and Biophysics Group, Theoretical Division, LosAlamos National Laboratory, Los Alamos, NM, USA.The Epidermal Growth Factor Receptor (EGFR) plays an important role in bothphysiological and cancer-related processes. To study the factors that influenceEGFR phosphorylation, we have coupled single-molecule microscopy experi-ments with rule-based modeling of EGFR signaling. We have made technicalimprovements over the previously described Single-Molecule Pull-down (SiM-Pull) assay to facilitate direct detection of the phosphorylation state of thou-sands of individual receptors. We monitored the phosphorylation of EGFR-GFP expressed in CHO cells. By counting the number of GFP molecules colo-calized with a red-emitting fluorescent antibody, the fraction of receptors phos-phorylated at a specific tyrosine residue was determined. We found that only asubpopulation of EGFR become phosphorylated under what is consideredmaximal activation conditions and that the extent of phosphorylation variesby tyrosine residue. Three-color imaging of EGFR-GFP with antibodiesdirected to two distinct phospho-sites revealed that multi-site phosphorylationfrequently occurs.To better understand the implications of these results, we created a computa-tional model of EGFR signaling. In our model, a phosphorylated site cannotbe dephosphorylated if it is bound by one of its protein binding partners,such as the adaptor protein Grb2. Our model predicted that an increase in theabundance of Grb2 would result in a higher percentage of receptors phosphor-ylated at sites to which Grb2 binds. In agreement with this prediction, overex-pression of Grb2 caused a dramatic increase in the phosphorylation levels of aGrb2-binding site in EGFR (Y1068), but not in a site which Grb2 does not bind(Y1173). These results demonstrate the importance of receptor:adaptor proteinratios in modulating receptor phosphorylation patterns. Since protein abun-dance varies across cell types and is often altered in cancer, we are currentlyextending these studies to cancer cells lines with markedly differentEGFR:Grb2 ratios.

2295-Pos Board B311Biophysical Control of Receptor Recycling Using Engineered LigandsAndre D. DeGroot, David J. Busch, Carl C. Hayden, Samuel A. Mihelic,Aaron T. Alpar, Marcelo Behar, Jeanne C. Stachowiak.Biomedical Engineering, University of Texas at Austin, Austin, TX,USA.Receptor internalization by endocytosis regulates diverse cellular processesfrom the rate of nutrient uptake to the timescale of essential signaling events.

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The established view is that internalization is tightly controlled by specific pro-tein binding interactions. However, recent work suggests that biophysical fac-tors influence the process in ways that cannot be explained by biochemistryalone. Specifically, work from several groups suggests that increasing the stericbulk of receptors may inhibit their uptake by multiple types of trafficking ves-icles. How do biochemical and biophysical factors work together to controlinternalization? Here we show that receptor uptake is well-described by a ther-modynamic tradeoff between receptor-vesicle binding energy and the entropiccost of confining receptors within endocytic vesicles. Specifically, using largeligands to acutely increase the size of engineered variants of the transferrin re-ceptor, we demonstrate that an increase in the steric bulk of a receptor dramat-ically decreases its probability of uptake by clathrin-coated structures. Inagreement with a simple thermodynamic model, all data follow a single trendrelating occupancy of the endocytic structure to occupancy of the surroundingplasma membrane. This fundamental scaling law provides a simple tool for pre-dicting the impact of biophysical factors on receptor uptake rate, including re-ceptor size, affinity for endocytic structures, and expression level. In agreementwith these predictions, our results demonstrate how receptors of varying sizeand affinity compete for limited space within crowded endocytic structures.Understanding this biophysical competition is important for predicting receptoruptake in settings where endocytosis is saturated by receptor overexpression, asis the case in many tumor cells. More broadly, this work demonstrates thatbulky ligands can drive the accumulation of specific receptors at the plasmamembrane surface, providing a biophysical tool for targeted modulation ofsignaling and metabolism from outside the cell.

2296-Pos Board B312Anionic Lipids Take Charge: Juxtamembrane Domain Interactions withCellular MembraneMichael J. Hallock1, Yekaterina A. Golubeva2, Taras V. Pogorelov1.1School of Chemical Sciences, Beckman Institute, University of Illinois,Urbana, IL, USA, 2Department of Microbiology, University of Illinois,Urbana, IL, USA.Nearly half of the human integral membrane proteome consists of single-passtransmembrane domains. Of these, receptor tyrosine kinases (RTKs), a classwith twenty families of cell-surface receptors, are key regulators of vitalcellular processes and targets of drug development efforts. The fibroblastgrowth factor receptors (FGFRs) a family of RTKs that influences cell growth,proliferation, differentiation, is also activated in a number of cancers. FGFRsdynamic architecture, that contains extracellular, single-pass transmembrane(TM), juxtamembrane (JM), and kinase domains, is tightly intertwined withcellular membrane and is a challenging target for structural studies. Recentexperimental studies revealed that highly charged FGFR3 JM domains areinvolved in stabilization of unliganded FGFR3 dimers, which is achieved inpart through interactions with charged lipids of the inner leaflet of the cellularmembrane. The mechanism of JM-lipid interaction is unknown. We use all-atom molecular dynamics with highly mobile membrane mimetic (HMMM)model to capture spontaneous JM-lipid encounter complexes. To investigatethe role of the anionic lipids we performed extensive simulations of multiplereplicas of pure phosphatidylcholine (PC), binary mixtures of phosphatidylser-ine (PS) and PC, and tertiary mixtures of PC, PS, and phosphatidylinositol 4,5-bisphosphate (PIP2) lipid bilayers. Simulations revealed formation of stableanionic lipid conformations coordinated by charged JM residues. Lipid type-specific geometries lead to characteristic modes of JM-lipid interactionsrevealing the role of the charged lipid headgroups in establishing specific inter-actions. Knowledge of these dynamic structures will facilitate our understand-ing of mechanisms of RTK activation.

2297-Pos Board B313Differential Association of Adenylyl Cyclase Isoforms with MembraneMicrodomains Regulates cAMP Compartmentation in Human AirwaySmooth Muscle CellsShailesh R. Agarwal1, Kathryn Miyashiro1, Htun Latt1, Chase Fiore1,Rennolds S. Ostrom2, Robert D. Harvey1.1Pharmacology, University of Nevada, Reno, Reno, NV, USA, 2School ofPharmacy, Chapman University, Irvine, CA, USA.Compartmentation of cAMP signaling is critical to explaining how different G-protein coupled receptors produce distinct responses within the same cell.Membrane microdomains have been suggested to play an important role byforming discrete receptor-specific signaling complexes. Key componentsinclude various isoforms of the cAMP producing enzyme adenylyl cyclase(AC). In human airway smooth muscle (HASM) cells, AC6 is preferentiallyfound in caveolar/lipid raft membrane domains while AC2 is found in non-raft domains. Here, we test the hypothesis that, b-adrenergic receptors(bARs) selectively couple to AC6 in lipid raft domains, while E-type

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prostaglandin receptors (EPRs) selectively couple to AC2 in non-raft domains.Using genetically encoded FRET-based biosensors targeted to the bulk cyto-solic compartment as well as lipid raft and non-raft membrane domains, wemonitored changes in cAMP activity in response to bAR and EPR stimulationin HASM cells overexpressing (OE) AC2 or AC6. We found that OE AC2selectively enhanced cAMP responses to EPR stimulation in non-raft locations,while having no effect on cAMP produced by bARs anywhere. These resultssuggest that AC2 is exclusively coupled to EPRs in non-raft domains of theplasma membrane. In sharp contrast, OE AC6 selectively inhibited cAMP re-sponses to bAR stimulation in lipid raft-associated locations, while havingno effect on cAMP produced by EPRs anywhere. Furthermore, the effect ofOE AC6 on bAR responses could be blocked by inhibition of phosphodies-terase type 4 (PDE4) activity, or by disrupting A kinase anchoring protein in-teractions with protein kinase A (PKA). These data suggest that AC6 isselectively coupled to bARs in lipid raft domains and that OE AC6 upregulatesPDE4 activity due to PKA-mediated phosphorylation or recruitment in lipidraft microdomains.

2298-Pos Board B314Effects of Cell Cortex-Based Transient Confinement on TransmembraneProtein Interactions in Intact CellsMichael Zucker, Arnd Pralle.Physics, University at Buffalo, Buffalo, NY, USA.Modifying the diffusion of membrane receptors transiently changes their localconcentration which affects their rate of interaction, and hence the cell signal.Underneath the plasma membrane bilayer is the cell cortex, a meshwork ofactin and related filaments, which actively and passively interact with proteinsin the plasma membrane. Diffusing transmembrane proteins are temporarilycorralled between these filaments, and traverse across these filaments via hopdiffusion. Here we demonstrate how binned-imaging fluorescence correlationspectroscopy (bimFCS) can quantify the corralling, and show via experimentsand simulation how this corralling affects the cell signaling. A novel techniquedeveloped by our lab, we use bimFCS to measure diffusive behavior at multiplelength scales simultaneously. Coupling this technique with the principles of to-tal internal reflection fluorescence (TIRF), excitation of fluorescently-taggedtransmembrane proteins are restricted near the basal membrane. Autocorrela-tion data is analyzed with two-component fit to separate fast short range diffu-sors from slower long range diffusors interacting with actin filaments. Data isinterpreted through FCS Law in order to derive the average confinementstrength and size of corrals. Through simulation, we also explore the effectthat corrals have on protein interactions. Because the cortical actin structureis constantly undergoing changes due to factors such as thermal fluctuations,ATP-driven remodeling, and intracellular and extracellular interactions, the in-fluence this variability has on protein interactions is of great interest. Parallel tocomputational results obtained by Kalay, et al. of Kyoto University, our simu-lation suggests that, while the overall quantity of protein dimerization events isindependent on how strongly the proteins are confined, the frequency of dimer-ization events is dependent on the confinement strength, which has implicationstowards amplification of a cell signal.

2299-Pos Board B315The Role of Cortical Actin in the Regulation of Eph Receptor SignalingAlessandro Bosco, Erik Benson, Bjorn Hogberg, Ana I. Teixeira.Medical Biochemistry and Biophysics (MBB), Karolinska Institutet,Stockholm, Sweden.Actin cytoskeleton filaments form a mesh lying in close proximity to membrane(cortical actin) that is able to compartmentalize receptors and other membraneproteins. It has been previously reported that the spatial organization of Eph re-ceptors and their ligands (ephrin) at the cell-cell contact interface modulatesignaling cascades in breast cancer cell lines, impacting tumor aggressiveness.In particular, it has been suggested that the formation of clusters of Eph recep-tors on the membrane correlates with an invasive phenotype. Following this ev-idence, we hypothesize that cortical actin regulates Eph receptor signaling bytuning the organization of receptor at the membrane. By employing ephrindecorated DNA nanostructures rendered from polyhedral flat sheets, we arestudying the role of the cortical actin in the stimulation of Eph-A2 receptorwith their ligands. The shape and size of the nanostructure will impact on thelateral association of the ligands and hence the receptors. DNA flat sheetsare functionalized with monomeric ephrin-A5 conjugates following two de-signs that allow or not the dimerization of ligand bound receptor, dependingon the proximity between ligands on the nanostructures. Proximity LigationAssay is used to monitor the phosphorylation levels of the receptor upon treat-ment with drugs (Latrunculin A, Jasplakinolide) that modify the size of thecortical actin mesh. High-resolution microscopies (AFM and STORM) areused to monitor the mesh size and the clustering of the receptors. All together,

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this approach will point out molecular mechanisms of spatial organization ofligands and receptors during clustering and the effects of cytoskeleton on mem-brane receptor-mediated signaling.

2300-Pos Board B316Control Neurotrophin Signaling using Light during PC12 Cell Differenti-ation and Xenopus Embryonic DevelopmentVishnu Krishnamurthy, John Khamo, Payel Mondal, Savanna Sharum,Kai Zhang.Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA.Growth factor-mediated signaling pathways regulate neuronal survival, prolif-eration, differentiation, and apoptosis. The mitogen-activated protein kinase(MAPK) signaling pathway is a primary signaling cascade downstream ofthe binding of neurotrophins to their receptors. Evidence suggests that signalingoutput of the MAPK pathway varies with its temporal kinetics. A quantitativedelineation of signaling kinetics is limited due to a lack of tools that allows pre-cise temporal control of the MAPK pathway. The emerging non-neuronal op-togenetics, which utilizes light to control intracellular signaling pathways,provides a new modality for spatiotemporal signaling control. We have devel-oped an optogenetic system that allows reversible activation of the MAPKsignaling pathway in intact cells and in developing Xenopus laevis embryos.In PC12 neuronal cell lines, light-controlled, intermittent MAPK activity re-veals a memory effect in light-induced neurite outgrowth. In Xenopus embryos,developmental stage-specific MAPK activation implies that this pathway canreprogram cell fate after germ layer specification, a crucial time window duringwhich destination of cell fate is set. Our strategy can be generalized to controlother kinase pathways with a similar activation mechanism. Results from ourresearch will help resolve intracellular mechanisms of neurotrophin-regulatedsignal transduction during cell differentiation and embryonic development.

2301-Pos Board B317Structural Basis for Growth Differentiation Factor 5 (GDF5) Signaling In-hibition by Repulsive Guidance Molecules (RGMs)Tomas Malinauskas1, Benjamin Bishop1, Thomas D. Mueller2,Christian Siebold1.1Division of Structural Biology, University of Oxford, Oxford, UnitedKingdom, 2Department of Molecular Plant Physiology and Biophysics,University of Wuerzburg, Wuerzburg, Germany.Repulsive guidance molecules (RGMs) are cell surface proteins that play keyroles in cell migration, neuron regeneration and iron homeostasis. RGMsmodulate two signaling pathways: the Neogenin and the Bone morphogeneticprotein (BMP)/Growth differentiation factor (GDF). Here, we present crystalstructures of the N-terminal domains of three human RGM family membersin complex with GDF5. All three RGMs occupy the binding site for GDF5type 1 transmembrane receptors. We show that mutations of RGM residuesthat cause the iron-overload disease are located in the GDF5-binding interfaceand weaken RGM-GDF5 interactions. Our RGM-GDF5 crystal structures,structure-guided biophysical binding studies and cellular assays suggest thatRGMs inhibit GDF5 signaling by competing with GDF5 type 1 receptors.

2302-Pos Board B318Cell Surface Calreticulin-LRP1 Binding and its Role in Apoptotic CellEngulfmentRomone M. Fancy1, Jun Li2, Huixian Hong2, John D. Mountz2,Joanne E. Murphy-Ullrich3, Santosh K. Katiyar4, Jianyi Zhang1,Yuhua Song1.1Department of Biomedical Engineering, The University of Alabama atBirmingham, Birmingham, AL, USA, 2Department of Medicine, TheUniversity of Alabama at Birmingham, Birmingham, AL, USA, 3Departmentof Pathology, The University of Alabama at Birmingham, Birmingham, AL,USA, 4Department of Dermatology, The University of Alabama atBirmingham, Birmingham, AL, USA.Phosphatidyl serine (PS) lipids in inner leaflet of cell membrane are external-ized and become exposed in cholesterol-rich domains during apoptosis andco-localized with cell surface calreticulin (CRT) (J Immunol. 148:2207-16,1992; Cell. 123:321-34, 2005). Association of cell surface CRT in apoptoticcells with LDL receptor-related protein (LRP1) in phagocytes plays an impor-tant role in apoptotic cell engulfment (Cell. 123:321-34, 2005). Whether cellsurface CRT directly binds to LRP1 in apoptotic cells and what role of CRT-LRP1 binding in apoptotic cell engulfment is needs to be addressed. Suchstudies could identify a potential site for regulation of apoptotic cell removalin tissue remodeling and tumor immunity. In this study, we characterizedCRT-LRP1 binding in apoptotic or viable wild type mouse embryonic fibro-blasts (K41 MEFs), apoptotic CRT-null MEFs (K42 MEFs) and apoptotic re-combinant CRT-rescued K42 MEFs with co-immunoprecipitationexperiments and determined engulfment of apoptotic wild type MEFs or

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CRT-null MEFs by RAW264.7 macrophages or non-professional phagocytes,NIH 3T3 fibroblasts, with phagocytosis assays. Both K41 MEFs and K42MEFs were treated with UVB (40 mJ/cm2) radiation for apoptosis induction.Results showed that apoptosis occurred to both K41 MEFs and K42 MEFswith 40 mJ/cm2 UVB radiation treatment that involved PS lipid externalizedto outer leaflet of cell membrane. CRT bound to LRP1 in apoptotic K41MEFs cells but not in viable K41 MEFs cells. For K42 MEFs rescued withwild type CRT recombinant protein, CRT-LRP1 binding was recovered inapoptotic CRT-rescued K42 MEFs, confirming direct CRT-LRP1 binding.Phagocytosis assays showed that engulfment of apoptotic wild type MEFs byRAW264.7 macrophages or non-professional phagocytes, NIH 3T3 fibroblasts,was significantly higher in comparison with uptake of apoptotic CRT-nullMEFs. These results show an important role for CRT-LRP1 binding inapoptotic cell clearance by phagocytes.

2303-Pos Board B319Peptoid JPT1A Reduces Rage Expression and Attenuates InflammatoryResponse: A Potential AD TherapeuticLauren M. Wolf1, Melissa A. Moss2, Shannon Servoss3.1Biomedical Engineering, University of South Carolina, Columbia, SC, USA,2Chemical Engineering, University of South Carolina, Columbia, SC, USA,3Chemical Engineering, University of Arkansas, Columbia, SC, USA.For several decades, amyloid-b (Ab) has been the key therapeutic target forAlzheimer disease (AD)related therapies, but more recent studies have recastAb as one of several participants in the disease rather than its sole etiology.AD therapies that aim to inhibit Ab production and/or aggregate formationalone have yielded little success in clinical trials. At the same time, recent clin-ical trials that target AD-associated inflammation using an antagonist for thereceptor for advanced glycation end products (RAGE) demonstrate improvedoutcomes in patients with mild-to-moderate AD.A peptoid mimic of Ab’s KLVFF hydrophobic core, JPT1a, was designed as apotential therapeutic for AD. Peptoids are peptidomimetics that attain invulner-ability to proteolytic degradation through repositioning of the sidechain fromthe acarbon to the amide nitrogen. This invulnerability to proteases, as wellas other qualities such as diminished immunogenicity, enhanced cellularpermeability, and capacity for intranasal administration, make peptoidsimmensely attractive as neurotherapeutic agents. We previously demonstratedthat JPT1a modulates Ab1-40 aggregation and alters the morphology of Ab1-40aggregates. In the current study, we examine JPT1a for additional anti-inflammatory capabilities toward AD.We have demonstrated that coincubation of the peptoid JPT1a with the proin-flammatory stimulus LPS significantly reduces RAGE upregulation in a dose-dependent manner within a chronic inflammation model that utilizes THP1macrophages. JPT1a also reduces production of pro-inflammatory cytokinesIL-1b, IL-6, and IL-8 within this model. When paralleled by the ability ofJPT1a to reverse RAGE expression, these results demonstrate the potentialof JPT1a as a dual-target therapy in AD, modulating both inflammation andAb aggregation. Moreover, as RAGE has also shown promise as a therapeutictarget in several pathologies, a RAGE inhibitor such as JPT1a may yield a newtherapeutic option to a wide array of illnesses.

2304-Pos Board B320Mechanotransduction through High-Affinity LFA-1 is a MinimumRequirement to Induce Kindlin-3/RACK1/OraI1 to Mediate IntracellularCalcium Flux and Outside-In SignalingVasilios A. Morikis, Scott I. Simon.Biomedical Engineering, University of California, Davis, Davis, CA, USA.Absence of Kindlin-3 impairs b2-integrin function on neutrophils, results inbleeding and resistance to arterial thrombosis and immunodeficiency.Kindlin-3 binds LFA-1 b-integrin tail independent of Talin-1 and is associatedwith integrin conversion to a high-affinity and the facilitation of micro-clusterformation that allow for long-lived shear resistance bonds with ICAM-1.However, the molecular and mechanical events that link LFA-1 to Kindlin-3 at spatially distinct sites to allow for subsequent engagement with scaf-folding protein RACK1 and calcium release activated channel Orai1 havenot been rigorously shown. We show that an allosteric shift of LFA-1 dueto force generation on the b2-integrin cytodomain promotes interaction withKindlin-3 F3 subdomain, and when sufficient force is transduced throughthis mechanical linkage induces calcium flux. We utilize real time two-linetotal internal reflection fluorescent and epifluorescence microscopy onneutrophil-like PLB-985 cells arrested to ICAM-1 within our microfluidicvascular mimetic we have been able to identify the stoichiometric bindingrate of Kindlin-3 and subsequent RACK-1 recruitment to high-affinity LFA-1 sites in response to increasing shear profiles prior to calcium flux. In addi-tion to outside-in signaling we have shown that force acting on LFA-1 am-

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plifies calcium influx during CXCR1/2 ligation in a Kindlin-3 dependentmanner. To assess the spatial proximity and association between Kindlin-3/RACK1 with focal adhesion clusters we are developing a FRET pair betweenthe Kindlin-3 PH domain and RACK1 scaffolds 5 to 7 to further elucidate thespatiotemporal events and requirements for outside-in signaling and neutro-phil progression to a migratory phenotype. This has allowed for us to identifykey forces and proteins required for outside-in signaling during neutrophiltransition from rolling to arrest which will help further glyocomimetic devel-opment to treat various disease states.

2305-Pos Board B321Development of Synthetic Notch Receptors for Photoactivatable GeneExpressionRyan E. Peace, John Ngo.Biomedical Engineering, Boston University, Boston, MA, USA.Notch is a cell-surface receptor that facilitates cell-to-cell signaling through amechanism requiring mechanical force. The receptor is activated upon trans-cellular binding to target ligands, which induces two successive proteolyticcleavages to liberate an intracellular transcriptional effector such that it cantranslocate to the nucleus. While it has largely been assumed that increasedcontact area between cells (i.e. increased opportunity for ligand-receptor inter-actions) leads to increased transcriptional output, several observations chal-lenge this assumption and highlight the importance of contact areadimension and stability in Notch-mediated juxtacrine signaling. Here, wedescribe the development of a synthetic Notch receptor that has been engi-neered to bind the small-molecule fluorescein (‘‘aFITC SynNotch’’) and pre-sent its capability as a platform for studying photo-activatable Notchsignaling with spatiotemporal control. Overall this system functions as apowerful probe for studying the natural mechanisms of Notch activation, andprovides a useful synthetic biology tool for investigating the sensing capabil-ities of mammalian cells. In future work, we will apply this receptor to detectspecific cell types with spatial and temporal control.

Posters: Calcium Signaling II

2306-Pos Board B322Changes in Calcium Behavior in Smooth Muscle Cells Induced by Bidirec-tional StretchAndroniqi Qifti.Chemistry & Biochemistry, Worcester Polytechnic Institute, Worcester, MA,USA.Muscle cells contain protein structures on their plasma membranes called cav-eoale that act like little springs which flatten and recoil when the cell isstretched and released. Caveolae are thought to provide mechanical strengthto membranes and to bind to and organize signaling protein complexes. Ourlab has shown that caveolae are responsible for stabilizing the activated ofthe protein Gaq which mediates Ca2þ signals in response to specific hormonesand neurotransmitters. Here, we tested the idea that stretching rat aorticsmooth muscle A10 cells will reduce Ca2þ signals and play a role in normalmuscle health. After conducting mechanical bi-directional static stretch of1-5% in A10 cells for different time periods, we obtained evidence of a struc-tural change in caveolae that leads to disruption of Cav/Gaq interactions andsignificant decrease of the Ca2þ signals intensity when Gaq is activated. Incontrast, oscillating stretch sensitizes calcium responses by impeding calciumrecovery mechanisms. Morphologically, when A10 cells undergo mechanicalbi-directional stretch only on the x-axis (static), the cells become longer andflatter without a change in morphology. In contrast, when cells undergo oscil-lating stretch, their morphology changes to becoming similar to cardiac mus-cle cells. These studies show that A10 cells are very sensitive in various formsof mechanical stress. We also studied mechanical stress due to local forcesinvolved in cell overcrowding. We find that high cell density with increasedcell-cell contacts, such as that found in normal muscle tissue, leads to anelevated levels of caveolae and a significant increase in calcium signals.Low confluency of cells has the opposite effect. In summary, we find thatdeformation of caveolae with mechanical stress allows cells to regulateCa2þ signals.

2307-Pos Board B323Non-canonical Role of Mitofusin 2 in Regulating Ca2D Homeostasis in theEndoplasmic Reticulum via Interaction with SERCA PumpMarina Balycheva, Stephen Hurst, Jyotsna Mishra, Gyorgy Csordas,Shey-Shing Sheu.Center of Translational Medicine, Thomas Jefferson University,Philadelphia, PA, USA.Mitofusin 2 (MFN2), originally recognized for mediating mitochondrialfusion, has been also established as a major player in the endoplasmic

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reticulum (ER) and mitochondria interaction. MFN2 ablation reduced the ef-ficacy of interorganelle Ca2þ signalling, which effect has been largely attrib-uted to MFN2’s ER- mitochondrial tethering role. However, the mechanismby which MFN2 regulates Ca2þ homeostasis between the organelles remainsunclear. Here, we introduced a non-canonical role of MFN2 in ER Ca2þ

regulation potentially via an interaction with sarco/endoplasmic reticulumCa2þ-ATPase (SERCA). To study the effect of MFN2-overexpression(OE) on ER Ca2þ homeostasis two sets of Myc-tagged MFN2 plasmidswere used: wild-type present in both ER and mitochondria (WT-MFN2)and MFN2 lacking the mitochondrial targeting sequence (ER-MFN2). West-ern blotting and confocal microscopy confirmed OE and localization oftagged MFN2. Effect of MFN2 knock-out (KO) and WT-, ER-MFN2-OEon Ca2þ dynamics was studied in permeabilized HEK293T and MEF cells.[Ca2þ] in the ER lumen ([Ca2þ]ER) was visualized using GEM-CEPIA1erCa2þ-biosensor. Basal [Ca2þ]ER was significantly lower in WT- and ER-MFN2-OE cells vs control and MFN2-KO cells where we observed highestlevel of the basal [Ca2þ]ER. Importantly, the rate of [Ca2þ]ER re-uptake bySERCA after its depletion by reversible inhibitor cyclopiazonic acid wassignificantly faster in MFN2-KO cells vs control. In WT- and ER-MFN2-OE [Ca2þ]ER re-uptake was significantly slower than in control with moredramatic effect in ER-MFN2-OE. Initial co-immunoprecipitation andFRET experiments suggest SERCA interaction with MFN2. Thus, our datashows that in addition to the ER-mito tethering role, MFN2 might interactwith and inhibit SERCA, and potentially regulate ER-mito calcium cross-talk via changing ER Ca2þ handling.

2308-Pos Board B324Cardiac Overexpression of Human Adenylyl Cyclase Type 8 Elicits WideSpread Adaptations to Limit Adrenergic Signaling in Sinoatrial NodalPacemaker Cells that Extend Well Beyond Desensitization of b-Adren-ergic ReceptorsKhalid Chakir1, Kirill Tarasov1, Yelena Tarasova1, Jack M. Moen2,Michael G. Matt3, Kenta Tsutui1, Ismayil Ahmet1, Thanh Huynh4,Karel Pacak4, Edward G. Lakatta1.1Laboratory of Cardiovascular Science, Intramural Research Program,National Institute on Aging, National Institutes of Health, Baltimore,Maryland, USA, Baltimore, MD, USA, 2Yale University, New Haven, CT,USA, 3University of Pittsburgh Medical School, Pittsburgh, MD, USA,4Section on Medical Neuroendocrinology, Eunice Kennedy Shriver NationalInstitute of Child Health and Human Development, NIH, Bethesda, MD,USA.Overexpression of the Human Adenylyl cyclase 8 (AdCy8) in mouse heartleads to a marked 40% increase in heart rate (HR) in vivo (telemetry) andto a marked reduction in heart rate variability (HRV); and the HR responseto dobutamine, a b1-AR selective agonist, was markedly blunted (BPJ.112(3) Sup1, p425a, 2017). To explore underlying potential mechanismsfor the reduced b-AR response, we assessed the expression of RNAseqof numerous genes that could potentially limit cAMP-PKA signaling. Hu-man AdCy8 was highly expressed in SAN tissue of TGAC8. Transcriptabundance for genes coding for proteins that desensitize b-adrenergic re-ceptor signaling (GRK2, GRK5, b-arrestin1/2, DAB2) and PDE3a,PDE3b, PDE4a, PDE4b, PDE4d, PDE5a and PDE7b were significantly(p<0.05%) upregulated in TGAC8 vs. WT. Transcripts of b-ARs, PKAand AKAPs did not differ in TGAC8 vs. WT. Furthermore, dopamine b-hy-droxylase (DBH) and phenylethanolamine N methyl transferase (PNMT)genes in SAN cells that code for enzymes that convert dopamine to norepi-nephrine, to epinephrine were suppressed in TGAC8 vs. WT. qRT-PCR orimmunolabeling performed to date validated aforementioned RNAseq re-sults for AdCy8, GRK2, GRK5, b-arrestin1/2, DAB2, DBH and PNMT.Interestingly, circulating plasma levels of dopamine and dopa were higher,and norepinephrine and epinephrine were lower in TGAC8 vs. WT. Thus,high AdCy8 overexpression in heart activates numerous adaptations inSAN cells to escape adrenergic surveillance. TGAC8 mouse breeder pairswere a kind gift of Jacques Hanoune and Nicole Defer at INSERM at Cre-teil, France.

2309-Pos Board B325Regulation of ATP Production by Mitochondrial Calcium Signals in HeartAndrew P. Wescott, Joseph P. Kao, W. Jonathan Lederer, Liron Boyman.The Center for Biomedical Engineering and Technology, University ofMaryland School of Medicine, Baltimore, MD, USA.Persistent and adaptable production of ATP is essential for the workingmyocardium. Cardiomyocytes undergo drastic alteration in work to meetthe demands of increased heart rate, blood pressure, or when stimulated bycirculating catecholamines. To meet these massive and dynamic demands,

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each cardiomyocyte contains �10,000 unique mitochondria, which are its pri-mary source of ATP. However, the physiological mechanisms that enable thisenergy homeostasis are largely unknown. It is widely accepted that calcium(Ca2þ) plays a critical role in the regulation of mitochondrial function, how-ever the mechanistic details remain controversial. Cytosolic Ca2þ ([Ca2þ]i)elevations that activate cellular contraction may enter the mitochondria tostimulate the regeneration of ATP supply. The ability of mitochondria totake up Ca2þ has been long known, with recent work identifying the molec-ular identities of critical proteins in the mitochondrial Ca2þ uniporter(MCU) complex. However, the concentration of free Ca2þ in the mitochon-drial matrix ([Ca2þ]m) in working cardiomyocytes and beat-to-beat dynamicsis controversial with current estimations ranging from 10 nM - 100s mM.These quantitative details are critical as [Ca2þ]m has been hypothesized toregulate Ca2þ sensitive proteins at every stage of mitochondrial metabolismincluding the tricarboxylic acid cycle, the electron transport chain, and ATPSynthase. In addition, the sensitivity of intra-mitochondrial processes to regu-lation by [Ca2þ]m and how [Ca2þ]m affects the net production of ATP remainsunclear. We present quantitative single cardiomyocyte experiments to revealthe [Ca2þ]m signaling range in intact cells, and further assess how the MCUcomplex is regulated by [Ca2þ]i and [Ca2þ]m. We also show a quantitativecharacterization of mitochondrial ATP production centered on the role playedby Ca2þ, cytosolic ADP, and phosphate. These data provide the first measure-ments detailing how [Ca2þ]m dynamics tune mitochondrial energy productionto match cellular demand.

2310-Pos Board B326Cross Talk between IP3 and Adenylyl Cyclase Signaling Pathways in Car-diac Atrial MyocytesDerek A. Terrar, Rebecca A. Capel, Thomas P. Collins,Skanda Rajasumdaram, Thamali Ayagamar, Rebecca AB Burton.Pharmacology, University of Oxford, Oxford, United Kingdom.IP3 receptors are present in junctional sarcoplasmic reticulum (SR) in atrialmyocytes, and Ca2þ release from the SR by IP3 acts in parallel with ad-enylyl cyclase signaling mechanisms that are also present in atrial cells.These two pathways are normally thought of as distinct, although theymay act synergistically. Here we present evidence for a more direct interac-tion in which IP3-mediated Ca2þ release from the SR activates Ca2þ-stimu-lated adenylyl cyclases, which in turn act via protein kinase A (PKA) toregulate the amplitude of the Ca2þ transient (CaT) in electrically stimulatedatrial myocytes.Immunohistochemical experiments showed that the Ca2þ stimulated ad-enylyl cyclase, AC8, and IP3 receptor proteins were both found to be pre-sent in close proximity in junctional SR just beneath the plasmalemma.Another Ca2þ stimulated adenylyl cyclase, AC1, was located in the spacebetween junctional and non-junctional SR. IP3-dependent pathways areactivated by alpha-adrenoceptor mechanisms. The alpha-agonist phenyl-ephrine (10 mM) increased the amplitude of CaT (3559%, n=8), andthe IP3 antagonist 2-APB (2.5 mM), the adenylyl cyclase inhibitorMDL-12,330 (10 mM) and the protein kinase A inhibitor H89 (1 mM)all reduced these effects of phenylephrine. Direct stimulation of IP3 recep-tors by photorelease of IP3 from a cytosolic caged compound alsoincreased CaT amplitude (60511% at 120s after photorelease, n=8),and the effects were significantly reduced by MDL-12,330 (�1353%change at 120s, n=6) and H89 (�1759%, n=9).Taken together, these observations are consistent with action of IP3 to initiateCa2þ release from junctional SR leading to stimulation of Ca2þ activated ad-enylyl cyclases which operate via PKA to increase the amplitude of Ca2þ tran-sients in electrically stimulated atrial myocytes. There thus appears to be afunctional interaction between IP3 and cAMP signalling pathways in cardiacatrial myocytes.

2311-Pos Board B327Identification of Cardiomyocytes’ Inner Workings Responsible forDynamical Changes in Calcium Profile in Response to Mechanical LoadZana Coulibaly1, Rafael Shimkunas2, Zhong Jian1, Ye Chen-Izu1,2,Leighton T. Izu1.1Pharmacology, University of California Davis, Davis, CA, USA,2Biomedical Engineering, University of California Davis, Davis, CA, USA.Background: We embedded cardiomyocytes in a cross-linked hydrogel tostudy the effect of increased afterload on in the heart. We observed thatmechanically-loaded cardiomyocytes undergo changes in their Ca2þ dy-namics that can compensate for the increase in load. We have shown thatthese changes are mediated by the upregulation of nitric oxide (NO) signal-ling, which in turns affects Ca2þ handling. Because many different Ca2þ

pathways could be affected we used an agnostic approach based on

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mathematical modelling to tease out changes in cell characteristics respon-sible for the differences in calcium and contraction profiles between load-free and after-load environment.Mathematical Method: We coupled the Shannon-Bers ventricular actionpotential model to a viscoelastic model to simulate the myocyte contractingin either a load-free condition (Tyrode solution) or under load (in the gelmatrix). The mathematical model establishes a closed feedback loop be-tween the calcium system and the extracellular environment that givesrise to the self-regulation we observed. We ran extensive simulations whereparameters associated with the influx and efflux of Ca2þ were modulatedsuch that they are either up-regulated or down-regulated by NO. In silicoresults are filtered out to qualitatively match cell-in-gel in vitro results.The filtering process is based on measures that capture multiple propertiesof calcium profiles.Conclusion: Our approach of identification hints that the upregulation of NOhas the effect of simultaneously modulating multiple parameters of the Ca2þ

handling pathway. Of the modulated parameters, the L-type current amplitudehas to be consistently increased. Coupled with the increase in L-type current,parameters associated with release and uptake of calcium by the SR have tobe modulated in opposite directions.

2312-Pos Board B328Pharmacological Targeting of Serca May Have Potential for CellularProtectionYuanzhao L. Darcy, Melanie Loulousis, Claudio G. Copello,Paula L. Diaz-Sylvester, Julio A. Copello.Pharmacology, Southern Illinois University, Springfield, IL, USA.We previously reported that CGP-37157 and K201, two benzothiazepines(BZT) with cardioprotective action, inhibit the Ca2þ ATPase of sarcoplasmicreticulum (SR) intracellular Ca2þ stores (SERCA). We tested if SERCA blockcould be part of the mechanism by which drugs protect cells from ischemicdamage. We also screened structural characteristics in BZT that could affecttheir drug potency as SERCA inhibitors. SR microsomes isolated from rabbitskeletal muscle (SkM) and pig heart ventricle were utilized to measure mod-ulation of SR Ca2þ loading and SERCA-mediated ATPase activity by drugs.Seven out of twenty cell-protective drugs tested (including pimozide, EGCG,KN-93 and carvedilol) inhibited, at least partially, SR Ca2þ loading and Ca2þ-stimulated ATPase activity in SkM and heart SR microsomes. We alsoscreened ten novel BZT derivatives and seven FDA-approved benzodiaze-pines (BZD; including bromazepam and clonazepam), which have close ho-mology to CGP37157. Ca2þ-dependent block of SERCA was found in fourBZT’s, which displayed higher (PH000995, PH000902) and similar potency(PH000902, PH006796) compared to CGP. BZD were all ineffective, whichsuggest that the sulfur atom in the BZT ring (substituted by nitrogen inBZD) is crucial for their SERCA blocking ability. All compounds above,were tested on ryanodine receptor (RyR) activity (planar bilayers, SR leak,[3H] ryanodine binding). None of these agents directly inhibited RyR functionin heart and muscle. In contrast, some agents (BZT and BZD) had mildagonistic action on channel function. We think that SERCA block by thesedrugs, which persists at pH �6.5, may benefit ischemic cells by preventingSR Ca2þ overload, known to trigger, upon reperfusion, abnormal RyR-mediated Ca2þ leak associated with cell death and tissue injury. BZT have po-tential as templates for therapeutic targeting of SERCA (Supported by AHAand Eskridge Foundation).

2313-Pos Board B329The Interplay between FGF23- and Angiotensin II- Mediated CalciumSignaling in Cardiac HypertrophyKetaki N. Mhatre1,2, Paulina Wakula1, Burkert Pieske1, Frank Heinzel3.1Cardiology, Charit�e – Campus Virchow-Klinikum, Berlin, Germany,2Cardiology, Medical University of Graz, Graz, Austria, 3Cardiology, Charit�e– Campus Virchow-Klinikum, Graz, Germany.Fibroblast growth factor 23 (FGF23) is a hormone strongly linked to heartfailure and cardiovascular mortality. It triggers pathological Ca2þ-regulatedtranscriptional pathways leading to left ventricular hypertrophy. In vivo,high circulating levels of FGF23 are associated with an altered renin-angiotensin-aldosterone system response. Here we investigated Ca2þ-depen-dent signaling of FGF23 and its interconnection with angiotensin II (ATII)in neonatal rat ventricular myocytes (NRVMs). Both ATII and FGF23induced hypertrophy in NRVMs as reflected by cell area and hypertrophicgene expression. In Ca2þ imaging experiments, an increase of cytoplasmic(2.4folds50.3) and nuclear (1.9folds50.3) Ca2þ transient amplitude wasobserved on acute treatment with FGF23 (p%0.01) similar to ATII. CaT in-tegral too was augmented significantly by both the treatments in cytoplasmand nucleus. A selective pro-hypertrophic enhancement of nuclear Ca2þ

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release as seen in ATII treatment was evident in FGF23-treated NRVMs(1.8folds50.2) when the nuclear integral was normalized to the correspond-ing cytoplasmic integral (ratio). Localised nuclear Ca2þ release involvesagonist (ATII)-led generation of inositol trisphosphate (IP3) and stimulationof nucleolemmal IP3-receptor (IP3R). IP3R inhibitor reverted the effect ofFGF23(p%0.01) on integral ratio implying the involvement of IP3 in theFGF23-mediated prolonged nuclear Ca2þ release. Our results reveal compa-rable response of NRVMs to FGF23 and ATII at multiple levels suggestinga crosstalk between their signaling. Interestingly, ATII receptor antagonistsignificantly attenuated FGF23-induced hypertrophy and changes in Ca2þ ho-meostasis. Long, as well as acute application of FGF23, increases intracellularexpression of ATII peptide (2.2folds50.1) vs. untreated NRVMs in a time-dependent manner, confirming ATII contribution. Nevertheless, results ofongoing mass spectrometry profiling of FGF23-induced intracellular andsecreted ATII in NRVM culture will be presented in detail. In conclusion,FGF23 may lead to a pathological activation of ATII signaling, which contrib-utes to hypertrophy in cardiomyocytes.

2314-Pos Board B330Calmodulin Mutations Associated with Congenital Cardiac DiseaseDisplay Novel Biophysical and Biochemical CharacteristicsMichail Nomikos1, Angelos Thanassoulas2, Brian L. Calver3, Konrad Beck4,Vyronia Vassilakopoulou2, Luke Buntwal3, Iris Konotgianni2, Adrian Smith3,Bared Safieh-Garabedian1, Evangelia Livaniou2, Egon Steen Toft1,George Nounesis2, F. Anthony Lai1.1College of Medicine, Qatar University, Doha, Qatar, 2National Center forScientific Research ‘‘Demokritos’’, Aghia Paraskevi, Greece, 3School ofBiosciences, College of Biomedical and Life Sciences, Cardiff University,Cardiff, United Kingdom, 4School of Dentistry, College of Biomedical andLife Sciences, Cardiff University, Cardiff, United Kingdom.Calmodulin (CaM) is a cytoplasmic multifunctional calcium (Ca2þ)-bindingmessenger that interacts with the cardiac ryanodine receptor (RyR2), a largetransmembrane Ca2þ channel that mediates Ca2þ release from the sarcoplasmicreticulum (SR) to activate cardiac muscle contraction. Recent genetic studieshave reported CaM missense mutations in patients with a history of severe car-diac arrhythmogenic disorders. Herein, we have investigated the effect of fournovel missense CaM mutations, identified in two patients presenting with longQT syndrome (LQTS) (N98I, D134H), and two patients with clinical featuresof both LQTS and catecholaminergic polymorphic ventricular tachycardia(CPVT), (D132E and Q136P), relative to the biophysical and biochemicalproperties of wild type CaM (CaMWT). We used CD spectroscopy to examinethe thermal stability of CaMWT and mutant proteins. In the absence of Ca2þ,thermodynamic values for all proteins were similar. In contrast, in the presenceof Ca2þ, there was a significant decrease in the stability of the five proteinsfollowing the order CaMWT> CaMN98I > CaMD132E > CaMQ136P >CaMD134H. Further Ca2þ-binding studies revealed that all CaM mutationssignificantly reduce the Ca2þ-binding affinity of CaMWT. CaMQ136P protein ex-hibited a �7-fold reduced Ca2þ-binding affinity compared to CaMWT, whileCaMD132E had a �14-fold reduction. Furthermore, biochemical analysis re-vealed that all four CaM mutants displayed dramatically reduced RyR2 inter-action and defective modulation of [3H]ryanodine binding to RyR2,regardless of LQTS or CPVT association. Our findings confirm our previousobservations suggesting that the clinical presentation of LQTS or CPVT asso-ciated with these four CaM mutations may involve both altered intrinsic Ca2þ-binding as well as dysregulation of RyR2-mediated Ca2þ release via aberrantinteraction of CaM with RyR2.

2315-Pos Board B331Dystonia-Associated Hippocalcin Mutants Dysregulate Cellular CalciumInfluxNordine Helassa1, Svetlana V. Antonyuk2, Lu-Yun Lian3, Lee P. Haynes1,Robert D. Burgoyne1.1Cellular and Molecular Physiology, University of Liverpool, Liverpool,United Kingdom, 2Molecular Biophysics Group, University of Liverpool,Liverpool, United Kingdom, 3NMR Centre for Structural Biology, Universityof Liverpool, Liverpool, United Kingdom.Dystonia is a neurological movement disorder that provokes muscle spasmsand contractions. It is characterized by sustained or intermittent muscle con-tractions causing abnormal, often repetitive movements and painful postures.Recently, mutations at positions T71N and A190T in the neuronal calcium-binding protein hippocalcin, have been shown to be critical in developmentof DYT2 dystonia. However, the effect of these mutations on the physiologicalrole of hippocalcin has not yet been elucidated. Using a multidisciplinaryapproach, we showed that mutations T71N and A190T in hippocalcin did notaffect stability, calcium-binding affinity, translocation to cellular membranes

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(Ca2þ/myristoyl switch) and three-dimensional structure of the protein. How-ever, the disease-associated mutations caused a defect in calcium-induced oli-gomerisation of hippocalcin. In KCl-stimulated SH-SY5Y cells expressingmutated hippocalcin, we observed an increased calcium influx, mostly drivenby N-type voltage-gated calcium channels. Our data demonstrate that thedystonia-associated mutations strongly affect hippocalcin cellular functionswhich suggest a central role for perturbed calcium signalling in DYT2 dystonia.This work has been funded by the Leverhulme Trust RPG-2014-194.

2316-Pos Board B332Role of NAADP for Calcium Signaling in the Salivary GlandJohn F. Imbery, David Giovannucci.Neurosciences, University of Toledo, Toledo, OH, USA.The secretion of saliva in response to physiological demand is under control ofthe autonomic nervous system. Coordination and regulation of intracellularCa2þ signaling following autonomic activation is crucial for proper salivarysecretion. Previous work from our lab has demonstrated parotid acinar cellscontain an abundant and polarized distribution of acidic organelles. Further-more, acidic organelles are recruited for agonist-induced Ca2þ signalingfollowing cAMP elevation. The current study interrogated a role for NAADP,a potent second messenger known to release Ca2þ from acidic organelles. Inparotid acinar cells, initial results demonstrated NED19, an inherently fluores-cent NAADP receptor inhibitor, was localized to acidic endosomes. Addition-ally, using a whole-cell patch clamp method to dialyze NAADP into parotidacini resulted in robust Ca2þ -activated Cl-currents. Peak activation occurredat low nanomolar concentrations of NAADP and higher concentrations showedan inhibitory effect. These observations suggest a role for NAADP in producingthe fluid component of saliva. Thus, this work identifies a NAADP signalingpathway as a potential therapeutic target for people suffering from salivaryhypofunction.

2317-Pos Board B333Spontaneous Ca2D Fluctuations Mediated by TRPM7 Channels in GrowthPlate ChondrocytesNianchao Qian1, Atsuhiko Ichimura1, Daisuke Takei1, Hua Zhu2,Miyuki Nishi1, Hiroshi Takeshima1.1Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto,Japan, 2Davis Heart and Lung Research Institute, The Ohio State University,Columbus, OH, USA.During embryonic bone outgrowth, round chondrocytes intensively proliferateand then sequentially differentiate into columnar and hypertrophic chondro-cytes in the growth plate cartilage. These processes might be regulated by intra-cellular Ca2þ signaling, however Ca2þ handling has been poorly explored ingrowth plate chondrocytes. We currently developed an experimental methodfor fluorometric Ca2þ imaging of intact chondrocytes in bone slice preparationsfrom E17.5 mouse embryos. In this imaging system, we found spontaneousintracellular Ca2þ fluctuations, characterized by small peak amplitudes rangingin estimated Ca2þ concentration of �50 nM, in round and columnar chondro-cytes. The spontaneous Ca2þ fluctuations were attenuated by FTY720 andNS8593, both of which inhibit the transient receptor potential melastatin sub-family 7 (TRPM7) channel, a non-selective cation channel that can conductCa2þ and Mg2þ. In contrast, the TRPM7 activators naltriben andNNC550396 facilitated the Ca2þ fluctuations. In accordance with these modu-lator effects, microarray analysis suggested that Trpm7 is expressed in growthplate chondrocytes. Furthermore, the phospholipase C (PLC) inhibitor U73122depressed the Ca2þ fluctuations, while the large-conductance calcium-acti-vated-Kþ (BK) channel activator NS1619 stimulated them. Therefore, growthplate chondrocytes generate spontaneous intracellular Ca2þ fluctuations, whichare likely mediated by TRPM7 channels and maintained by PLC and BK chan-nel activities. We now attempt to address the role of TRPM7-mediated Ca2þ

fluctuations in bone development.

2318-Pos Board B334Calmodulin Interaction with Gap Junction Intracellular Loop PeptidesSilke Kerruth1, Catherine Coates1, Syed Alireza Rezavi1,Camillo Peracchia2, Katalin Torok1.1Molecular and Clinical Sciences Research Institute, St. George’s Universityof London, London, United Kingdom, 2School of Medicine and Dentistry,University of Rochester Medical Center, Rochester, NY, USA.Connexins (Cx) are membrane-spanning proteins that form gap junctionswhich allow the exchange of small molecules between cells, e.g. ATP, IP3and ions. Although calmodulin (CaM) binding sites have been identified inthree regions, the N- and C-terminal tails1,2 and the intracellular loop(ICL)3, moreover CaM blocks Cx45 gap junctions4, the role of CaM in gapjunction function is not well understood. Thus, CaM interaction with syntheticpeptides of ICL from representatives of each the three Cx subgroups was

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investigated. Fluorescence changes of double labelled FRET-probe DA-CaM and Ca2þ sensitive TA-CaM were monitored by fluorescence spectros-copy and stopped-flow fluorimetry at physiological ionic strength and pH 7.5and 20 �C. Both Ca2þ-dependent and -independent interactions were identi-fied. Kd values of 40 5 4 nM, 31 5 3 nM, 75 5 4 nM and 60 5 7 nM inCa2þ and 924 5 223 nM, 3.2 5 0.5 mM, 849 5 105 nM and 625 5 123nM in the absence of Ca2þ were obtained for ICL of Cx32, Cx35, Cx45,Cx57, respectively. FRET measurements revealed partial compaction ofDA-CaM (54-70% quenching in the presence of Ca2þ and 33-62% quenchingin the absence of Ca2þ). The kinetic data revealed a two-step process of rapidbinding followed by isomerisation. Evidence for both Ca2þ-dependent and in-dependent binding indicates that CaM may be anchored to gap junctions inresting cells and becomes fully bound upon stimulation. Thus, our datastrongly suggest a modulatory role for CaM-ICL interactions in gap junctionregulation.1Torok et al., 1997 Biochem J 326, 479-4832Dodd et al., 2008 J Biol Chem 283, 26911-269203Zhou et al., 2007 J Biol Chem 282, 35005-350174Peracchia et al., 2003 J Membr Biol 195, 53-62This work is funded by BBSRC grant BB/M02556X/1 to K.T.

2319-Pos Board B335Mathematical Modeling of Calcium Signaling in MicrogliaPeter M. Kekenes-Huskey1, Brad D. Stewart1, Darin D. Vaughan2.1Chemistry, University of Kentucky, Lexington, KY, USA, 2Chemistry,Morehead State, Morehead, KY, USA.Microglia function is orchestrated through highly-coupled signaling pathwaysthat depend on calcium. In response to external stimuli, microglia commonlypresent elevated intracellular calcium (Ca2þ), stemming from either externalsources, e.g. through plasma membrane Ca2þ channels, or internal sources,including IP3 mediated endoplasmic reticulum Ca2þ release. Numericalmodels of Ca2þ -signaling processes in microglia could provide a basis for in-terpreting wide-ranging experiments and offering testable hypotheses. Here, wehave created a computational model of microglial Ca2þ handling, includingATP-dependent P2X activation, p38/ERK kinase activity, activation ofNFAT transcription factors, as well as tumor necrosis factor alpha secretion.The model was trained using published data for each of these processes.With this model, we have probed the extent to which triggering of Ca2þ influxvia activation of plasma membrane Ca2þ channels, namely P2X channels, con-trols intracellular Ca2þ , and its propensity to influence transcription. Given thatCa2þ dysregulation in microglia is common to many neurological diseases, weanticipate that our model may provide a framework to better understand andprobe microglial pathophysiology.

2320-Pos Board B336Effect of Calcium Flux on Filopodia of Epithelial CellsOmolade M. Ademuyiwa, Carol A. Heckman.Biological Sciences, Bowling Green State University, Bowling Green, OH,USA.Filopodia are the sensors of a cell and are responsible for directing cell motility.Like ‘‘antennae,’’ they receive signals from both soluble ligands in the extracel-lular mileau and those attached to the substrate. In the nerve axon, these twoaspects of signaling are integrated so as to modulate chemotactic signaling ac-cording to the composition of the substrate. The role of filopodia in the modu-lation is not clear. Although epithelial cells differ from nerve cells in theirorganization, the same second messengers mediate the filopodia’s sensoryfunction. In both cell types, activation of a PKC (protein kinase C) with a tumorpromoter inhibited filopodia or caused their dissolution. Because Caþþ selec-tively activates one subset of PKCs, local Caþþ elevation could activate theseisozymes and also thereby decrease filopodia. Previous reports showed contra-dictory results for Caþþ second messenger, however, in the axon, i.e. positiveor negative effects on filopodia. Here, we investigate how filopodia dynamicsare affected by calcium. We measured percentage of the cell periphery coveredwith filopodia and percentage of cells showing filopodia. There is an inhibitorof filopodia in culture media, so that replacing the medium with a bufferincreased filopodia. This occurred regardless of whether calcium was presentin the buffer or absent. When cells remained in the medium, however, inhibitorsof calcium transport caused a decrease in filopodia. Cyclopiazonic acid inCaþþ-free buffer typically blocks uptake of Caþþ into the endoplasmic reticu-lum (ER) and causes net Caþþ efflux. This enhanced filopodia. Restoring extra-cellular Caþþ in the extracellular buffer after depleting the ER store alsoenhanced filopodia production. We conclude that filopodia display was sensi-tive to calcium flux but not to absolute physiologically relevant calcium con-centrations. It remains to be determined how calcium flux is detected inepithelial cells.

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Posters: Excitation-Contraction Coupling I

2321-Pos Board B337Architecture of Transverse Tubules and Triads in Huntington’s DiseaseSkeletal MuscleShannon H. Romer1, Melissa Bautista1, Daniel E. Hutcherson1,Robert J. Talmadge2, Andrew A. Voss1.1Biological Sciences, Wright State University, Dayton, OH, USA,2Biological Sciences, California State Polytechnic University, Pomona,Pomona, CA, USA.Huntington’s disease (HD) is a fatal and progressive condition with severedebilitating motor defects and muscle weakness. Although classically recog-nized as a neurodegenerative disorder, there is increasing evidence of cellautonomous toxicity in skeletal muscle. Our laboratory recently demonstratedthat, despite being fully innervated, skeletal muscle fibers from R6/2 transgenicmice are hyperexcitable due to reduced chloride and potassium currents. Thesedefects coincide with aberrant mRNA processing. We also report a decrease inspecific membrane capacitance, suggesting a loss of transverse tubular (t-tubular) membrane in R6/2 muscle. Furthermore, previous reports indicatethat both Cav1.1 currents and RyR1 Ca2þ release are reduced in R6/2 skeletalmuscle. Thus, we hypothesis that a loss and/or disruption of the skeletal musclet-tubule system contributes to changes in EC coupling in R6/2 skeletal muscle.To assess the t-tubule architecture in late-stage R6/2 muscle and age-matchedwild type muscle, we used live-cell imaging with multiphoton confocal micro-scopy and transmission electron microscopy (TEM). Using AutoTT software toprovide unbiased and consistent quantification of t-tubule system, we found nosignificant differences in the t-tubule system density, regularity or integrity. Wedid, however, find changes in the ultrastructure of the triads, the location ofwhere t-tubules interact with the sarcoplasmic reticulum in EC coupling. Addi-tionally, to link this to other established molecular mechanisms, we found sig-nificant alternations in the R6/2 splicing of BIN1, a protein involved in t-tubularmaintenance. Understanding the architectural and molecular changes of the t-tubule system is necessary to fully interpret alterations in Ca2þ homeostasisand muscle force generation in HD.

2322-Pos Board B338Characterisation of a Novel Mouse Model Carrying a Non-sense Mutationin RYR1 EX36Francesco Zorzato.Dept Anesthesiology, Basel, Basel, Switzerland.The ryanodine receptor 1 (RyR1) plays a fundamental role in excitation-contraction coupling. Dominant and recessive mutations in RYR1, the gene en-coding the RyR1, are associated with a range of neuromuscular disordersincluding the core myopathies central core disease and multi-minicore disease,congenital fiber type disproportion, centronuclear myopathy as well as thepharmacogenetic disorder malignant hyperthermia. For many mutations thefunctional effect has been studied, however, to date the effect of non-sense mu-tations leading to a premature stop codon have not been investigated in depth.Using the CRISPR/CAS9 technology we created a mouse model knocked-in fora premature stop codon in RYR1 exon 36 (RYR1ex36 KI) and compared itsphenotype at 3 months with that of age-matched wild-type littermates. Inmice co-expression of the WT RYR1 allele and of the prematurely truncatedRYR1 allele leads to a decrease in both transcript and RyR1 protein expression.No significant changes were observed in the expression levels of other sarco-plasmic reticulum proteins. Spontaneous motor activity assessed with a volun-tary running wheel revealed that heterozygous RYR1ex36 KI mice runsignificantly less compared to age-matched WT. In vitro analysis of the me-chanical properties of muscles showed lower twitch and tetanic specific forceboth in EDL and soleus from RYR1ex36 KI mice compared to age-matchedwild-type. No differences in SR Ca2þ release in FDB fibers from RYR1ex36KI and WT mice were observed. Our results show that in mice the expressionof 50% of the RyR complex has a small but significant effect on skeletal musclefunction.

2323-Pos Board B339Altered Eye Muscle Function in RYR3KO MiceJan Eckhardt1, Hiroshi Takeshima2, Miyuki Nishi2, Jianjie Ma3,Francesco Zorzato1,4, Susan Treves1,4.1Departments of Biomedicine and Anaesthesia, Kantonsspital Basel, Basel,Switzerland, 2Department of Biological Chemistry, Graduate School ofPharmaceutical Sciences, Kyoto University, Kyoto, Japan, 3Davis Heart andLung Research Institute, The Ohio State University Wexner Medical Center,Columbus, OH, USA, 4Life Sciences, University of Ferrara, Ferrara, Italy.Ophthalmoplegia or paralysis of the muscles controlling eye movement is theunderlying feature of a number of neuromuscular conditions including multiple

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sclerosis, characteristically affecting the nerve, mitochondrial myopathies dueto mutations in genes encoded by mitochondrial DNA and congenital myopa-thies such as Multiminicore disease, myotubular myopathy and Native Amer-ican Myopathy. Although it is not a life threatening condition it can causedouble vision and thus severely impair vision. Extraocular muscles (EOMs)are among the fastest and most fatigue resistant skeletal muscles; they are cate-gorized as a separate group of muscles or ‘allotype’ since they represent aunique group of highly specialized muscles anatomically and physiologicallydifferent from other skeletal muscles. The distinct origin and innervation ofEOMs are probably responsible for their different gene and protein expression.Indeed, detailed biochemical characterization of the excitation-contractioncoupling (ECC) machinery has demonstrated that they are different from quad-riceps muscles in that they express, in addition to the skeletal muscle isoformsof proteins involved in ECC, Cav1.2, CASQ2 and RyR3. In mouse limb mus-cles, RyR3 is present at birth and during muscle development but subsequentlydisappears and is almost undetectable in most adult muscles, except for the dia-phragm where it is present in approximately 15% of the fibers. Studies on flexordigitorum brevis (FDB) fibers isolated from ryr3-/-mice have indicated that theabsence of RyR3 causes subtle changes in Ca2þ homeostasis. We will presentdata showing that RYR3KO mice have a visual impairment and that this is dueto change in function of their extraocular muscles.

2324-Pos Board B340Mitochondrial Dysfunction in Malignant Hyperthermia Susceptible Skel-etal MuscleLeon Chang1, Katie Nicoll Baines1, Paul Denney Allen1,Philip Morgan Hopkins1, Marie-Anne Shaw1, John Peter Boyle2.1UK Malignant Hyperthermia Unit, St James University Hospital, Leeds,United Kingdom, 2Faculty of Medicine & Health, University of Leeds,Leeds, United Kingdom.Malignant Hyperthermia (MH) is a pharmacogenetic condition primarilycaused by mutations in RYR1 and CACNA1S which lead to dysregulation inintracellular calcium handling. Individuals with these mutations suffer frompotentially fatal, hypermetabolic reactions when exposed to volatile anaes-thetics and/or depolarizing muscle relaxants. Several studies have suggesteda possible link between MH susceptibility and mitochondrial dysfunction.Here we investigated skeletal muscle mitochondrial function by measuring ox-ygen consumption rates (OCR) in six respiratory states through high resolutionrespirometry using the Oroboros Oxograph-2k analyser. Human skeletal mus-cle biopsies from MH susceptible (MHS) and MH normal (MHN) individualsconfirmed by IVCT, and skeletal muscle from wild type and MHS RYR1G2435Rknock-in mice were permeabilized and subjected to a substrate-inhibitor-titration (SUIT) protocol to analyze different stages of oxidative phosphoryla-tion (OXPHOS).In comparison to MHN controls, human MHS skeletal muscle showed a signif-icantly reduced OCR in two different respiratory states, both involving com-plex II (succinate dehydrogenase) of the electron transport chain. In contrast,when compared to wild type mice, MHS RYR1G2435R knock-in murine mito-chondria showed evidence of defect in complex I (NADH dehydrogenase),in addition to an overall reduction in the maximum capacity of the electrontransport system (ETS). We conclude that based on these data that there is clearevidence of reduced OXPHOS capacity in MHS human and MHS RYR1G2435Rmurine mitochondria using permeabilized skeletal muscle. The significantlyreduced OCR seen in both datasets, provide additional evidence towards aconnection between MH susceptibility and mitochondrial dysfunction, prompt-ing the need for further research in this area.

2325-Pos Board B341Aerobic Training Prevents Heat-Strokes in Calsequestrin 1 KnockoutMice by Reducing Oxidative StressFlavia A. Guarnier1,2, Matteo Serano1, Antonio Michelucci1,Laura Pietrangelo1, Simona Boncompagni1, Feliciano Protasi1.1University G. d’Annunzio, Chieti, Italy, 2Univ. Estadual, Londrina, Brazil.Calsequestrin-1 knockout (CASQ1-null) male mice suffer lethal episodes whenexposed to high environmental heat, crisis known as environmental heat stroke(HS). During lethal HS crises excessive oxidative stress plays a key role in theevents that lead to contractures and rhabdomyolysis of skeletal fibers. We havepreviously demonstrated that exogenous antioxidants (such as N-acetylcys-teine) are sufficient to reduce mortality of CASQ1-null mice, thanks to theirability to normalize Ca2þ handling in skeletal fibers. As aerobic training hasbeen demonstrated to boost endogenous antioxidant protection, we subjectedCASQ1-null mice to treadmill running for 2 months (at 60% of their maximalspeed, 1h 5 times/week), a training that increased significantly their aerobic ca-pacity (70% less final lactate accumulation during constant load test). At theend of training (4 months of age), mice were exposed to a heat-stress protocol

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(41�C/1h): interestingly mortality rate was dramatically decreased in trained (t)CASQ1-null mice compared to un-trained (un-t) controls: 16% vs. 86%,respectively. In tCASQ1-null mice, the increase in core temperature duringexposure to heat-stress (i.e. hyperthermia) was lower than in un-t mice, andtheir EDL muscles displayed a lowered threshold of response when exposedto increasing [caffeine] during in-vitro contracture test (IVCT). Several otherparameters were assessed in muscle samples: aerobic training succeeded indecreasing mitochondrial damage (14.853.9% vs. 752.3%), mitochondrialefficiency by increasing of 34% the cytochrome-c oxidase activity, whiledecreasing SERCA’s and also Ca2þ-dependent proteolytic activity. Addition-ally, training also reduced lipid peroxidation (elevated in un-tCASQ1-nullmice) in isolated sarcoplasmic reticulum and mitochondria membranes: �45and �35 %, respectively. In conclusion, aerobic training protects CASQ1-null mice from HS, an effect essentially mediated by a significant reductionin oxidative stress and fiber damage.

2326-Pos Board B342Exercise Prevents Formation of Tubular Aggregates in Ageing SkeletalMuscle Fibers of Wild-Type MiceClaudia Pecorai, Antonio Michelucci, Laura Pietrangelo, Feliciano Protasi,Simona Boncompagni.University of G. D’Annunzio, Chieti, Italy.Tubular aggregates (TAs) are ordered arrays of sarcoplasmic reticulum (SR)tubes found in muscle biopsies from patients affected by TA myopathy(TAM). TAM is a disorder linked to mutations in STIM1 and Orai1, the twomain players in store-operated Ca2þ entry (SOCE), a mechanism that allowsrecovery of extracellular Ca2þ during repetitive muscle activity. TAs are alsofound in ageing 24 month old C57Bl6 mice in about 50% of fast-twitch EDLfibers (while they are not present in adult 4 month old mice) and, using immu-nofluorescence, we verified that such TAs are strongly positive for both STIM1and Orai1. Measurements of contractile force during repetitive stimulation (30consecutive tetanic pulses of 1 sec at 60 Hz applied every 5 seconds) revealedthat 24 month old EDL muscles exhibit a faster decay of contractile force thanadult (relative force at the 15th tetanus: 40.752.1% vs. 48.3.652.8% respec-tively). In addition, experiments performed in presence or absence of 2.5 mMextracellular Ca2þ suggest that the faster force decay of ageing EDLs is likelycaused by their inability to recruit extracellular Ca2þ. We finally analyzed EDLmuscles from 24 month old mice that exercised voluntarily in wheel cages for15 months (average of 396529 km; n=3): formation of TAs was greatlyreduced in trained vs. untrained age-matched mice (found in only 7% of fibers),while the capability of EDL muscles to maintain contractile force during repet-itive stimulation was significantly rescued (relative force after 15th tetanus:52.454.7%). Our findings suggest that: a) TAs accumulate dysfunctionalSTIM1 and Orai1; b) exercise limits formation of TAs and improves musclefunction during repetitive stimulation, possibly improving the capability ofaged muscle to use external Ca2þ via SOCE.

2327-Pos Board B343Atorvastatin Activates Skeletal RyR1 Channels: Towards Reducing StatinSide-EffectsChris Lindsay1,2, Abigail D. Wilson1, Elisa Venturi1, Angela J. Russell1,2,Rebecca Sitsapesan1.1Department of Pharmacology, University of Oxford, Oxford, UnitedKingdom, 2Chemistry Research Laboratory, University of Oxford, Oxford,United Kingdom.We have previously reported that simvastatin can activate single skeletal mus-cle ryanodine receptor (RyR1) channels gating in artificial membranes andstimulate sarcoplasmic reticulum (SR) Ca2þ release from isolated skeletal mus-cle cells. We suggested that the interaction of statins with RyR1 may contributeto their muscle-related side effects, including fatal rhabdomyolosis. We there-fore investigated whether RyR1 channel activation is a common property ofclinically relevant statins, focusing on atorvastatin, a more recently developedand fully synthetic drug.Sheep skeletal RyR1 were incorporated into planar phospholipid bilayers undervoltage-clamp conditions as previously described (Sitsapesan et al., 1991, JPhysiol., 434:469-488). Low concentrations of cytosolic atorvastatin signifi-cantly increased Po from 0.02950.0098 (mean5SEM, n=12) in control con-ditions to 0.07750.023 (mean5SEM, n=12, p<0.05) with 100 nMatorvastatin and 0.08350.027 (SEM, n=11, P<0.05) with 1mM atorvastatin.Atorvastatin appears to sensitise RyR1 channels to the effects of cytosolicCa2þ since it was unable to activate RyR1 at sub-activating cytosolic [Ca2þ].The addition of atorvastatin to the luminal side of RyR1 did not activate thechannels even at high concentrations (%100 mM), indicating that atorvastatindoes not cross the bilayer and that it interacts with RyR1 via cytosolic bindingsites. Atorvastatin also significantly increased [3H]ryanodine binding to sheep

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skeletal SR membranes, which further demonstrates its potential to promoteRyR1 channel opening.As a means of developing a statin with fewer side effects, we now aim to utilisea medicinal chemistry approach to design an effective 3-hydroxy-3-methylglu-taryl coenzyme A (HMG-CoA) reductase inhibitor that can lower blood choles-terol levels yet does not activate RyR1.Funded by the BHF Centre of Research Excellence, Oxford

2328-Pos Board B344Cross-Influence of Halothane and Voltage on Intracellular Ca2D in Iso-lated Muscle Fibers of Mice Expressing Human RyR1 Mutation Y522SStefan Mall1, Philipp Elischer1, Alberto Zullo2,3, Martin Textor1,Andreas Alt4, Werner Klingler5,6, Werner Melzer1.1Institute of Applied Physiology, Ulm University, Ulm, Germany,2Biotecnologie Avanzate s.c. a r.l., CEINGE, Napoli, Italy, 3Department ofSciences and Technologies, University of Sannio, Benevento, Italy, 4Instituteof Legal Medicine, Ulm University, Ulm, Germany, 5Department ofNeuroanaesthesiology, Ulm University, G€unzburg, Germany, 6QueenslandUniverstity of Technology, Brisbane, Australia.We studied the effect of halothane and its interference with membrane voltagein malignant hyperthermia (MH) susceptible muscle by using enzymaticallyisolated single fibers (m. interosseus) of knock-in mice with the RyR1 mutationY524S (human MH mutation Y522S). To investigate anesthetic- and voltage-triggered changes of Ca2þ concentration, we measured fluorescence from fibersloaded with Fura-2-AM. Similar as in the diagnostic in-vitro contracture test(IVCT) for MH susceptibility, cells were exposed to different halothane-containing solutions using a vaporizer system. The percentage of halothanein the gas phase was determined using infrared photometry. The temporalchange of the halothane concentration in the recording chamber was checkedusing gas chromatography. In Krebs-Ringer’s solution at room temperature,a step from 0 to 0.5% halothane in the gas phase caused a clear increase inresting Ca2þ concentration in most of the investigated fibers of mutant micebut very little response in fibers of WT littermates. The Ca2þ response wasphasic: after reaching a peak the Ca2þ signal decreased at a rate of about10%/min indicating either reduced sensitivity to halothane or inhibition ofCa2þ release after activation. A likely explanation is Ca2þ-induced inactivationof RyR1. In further experiments, we applied halothane to single fibers whichwere voltage-clamped using two intracellular microelectrodes. Depolarizingpulses to different membrane potentials from a holding potential of �80 mVrevealed a strong shift in the voltage threshold for activation of Ca2þ releaseto more negative potentials. Correspondingly, hyperpolarizing steps led to arapid partial recovery from the halothane-induced increase in basal Ca2þ con-centration. These results demonstrate reciprocal effects of a volatile anestheticdrug and membrane voltage on Ca2þ release in MH-susceptible skeletalmuscle.

2329-Pos Board B345Role of Transverse Tubule Plasticity in Calcium Entry Unit DisassemblyFollowing Acute ExerciseAntonio Michelucci1,2.1Pharmacology and Physiology, University of Rochester, Rochester, NY,USA, 2CeSI-Met, Center for Research on Ageing and TranslationalMedicine, University G d’Annunzio of Chieti, Chieti, Italy.Acute treadmill exercise in-vivo drives remodeling of the sarcotubular mem-branes and formation of new junctions between stacks of sarcoplasmic reticu-lum (SR) cisternae and transverse-tubules (TTs) in fast twitch skeletal musclefibers. These newly formed SR-TT junctions: i) contain STIM1 and Orai1, themolecular machinery of store-operated Ca2þ entry (SOCE); ii) function as‘‘calcium entry units’’ (CEUs) that promote resistance to fatigue during repet-itive muscle activity.Here we evaluated the time course of CEU disassembly in extensor digitorumlongus (EDL) fibers from wild type mice subjected to 1h of running atincreasing speed (from 5 m/min to 25 m/min). Using electron microscopy,we quantified the number of SR-stacks/100mm2 and the TT-length/100mm2 atthe I-band in EDL muscles from control and exercised mice, as well as frommice that were exercised and then allowed to recover for either 6 or 24h. A sin-gle bout of treadmill exercise increased both the number of SR-stacks/area(from 2.050.3 to 9.950.7) and TT-length/area (from 2.450.8mm to6.150.8mm). While the number of SR-stacks further increased (18.752.6)6h after exercise, average TT-length returned to control values (2.050.5mm).After 24h of recovery, both parameters returned to control levels. To assessthe correlation between number of SR-stacks and TT-length to function,EDL muscles were subjected to a high-frequency stimulation protocol(30x1s-60Hz pulses every 5 seconds), in presence of external Ca2þ. EDL mus-cles removed immediately from exercised mice exhibited a higher capability to

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maintain contractile force compared to those from controls and those removedand evaluated 6h after exercise. These results suggest that: i) plasticity of TTretraction from CEUs occurs prior to SR-stack disassembly after exerciseand ii) stacks of SR membranes at the I-band function as CEUs only while be-ing coupled to extensions of the TT.

2330-Pos Board B346STIM2 and STIM1 have Similarities and Differences, but Both RegulateCa2D Movement in Skeletal MuscleMi Ri Oh1, Keon Jin Lee1, Mei Huang1, Jin Ock Kim2, Do Han Kim2,Chung-Hyun Cho3, Eun Hui Lee1.1Dept. of Physiology, College of Medicine, The Catholic Univ. of Korea,Seoul, Korea, Republic of, 2School of Life Sciences, GIST, Gwangju, Korea,Republic of, 3Department of Pharmacology, College of Medicine, SeoulNational University, Seoul, Korea, Republic of.Stromal interaction molecule 1 (STIM1) along with Orai1 mediates extracellularCa2þ entry into the cytosol through a store-operated Ca2þ entry (SOCE) mech-anism in various tissues including skeletal muscle.However, the role(s) ofSTIM2, a homolog of STIM1, in skeletal muscle has not been well addressed.The present study, first, was focused on searching for STIM2-binding proteinsfrom among proteins mediating skeletal muscle functions. This study used abinding assay, quadrupole time-of-flight mass spectrometry, and co-immunoprecipitation assay with bona-fide STIM2- and SERCA1a-expressingrabbit skeletal muscle. The region for amino acids from 453 to 729 of STIM2binds to sarcoplasmic/endoplasmic reticulum Ca2þ-ATPase 1a (SERCA1a).Next, oxalate-supported 45Ca2þ-uptake experiments and various single-myotube Ca2þ imaging experiments using STIM2-knockdown mouse primaryskeletal myotubes have suggested that STIM2 attenuates SERCA1a activity dur-ing skeletal muscle contraction, which contributes to the intracellular Ca2þ dis-tribution between the cytosol and the SR at rest. In addition, STIM2 regulatesCa2þ movement through RyR1 during skeletal muscle contraction as well asSOCE. Therefore, via regulation of SERCA1a activity, STIM2 regulates bothintracellular Ca2þ distribution and Ca2þ movement in skeletal muscle, whichmakes it both similar to, yet different from, STIM1.

2331-Pos Board B347Dysferlin C2A Domain Is Involved in Recovery of Voltage-Induced SRCalcium Release after Osmotic Shock in Murine Muscle FibersValeriy Lukyanenko, Joaquin Muriel, Robert J. Bloch.Physiology, University of Maryland, Baltimore, MD, USA.Osmotic shock injury (OSI) decreases the amplitude of voltage-induced Ca2þ-transients (VICTs) in dysferlin-null (A/J) but not control (A/WySnJ) myofibers(Kerr et al., Proc. Natl. Acad. Sci. USA, 2013). Using the OSI assay, werecently showed that dysferlin modulates the coupling of excitation to Ca2þ

release in murine muscle fibers (Lukyanenko et al., J. Physiol. 2017). Herewe studied dysferlin-null A/J mouse muscle fibers expressing mutatedVenus-dysferlin chimeras to reveal the region of the dysferlin molecule respon-sible for that coupling. We examined the effects of deletion of the domain C2Aand point mutations, V67D, R1022Q, I1298V, R1331L, I1298V, and K1598N,on VICT following OSI. Deletion of C2A and the V67D mutant, located in theC2A domain, inhibited recovery of VICTs after OSI (37% and 31%, respec-tively), while the R1022Q (77%), R1331L (68%), I1298V (61%), andK1598N (56%) mutations were statistically identical to wild type Venus-dysferlin chimera (69%). Dysferlin localization to transverse (t) tubules withthe C2A deletants and each of the point mutations was examined with C-termi-nal pHluorin constructs by changing extracellular pH from 7.4 to 6.5. The pos-itive control showed �60% reduction of fluorescence at pH=6.5, whereasnegative controls showed 10-15% reduction; V67D and R1022Q values wereabout 35% and R1331L, I1298V, K1598N and C2A about 60%. Thus the mu-tants incorporated into T-tubules either partially (V67D, R1022Q) or as effi-ciently as wild type (R1331L, I1298V, K1598N, C2A). We conclude thatdomain C2A modulates the coupling of excitation to Ca2þ release in murineskeletal muscle, and that C2A deletion or mutation is pathogenic. Supportedby the Jain Foundation, MDA and NIH (RO1 AR064268).

Posters: Cardiac, Smooth, and Skeletal MuscleElectrophysiology I

2332-Pos Board B348A Mathematical Model of a Pig Ventricular MyocyteBardia Ghayoumi1, Bence Hegyi2, Ye Chen-Izu2, Daisuke Sato2.1University of California, Davis, Davis, CA, USA, 2Pharmacology,University of California, Davis, Davis, CA, USA.Over the last few decades, computational modeling and simulations have beenwidely used to reveal fundamental mechanisms underlying non-trivial phenom-

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ena in arrhythmias, such as discordant alternans, tachycardia, and fibrillation.Especially, multiscale models are useful to understand how single channel levelmodifications affect cellular and tissue scale phenomena. A pig is one of thebest animal models to investigate cardiac phenomena since many aspects ofthe pig heart are similar to those of the human heart; however, few mathemat-ical models have been developed. Here, we built a physiologically detailedmathematical model of a pig ventricular myocyte based on experimental datameasured with the physiological action potential (AP)-clamp SequencialDissection (also called ‘onion-peeling’) method. The base model is a rabbitventricular myocyte model developed by Mahajan et al (Biophys J. 2008 Jan15;94(2):392-410). We removed currents, which do not exist in the pig ventric-ular myocyte and added several currents such as calcium (Ca)-dependent chlo-ride (Cl) current and small-conductance Ca-activated potassium (K) current,which are important players in the pig ventricular myocyte. We varied modelparameters to fit amplitudes and kinetics of the pig myocyte currents and actionpotential. We made sets of parameters for the normal myocyte, heart failuremyocytes in the border zone and remote zone of the infarct. With these param-eters, our mathematical models can recapitulate experimental observations ofthese myocytes. This computational model provides a basis to explore actionpotential and calcium dynamics in the pig cardiac myocyte and also at tissuelevel under various pathophysiological conditions.

2333-Pos Board B349A Novel Approach to Measure Short Term Cardiac Ventricular ActionPotential Memory: Comparison between Five Numerical ModelsMassimiliano Zaniboni.Chemistry, Life Sciences and Environmental Sustainability, University ofParma, Parma, Italy.Rate-dependence (RD) and electrical restitution (ER) describe the dependenceof cardiac action potential duration (APD) from the average or from the previ-ous pacing cycle length (CL). We know however that in certain dynamic con-ditions APD depends not only from the average of from the previous, but from agiven small number (3 to 5) of CLs preceding each beat. Such dependence isusually called short term AP memory (STAM) and has been shown to controlrepolarization stability. Despite the relevance of STAM for the transition to ar-rhythmias, a clear definition of it and a way to measure it are still lacking, mak-ing it difficult to compare findings from different preparations/laboratories. Bymeans of simulations carried out on 5 different human cardiac ventricular APmodels, I provide a novel general definition of STAM which is substantiallydifferent from those proposed in literature so far, and is based on the collectivebehavior of the family of ER curves that describe the system during dynamicpacing. I show how STAM can predict repolarization stability under high-and beat-to-beat-variable pacing rate after singular perturbations of the rhythm.The measure of STAM in the 5 AP models shows that memory involves a num-ber of preceding beats up to 15 at high pacing rate (CL = 320 ms). I monitoredSTAM over consecutive beats during periodic and random pacing trains in acompact representation that allows comparison between models. STAM, asmeasured here, compactly represents otherwise hidden dynamic properties ofcardiac AP at high pacing rate, which is of great interest in a variety of cardiacelectrophysiological issues, like the mechanism underlying latent instability ofrepolarization in LQT1 syndrome, to cite one.

2334-Pos Board B350Ionic Currents Assessed by Voltage Clamping a Myocyte Diffusion-Reaction Model with Experimental Action PotentialsJuan I. Felice1, Ariel L. Escobar2.1CIC, Universidad Nacional de La Plata - CONICET, La Plata, Argentina,2Engineering, UC Merced, Merced, CA, USA.The time course of ventricular action potentials (AP) largely differ betweendifferent mammals. Recently, it was shown that the Ca2þ influx that triggerssarcoplasmic reticulum (SR) Ca2þ release in mouse hearts occurs during APphase 1 and that phase 2 is defined by a Naþ influx through the Naþ-Ca2þ

exchanger (NCX). The likelihood of these new ideas was assessed using amathematical model. Local changes in cytosolic and luminal Ca2þ concentra-tion were simulated using a diffusion-reaction framework. Two adjacent sec-tions that represent the myoplasm and the SR lumen were sliced into Ndiffusional connected sub-compartments. The Ca2þ exchange between cytosoland SR occurs only via ryanodine receptors (RyR) and SERCA pumps. SRCa2þ efflux is governed by the Ca2þ gradient and the RyR open probabilitydescribed by a Markov model having a luminal Ca2þ regulation mediated byCa2þ calsequestrin (CSQ). Ca2þ buffering in the SR was also defined byCSQ, modeled as an allosteric protein. Myoplasmic Ca2þ buffering wasmodeled including several mobiles (i.e. ATP, Ca2þ indicator) and one fixedbuffer (Troponin C). Ca2þ extrusion from the cytosol was defined by theNCX. Sarcolemmal Ca2þ influx is mediated by Ca2þ channels that have voltage

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and Ca2þ dependent inactivation. In order to minimize the number of variableparameters, experimentally recorded epicardial APs were used to electricallydrive the model. The parameters of the model were adjusted by comparingthe simulated data with Ca2þ transients recorded experimentally in intacthearts. Using this AP voltage clamp framework we were able to predict thatCa2þ influx to myocytes does occur during phase 1 and that phase 2 is definedby a Naþ influx through NCX.

2335-Pos Board B351Memory Alters Formation of Voltage- and Calcium-Mediated Alternans ina Fractional-Order Cardiomyocyte modelTien Comlekoglu, Seth H. Weinberg.Virginia Commonwealth University, Richmond, VA, USA.Cardiomyocyte electrical activity is typically modeled using ideal parallelcapacitor-resistor circuit networks. However, studies have suggested that thepassive properties of cell membranes may be more appropriately modeledwith non-ideal circuit elements, in which the current-voltage relationship isgoverned by fractional-order dynamics. Fractional-order dynamics representcapacitive memory effects, which confer the history-dependence on the trans-membrane potential history. Our recent work demonstrated that fractional-order transmembrane potential dynamics typically shortens the action potentialduration (APD) and suppresses alternans, a beat-to-beat alternation in the APD,in a minimal model for which alternans is solely driven by voltage-mediatedinstabilities. Here, we investigate the effects of capacitive memory in a modelfor which alternans can be driven by both voltage- and calcium-driven mech-anisms. We considered four parameterizations, corresponding to voltage-driven, calcium-driven, and two mixed mechanism regimes. We ran simula-tions for fractional-orders between 0.85 and 1 with varying cycle lengths(CLs) between 200 and 600 msec. For all parameterizations, capacitive mem-ory shortened APD, consistent with our prior work. We found that capacitivememory suppressed voltage-mediated alternans that was induced at shortCLs (<300 msec). However, for intermediate CLs, a small degree of capacitivememory (fractional-order of 0.95) promoted alternans, while larger capacitivememory effects suppressed alternans (fractional-order below 0.9). We foundthat capacitive memory suppressed calcium-mediated alternans for all cyclelengths, while decreasing the cycle length for the onset of alternans. Interest-ingly, capacitive memory suppressed mixed mechanism-mediated alternansat short CLs, while promoting alternans at longer CLs. Our results reflectthat capacitive memory can play a role in alternans formation and suppressionin both voltage- and calcium-mediated mechanisms.

2336-Pos Board B352Heart Rate Variability Alters Cardiac Alternans and ElectromechanicalDynamicsVrishti M. Phadumdeo, Seth H. Weinberg.Biomedical Engineering, Virginia Commonwealth University, Richmond,VA, USA.Heart rate variability (HRV) occurs under normal physiological conditions, re-sulting from circadian rhythm, stochasticity in pacemaking, and autonomic regu-lation. HRV influences the cardiac action potential duration (APD), intracellularCa signaling, and the interplay between APD and Ca. Clinically, reduced HRV isassociated with an increased risk of arrhythmias. In this work, we investigate theeffects of HRV on alternans, an arrhythmogenic beat-to-beat alternation in APDor Ca, which occur at very fast heart rates. The effects of HRV on the APD-Carelationship and electromechanical characteristics is not well studied. Weemploy a nonlinear discrete time map model that governs APD and intracellularCa signaling in a cardiac cell. HRV is simulated using a variable pacing period,with the pacing period specified by a constant value and a Gaussian random var-iable with specified magnitude. At fast pacing periods for which alternans is pre-sent, we find that HRV decreases the variability in both APD and peak Ca. Wefind that the correlation between APD on successive beats decreases withincreased HRV, and similarly for peak Ca, which demonstrates that HRV dis-rupts the alternation associated with alternans. These results suggest that HRVplays an important role in preventing arrhythmias. The effect of HRV on cardiactissue will be further investigated using a cable model, to understand how HRVinfluences the spatial pattern of alternans, in particular spatially discordant alter-nans, which can be a direct precursor to arrhythmias.

2337-Pos Board B353Novel T1 Mapping-Based Preclinical Models for Cardiac Electrophysi-ology: A Combined Experimental and Theoretical StudyMengyuan Li1, Maxime Sermesant2, Sebastian Ferguson3, Jen Barry3,Graham Wright3,4, Mihaela Pop3,4.1University of Toronto, Toronto, ON, Canada, 2Inria, Sophia-Antipolis,France, 3Sunnybrook Research Institute, Toronto, ON, Canada, 4MedicalBiophysics, University of Toronto, Toronto, ON, Canada.

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Computational models are powerful tools for electrophysiology (EP), helpingus understand and predict arrhythmia associated with structural heart disease,a major cause of sudden cardiac death. We have developed preclinical modelsusing advanced Magnetic Resonance Imaging (MRI) methods that efficientlyprobe the biophysical MR signals in chronically infarcted myocardium. Ouraim is to couple such biophysical models with computational modelling, toenable accurate predictions of electrical wave propagation and to test thesemodels in a preclinical framework prior to clinical translation. In this studywe used n=5 swine with chronic infarct (�6 weeks following the occlusion-reperfusion of a major artery) and one healthy swine. Each animal underwentMR imaging followed by conventional X-ray guided EP study using catheter-based electrical mapping systems. For infarct imaging, we used our custom-developed T1-mapping MR method (i.e., Gadolinium-based multi-contrastlate enhancement, 1x1x5mm spatial resolution). We used these images as inputto a robust fuzzy-logic algorithm, and segmented the infarcted zone into: unex-citable dense fibrosis and slow conductive peri-infarct (arrhythmia substrate), amixture of viable and non-viable myocytes. From these segmented images wefurther built high-fidelity 3D predictive heart models, integrating the three tis-sue zones: healthy myocardium, dense fibrosis and peri-infarct into detailedcomputational meshes (1mm element size). Finally, we investigated the accu-racy of model predictions by comparing simulated and measured isochronalmaps (i.e., depolarization time maps) under pacing conditions. For simulationswe used reaction-diffusion type of equations and a macroscopic formalism. Re-sults showed that our predictive T1-based heart models are sufficiently accu-rate; the mean absolute error between the simulated and measureddepolarization times was small: �7ms for control heart and 10ms in infarctedhearts (in average). Future work will focus on refining the T1-mapping spatialresolution and simulating the arrhythmia inducibility.

2338-Pos Board B354Population-Based Mechanistic Modeling Allows for Quantitative Predic-tions of Drug Responses across Cell TypesJingqi Q.X. Gong, Eric A. Sobie.Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, NewYork, NY, USA.Quantitative mismatches between human physiology and experimental modelscan be problematic for the development of effective therapeutics. When the ef-fects of drugs on human adult cardiac electrophysiology are of interest, pheno-typic differences with animal cells, and more recently stem cell-derivedmodels, can present serious limitations. We addressed this issue through a com-bination of mechanistic mathematical modeling and statistical analyses. Phys-iological metrics were simulated in heterogeneous populations of modelsdescribing cardiac myocytes from adult ventricles and induced pluripotentstem cells derived cardiac myocytes (iPSC-CMs). These simulated measureswere used to construct a cross-cell type regression model that predicts adultmyocyte drug responses from iPSC-CM behaviors. We found that: (1) quanti-tatively accurate predictions of adult myocyte responses to selective or non-selective ion channel blocking drugs could be generated based on iPSC-CM re-sponses under multiple experimental conditions; (2) altering extracellular ionconcentrations is an effective experimental perturbation for improving themodel’s predictive strength; (3) the method can be extended to predict andcontrast drug responses in diseased as well as healthy cells, indicating a broaderapplication of the concept. This cross-cell type model can be of great value fordrug development, and the approach, which can be applied to other fields, rep-resents an important strategy for overcoming experimental model limitations.

2339-Pos Board B355A Computational Analysis of Inter-Subject Variability in InducedPluripotent Stem Cell-Derived CardiomyocytesDivya C. Kernik1, Stefano Morotti2, Henry J. Duff3, Junko Kurokawa4,Jose Jalife5, Joseph C. Wu6, Eleonora Grandi2, Clancy E. Colleen1.1Physiology and Membrane Biology, UC Davis, Davis, CA, USA,2Pharmacology, UC Davis, Davis, CA, USA, 3Cardiac Sciences, Universityof Calgary, Calgary, AB, Canada, 4Bio-informational Pharmacology,University of Shizuoka, Shizuoka, Japan, 5Center for Arrhythmia Research,University of Michigan, Ann Arbor, MI, USA, 6Cardiovascular Medicine,Stanford University, Stanford, CA, USA.There is a profound need to develop a strategy to predict the effect of patient-to-patient variability on mechanisms of cardiovascular disease. A promisingemerging experimental method utilizes induced pluripotent stem cell-derivedcardiomyocytes (iPSC-CMs), which are patient-derived cells that recapitulatethe electrical attributes of normal and diseased adult cardiomyocytes. Thesecells capture not only patient-specific genotype-phenotype relationships, butalso cell-to-cell variability of cardiac electrical activity. Additionally, compu-tational models of cardiac cells have been used to complete systematic analysis

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to elucidate mechanisms of cardiac rhythm disorders. Harnessing the geneticdiversity observed experimentally in iPSC-CMs in a computational model al-lows for computational ‘‘experiments’’ to analyze the effect of genetic varia-tion on rhythm disorders. We have developed a whole-cell model of iPSC-CMs composed of single-exponential voltage-dependent gating variable rateconstants, parametrized to fit experimental iPSC-CM steady-state and kineticvalues for all major ionic currents. By optimizing the model parameters to mul-tiple experimental datasets for each ionic current, we have implementedexperimentally-observed variability in the driving ionic currents by varyingboth gating parameters and current densities in the model. The model predictspacing frequencies from 25 to 200 bpm, action potential durations (90%) from150 to 650 ms, and resting potentials from�50 to�80 mV, thus encompassingthe experimental range of inter-subject variability in iPSC-CMs. The resultingpopulation of cells is used to relate specific iPSC-CM phenotype variations toproclivity for arrhythmia. This methodology effectively links molecular mech-anisms to cellular-level outputs by revealing unique subsets of model parame-ters linked to known iPSC-CM phenotypes, including proarrhythmic behavior.This study is the first step towards a computational method to integrate clinicaland experimental data into a high throughput methodology to link patient-specific genotype-phenotypes relationships, and examine these relationshipsthe presence of pharmacological intervention.

2340-Pos Board B356Calcium-Dependent Regulation of PotassiumChannels in Cardiac Electro-physiology: A Computational StudyHenry Sutanto1, Dobromir Dobrev2, Eleonora Grandi3, Paul G.A. Volders1,Jordi Heijman1.1Department of Cardiology, Maastricht University, Maastricht, Netherlands,2Institute of Pharmacology, University Duisburg-Essen, Essen, Germany,3Department of Pharmacology, University of California, Davis, CA, USA.Calcium-dependent regulation of several potassium channels has recentlybeen identified, but its relative importance for atrial and ventricular electro-physiology, and potentially arrhythmogenesis, is incompletely understood.Here, we employed computational modeling to study calcium-dependentregulation of potassium channels in cardiac electrophysiology. Calcium-dependent regulation of the basal inward-rectifier (IK1), slow delayed-rectifier (IKs), and small-conductance calcium-activated (ISK) potassium cur-rents was incorporated into established atrial (Grandi-Bers) and ventricular(O’Hara-Rudy) cardiomyocyte models and compared to simulations withoutcalcium-dependent regulation. Calcium-dependent activation of IK1 and ISKshortened atrial and ventricular action potential (AP) duration (APD), buttheir regulation by rate-dependent intracellular calcium loading onlymodestly affected APD rate dependence. The impact of calcium-dependentpotassium current regulation was limited because 1) IKs has a minor impacton repolarization under basal conditions and 2) calcium-dependent activationof IK1, IKs and ISK is already almost saturated at slow rates. Accordingly, afterincorporating a gain-of-function mutation that increased IKs, calcium-dependent IKs activation reduced APD without modulating APD rate depen-dence. Furthermore, simulated L-type calcium channel inhibition loweredintracellular calcium, decreasing calcium-dependent regulation of potassiumcurrents and significantly prolonging APD. Finally, simulated diastolic sarco-plasmic reticulum calcium releases produced delayed afterdepolarizations inthe ventricular model and triggered APs in the atrial model. Calcium-dependent augmentation of potassium currents did not affect the thresholdfor triggered atrial APs, but significantly reduced afterdepolarization ampli-tude in the ventricle due to a calcium-dependent increase in IK1. This effectwas not visible in the atrial model due to the smaller IK1 and the higher sensi-tivity for triggered APs. Taken together, physiologic calcium-dependent po-tassium-channel regulation has a modest impact on basal atrial andventricular electrophysiology, but may be further enhanced during conditionsof abnormal repolarization or impaired calcium handling, and as such mayplay a role in cardiac arrhythmogenesis.

2341-Pos Board B357Effects of Modulation of Small-Conductance Calcium-Activated Potas-sium Current on Atrial Electrophysiology and Arrhythmogenesis: APopulation-Based Computational StudyStefano Morotti1, Nicholas Ellinwood1, Haibo Ni1, Jussi T. Koivum€aki2,Mary M. Maleckar3, Jordi Heijman4, Dobromir Dobrev5, Eleonora Grandi1.1Department of Pharmacology, University of California Davis, Davis, CA,USA, 2A.I. Virtanen Institute for Molecular Sciences, University of EasternFinland, Kuopio, Finland, 3Allen Institute for Cell Science, Seattle, WA,USA, 4Department of Cardiology, Maastricht University, Maastricht,Netherlands, 5Institute of Pharmacology, University Duisburg-Essen, Essen,Germany.

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Given their higher abundance in atria vs. ventricles and suggested involvementin atrial fibrillation (AF) mechanisms, small-conductance Ca2þ-activated Kþ

channels (SKs), carrying IK,Ca, are promising atrial-selective pharmacologicaltargets against AF, but their role in atrial arrhythmogenesis remains incom-pletely understood. We investigated the consequences of IK,Ca modulation onhuman atrial myocyte electrophysiology and arrhythmogenesis in silico. Tothis aim, we incorporated a new mathematical formulation of IK,Ca, based onexperimental observations in atrial myocytes isolated from normal sinusrhythm (nSR) and AF patients, in our established human atrial myocyte model.We investigated the impact of systematically varying SK conductance andapparent Ca2þ-affinity (to simulate positive and negative IK,Ca modulators)on atrial electrophysiological biomarkers (e.g., action potential duration andeffective refractory period, ERP) and occurrence of early- and delayed-afterdepolarizations, triggered activity, and alternans, which permitted calcula-tion of an arrhythmia score. We found that IK,Ca inhibition favors focalarrhythmia (afterdepolarizations and triggered activity), but minimizes the sub-strate for reentry (ERP shortening and alternans). For the SK modulators withthe largest antiarrhythmic effect (i.e., lowest arrhythmia scores), we then gener-ated populations of hundreds of model variants. Multivariable regression anal-ysis allowed quantifying how SKs impact cellular electrophysiologicalparameters, and logistic regression analysis was used to investigate how vari-ability within the populations affects the ability of SK modulation to preventarrhythmias in both nSR and AF-remodeled atrial myocytes. By identifyingthe main factors responsible for arrhythmia occurrence or prevention at variousdegrees of AF-remodeling, our systematic investigation of IK,Ca parametersprovides potentially useful insight for developing new mechanism-based ther-apeutic strategies to treat AF.

2342-Pos Board B358Increased Contribution of Funny Current to Sinoatrial Node Firing Activ-ity at Slow Heart RatesStefano Morotti1, Emily Sharpe2, Pin W. Liu3, Nicholas Ellinwood1,Bruce P. Bean3, Catherine Proenza2, Eleonora Grandi1.1Pharmacology, University of California Davis, Davis, CA, USA,2Department of Physiology and Biophysics, University of Colorado Denver-Anschutz Medical Campus, Denver, CO, USA, 3Department ofNeurobiology, Harvard University, Boston, MA, USA.b-adrenergic receptor (b-AR) stimulation speeds up the rate of action potential(AP) firing in sinoatrial node myocytes (SAMs) via increased cAMP signaling.Although cAMP-dependent regulation of hyperpolarization-activated cyclicnucleotide-gated channels, carrying the funny current If, is thought to play arole in this process, how b-AR stimulation alters If properties during theSAM AP is incompletely understood. Here, we combine in vitro and in silicoapproaches to investigate the effect of isoproterenol (ISO) on If availabilityand kinetics at varying SAM firing rates. We developed a mathematical modelof If that mimics the ISO-induced positive shift in voltage-dependence of acti-vation and slight increase in time constants of activation and deactivation atphysiologic membrane potentials seen in voltage-clamp experiments in isolatedmurine SAMs. The model also recapitulates characteristics of If measured inAP-clamp experiments that revealed inward and outward components of If(at a basal firing rate of 400 bpm), both increased in the presence of ISO.Our simulations showed that the ISO-induced enhancement in If amplitude isprimarily due to its increased availability, whereas the slowdown of If kineticshas a limited (and opposite) effect, at least in our experimental conditions. Wethen simulated AP-clamp experiments using stylized APs at varying firing rates(100-600 bpm) and found that If amplitude increases at slow firing rates, whenmore time is available for channel activation between beats. Similar frequency-and voltage-dependent behavior was observed in control condition and duringISO administration, confirming the limited role of ISO-induced changes in Ifkinetics. Our results suggest that If plays a greater role in regulating firing ac-tivity at slow heart rates (perhaps more in human than mouse?), and enhancedavailability due to b-AR stimulation is the main mechanism involved.

2343-Pos Board B359In Silico Assessment of Atrial Fibrillation-Selectivity of Ikur Inhibitors:Role of Variability in Disease-Associated RemodelingNicholas Ellinwood1, Dobromir Dobrev2, Stefano Morotti1,Eleonora Grandi1.1Department of Pharmacology, UC Davis, Davis, CA, USA, 2Institute ofPharmacology, West German Heart and Vascular Center, UniversityDuisburg-Essen, Essen, Germany.The ultra-rapid delayed-rectifier current (IKur), carried by the atrial predomi-nant (vs. ventricle) alpha subunit KV1.5, has been studied as a promising targetto treat atrial fibrillation (AF). However, while numerous KV1.5-selective com-pounds have been screened in vitro and in animal models of AF, evidence of

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antiarrhythmic efficacy in human clinical trials is lacking, perhaps because pre-clinical assessment of candidate drugs overly relies on steady state drug con-centration response curves rather than accounting for channel conformationalstate specificity and kinetics of drug binding. Here, we simulated a Markov-type model of IKur gating and drug-channel interaction within our comprehen-sive atrial cell model to reveal the ideal binding properties of IKur inhibitors thatmaximize AF-selectivity in normal sinus rhythm (nSR) and chronic AF (cAF).Specifically, we identified drugs exhibiting anti-AF properties at fast-pacingrates (prolongation of effective refractory period, ERP), while having little ef-fect during normal heart rhythm (limited prolongation of action potential dura-tion, APD). We also found that despite being downregulated in our simulations(by 50%), IKur contributes more prominently to APD and ERP in cAF than innSR, and block of IKur in cAF has less cardiotoxic effects and increased effi-cacy. Finally, we studied how inter-subject variability and/or variable degreesof ionic remodeling modulate the response to IKur anti-AF therapy, by revealingthe efficacy and toxicity of IKur inhibitors in populations of nSR and cAF atrialmyocyte models, where ion channel conductances are randomly varied in eachcell. Our in silico strategy can be combined with in vitro and in vivo assays toidentify the complex net impact of IKur inhibitors in different AF-remodelingconditions, and to facilitate the ongoing search for novel agents against AF dur-ing the pre-clinical drug development process.

2344-Pos Board B360Facilitation by hERG Channel Blockers Suppresses Early Afterdepolari-zation of Simulated Cardiac Action PotentialsKazuharu Furutani1,2, Kunichika Tsumoto2, Jon T. Sack1,Yoshihisa Kurachi2.1Department of Physiology and Membrane Biology, University of California,Davis, Davis, CA, USA, 2Department of Pharmacology, Osaka UniversityGraduate School of Medicine, Suita, Osaka, Japan.Block of the cardiac rapid delayed rectifying potassium current (IKr) throughthe human ether-a-go-go-related gene (hERG) channel is the most commoncause of acquired, or drug-induced, long QT syndrome. Current regulatoryguidelines focus on hERG channel block as a surrogate marker to assessthe risk of drug-induced torsades de pointes. We have demonstrated thatseveral clinically-used drugs that block hERG channels also possess anothereffect; they increase the hERG current amplitude in response to low-voltagedepolarization. We refer to this phenomenon as facilitation. To assess thepotential clinical relevance of hERG modulation by these drugs, we con-ducted action potential simulations in a human ventricular myocyte model,incorporating the dual effects of hERG block and facilitation. We calibrateda model of IKr modulation by nifekalant, a Class III antiarrhythmic agent,that was constrained by voltage clamp recordings. Nifekalant’s antiar-rhythmic mechanism of action is to prolong cardiac action potential durationand the relative refractory period. Our simulations likewise indicated thatnifekalant block prolonged the simulated action potential duration and re-fractory period. Inclusion of facilitation in the models had little effect onthese outputs, suggesting that facilitation has little impact on the anti-arrhythmic efficacy of nifekalant. Excessive IKr block can cause early after-depolarization, and we found that modeling nifekalant block resulted in suchearly afterdepolarizations. When facilitation was introduced, early afterde-polarizations were suppressed. This modeling suggests the facilitationmechanism prevents early afterdepolarization by accelerating repolarizationvia an IKr increase that occurs only during the prolonged phase-3 of actionpotentials. Thus, while hERG blockers achieve antiarrhythmic effects byprolonging phase-2 and 3 of action potentials, blockers that also facilitatemay prevent hazardous early afterdepolarizations by shortening latephase-3.

2345-Pos Board B361Unperceived Properties of the Relationship between Net MembraneCurrent and Action Potential DurationAntonio Zaza.Biotechnology and Biosciences, Universita Milano, Bicocca, Milano, Italy.Net membrane current (Im) linearly determines membrane potential velocity(dV/dt=Im/Cm); however, the latter non-linearly translates into action potential(AP) duration (APD=aV/dV/dt). Here we test how non-linearity of the APDvs dV/dt relationship may affect APD response to a ‘‘local’’ Im perturbation,i.e. that occurring at a specific time during repolarization. Methods: Im wasobtained by differentiating a ventricular AP waveform. Random ‘‘noise’’was added to Im, uniformly throughout repolarization; Im noise (ImN) wassymmetrical about mean Im (ImN=0). ‘‘APD noise’’ (APDN) was calculated,point by point, as the change in global repolarization time that would be pro-duced by each local ImN value. Global AP waveforms were recalculated byintegrating the Im and the ImþImN functions respectively. Results: APDN

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was characteristically distributed during repolarization, achieving a maximumduring phase 2 (lowest dV/dt); the relationship APDN vs ImN was well fittedby an inverse square function (APDN=-1/ImN

2), i.e. the derivative of the hy-perbolic function relating APD to dV/dt. In spite of ImN symmetry, APDNwas substantially larger for prolonging than for shortening perturbations(P

APDN > 0). Thus, albeit mean Im was unchanged, ImN always prolongedAPD. Conclusions: according to the numerical APD vs Im relationship, 1) theimpact of an Im change on APD closely depends on the repolarization phaseaffected; 2) at any repolarization time, Im perturbations prolonging APD pre-vail over those shortening it. Divergence from these numerical predictionsmust be attributed to the feed-back between membrane potential and the indi-vidual components of Im.

2346-Pos Board B362Slow Delayed Rectifier KD Current Stabilizes Ventricular Action Poten-tials across SpeciesMeera Varshneya, Ryan A. Devenyi, Eric A. Sobie.Department of Pharmacological Sciences, Icahn School of Medicine atMount Sinai, New York City, NY, USA.The slow (IKs) and rapid (IKr) delayed rectifier Kþ currents are responsiblefor action potential repolarization in ventricular myocytes. Although sub-stantial differences in the voltage-dependent gating kinetics of the two cur-rents have been carefully documented, differences in the physiological rolesof IKr and IKs are less established. A recent study comparing simulationswith experimental recordings in guinea pig myocytes proposed the hypoth-esis that IKs might be superior to IKr at stabilizing the ventricular action po-tential (AP) and protecting cells against arrhythmias. We sought todetermine whether IKs is uniformly protective across species and to uncovermechanisms underlying this protective effect. To address this, we performedsimulations using 10 ventricular myocyte models describing electrophysi-ology of human, rabbit, canine, and guinea pig cells. We assessed modelsusing several methods including simulations of heterogeneous populations,susceptibility to action potential prolongation, and simulations of action po-tential clamps. We found that: (1) models with higher baseline IKs exhibitedless cell-to-cell variability in action potential duration; (2) models withhigher baseline IKs were less susceptible to early afterdepolarizations(EADs) induced by depolarizing perturbations; (3) as action potential dura-tion (APD) is extended, IKs increases more profoundly than IKr, therebyproviding negative feedback that resists excessive APD prolongation; and(4) the increase in IKs that occurs during beta-adrenergic stimulation is crit-ical for protecting cardiac myocytes from EADs under these conditions.Overall, the results confirm a uniformly protective role of IKs across a vari-ety of cell types and support the idea that augmentation of IKs could poten-tially be an effective antiarrhythmic strategy.

Posters: Voltage-gated K Channels andMechanisms of Voltage Sensing and Gating III

2347-Pos Board B363A Universal Molecular Mechanism for C-type Inactivation in PotassiumChannelsJing Li, Jared Jostmey, Eduardo Perozo, Benoit Roux.Biochemistry and Molecular Biology, University of Chicago, Chicago, IL,USA.C-type inactivation of Kþ channels is an elusive molecular process that isthought to involve a subtle conformational change at the level of the selec-tivity filter. C-type inactivation is also of great physiological significance, asit affects the firing patterns of neurons in the central nervous system, and therepolarization of cardiac cells in the heart. In the present study, extendedmolecular dynamics simulation of the hERG channel with an open innergate converged toward a stable constricted conformation for the selectivityfilter. This conformation displays both similarities and differences with theknown constricted conformation observed in X-ray structures of the KcsAchannel. The constricted hERG filter involves a considerable reorientationof phenylalanine at position 627 along the selectivity filter to dock intothe binding pocket occupied by the inactivating water molecules in the con-stricted KcsA structure. This observation supports the notion that constric-tion of the filter is the molecular basis of inactivation, but with alternativestructural factors stabilizing the conformation. Additional MD simulationsand free energy calculations carried out on the KcsA channel show thatthe filter can remain conductive as long as the intracellular gate is not fullyopen, but rapidly transitions to a constricted non-conductive conformationon a microsecond timescale once the inner gate is fully open. The shortmicrosecond lifetime of the conductive filter with a fully open gate, cannotexplain the relatively long periods of sustained ion conduction observed

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experimentally, suggesting that the conduction period must predominantlycorrespond to long-lived states of the channel with a partially open activa-tion gate. In summary, C-type inactivation is linked to a conductive-to-constricted transition of the selectivity filter that is allosterically driven bythe slow opening of the intracellular gate.

2348-Pos Board B364Converting the Depolarization-Activated Shaker KV Channel into aHyperpolarization-Conducting Cation-Selective Channel by Two PoreMutationsEvelyn Martinez-Morales, Laura C. Coonen, Dieter V. Van de Sande,Dirk J. Snyders, Alain J. Labro.Biomedical Sciences, University of Antwerp, Antwerpen, Belgium.Substituting the pore residue W434 in the Shaker Kv channel by a phenylal-anine (W434F) accelerates its C-type inactivation, which involves thecollapse of the selectivity filter (SF). In Shaker-W434F this collapse precedesS6-gate opening upon activation and no ionic currents are recorded. However,a valine mutation for the residue T449 (T449V) slows inactivation and intro-ducing this T449V substitution in the Shaker-W434F rescues ionic conduc-tion. The human Shaker-type Kv1.2 channel has a valine at the homologousT449 position (V381). Interestingly, the Kv1.2-W366F mutant yielded ioniccurrent (similar to the Shaker -W434F-T449V combination). Accordingly,substituting V381 in Kv1.2-W366F by a threonine resulted in a similar(non-conducting) phenotype as Shaker-W434F. Thus the rate of SF collapsecan be controlled by specific combinations of pore residue mutations.Patch-clamp analysis of the ionic currents of Kv1.2-W366F and Shaker-W434F-T449V showed that the channels remained Kþ selective. Thus, theSF of Shaker-W434F-T449V adopts a Kþ-selective conformation when theS6-gate opens. To study the SF conformation of Shaker-W434F when theS6-gate closes, we combined Shaker-W434F with the mutation P475D, thatby itself prevents complete closing of the S6-gate. We expected to recordionic currents of the double Shaker-W434F-P475D mutant at hyperpolarizedpotentials when the S6-gate closes and the SF recovers from its collapsed in-activated state. Indeed, Shaker-W434F-P475D resulted in functional voltage-dependent channels that were conducting at hyperpolarized potentials andceased conducting at depolarized potentials. Interestingly, this conductivestate of Shaker-W434F-P475D was both Naþ and Kþ permeable, i.e. thehigh Kþ selectivity was lost. The mutant remained, however, sensitive toexternal TEA block. Thus, preventing full closure of the S6-gate appears toaffect the recovery process of the SF such that it is trapped in a conformationconducting both Naþ and Kþ.

2349-Pos Board B365Observation of Structural Changes in Closed KD Channels by VoltageClamp SpectroscopySebastian Fletcher-Taylor1, Parashar Thapa2, Jon T. Sack2, Bruce E. Cohen1.1The Molecular Foundry, Lawrence Berkeley Laboratory, Berkeley, CA,USA, 2Department of Physiology and Membrane Biology, University ofCalifornia Davis, Davis, CA, USA.Ion channels are polymorphic membrane proteins whose states and transitionshave been identified by electrophysiology, and whose high-resolution staticstructures have begun to yield to X-ray and EM techniques. These structureshave offered images of individual states, giving us starting points for identi-fying the complex and transient structural changes that give rise to channelphysiology. To understand the structural changes that underlie the gating ofvoltage-gated Kþ channels, we have synthesized fluorescent probes of Kv2channel activity and used spectral imaging to identify structural changes inthe Kv2 complex. We have synthesized chemoselective point mutants ofthe tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot pep-tide that binds selectively to Kv2 channels, and labeled them with a novelenvironment-sensitive far-red fluorophore, JP, whose emission is sensitiveto the polarity of its surroundings. JP-GxTX fluorescence measured in livecell membranes is dependent on the presence of Kv2 channels, the labelingsite on the toxin, and the membrane potential. We collect full emission spectrawhile varying the membrane potential of patch clamped cells (i.e., voltageclamp spectroscopy, VCS) and have developed curve fitting techniques toidentify structural changes of the complex by identifying changes to the envi-ronment of the toxin during voltage changes. Emission spectra of the JP27GxTX mutant comprise 2 major species, polar and non-polar, whose popula-tions change as a function of voltage. These changes occur at far more nega-tive potentials than channel opening, suggesting that they reflectconformational changes of the complex while the channel is closed. VCS ofchannel-bound fluorophores can bridge the gap between electrophysiologyand static structures to offer insight into structural changes of functional chan-nels in live cells.

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2350-Pos Board B366A Characteristic Extracellular Loop of Prestin Modulates its VoltageOperating PointMakoto F. Kuwabara1, Koichiro Wasano2, Satoe Takahashi2,Justin Bodner3, Tomotaka Komori1, Sotaro Uemura1, Jing Zheng2,Tomohiro Shima1, Kazuaki Homma2.1Department of Biological Sciences, Graduate School of Science, TheUniversity of Tokyo, Tokyo, Japan, 2Feinberg School of Medicine,Department of Otolaryngology, Head and Neck Surgery, NorthwesternUniversity, Chicago, IL, USA, 3DePaul University, Chicago, IL, USA.Prestin is a member of the solute carrier 26 (SLC26) family of anion trans-porters and expressed in the outer hair cells in the cochlea. It provides electro-mechanical feedback to amplify sound signals at specific frequencies. Despitehigh sequence similarity among the SLC26 family members, only prestin isthought to exhibit motile activity driven by changes in the membrane potential(electromotility). In order to identify the key structural element responsible forthe electromotility, we generated molecular models for prestin and anotherSLC26 protein, pendrin. These models show the presence of an extracellularloop whose net charge is opposite for prestin (net positive) and pendrin (netnegative). By employing a mutagenesis approach combined with whole-cellpatch clamp measurements, we examined how the electrostatic property ofthe extracellular loops contribute to the functions of prestin and pendrin. Wefound that pendrin exhibits a sign of voltage-sensing ability at highly hyperpo-larized potentials within experimentally measurable voltage range. Pendrin mu-tants in which negatively charged residues in the extracellular loop werereplaced with noncharged or positively charged ones exhibited robustvoltage-sensing ability. These observations suggest that pendrin is also ableto respond to the membrane potential, and that the operating voltage range ofwild-type pendrin is highly hyperpolarized due to the electrostatic propertyof the extracellular loop. Consistent with this idea, a chimeric prestin constructthat contains the extracellular loop of prendrin showed hyperpolarizing shifts inits operating voltage range. Our results challenge the current view that onlyprestin possesses voltage-sensing ability among the SLC26 family, and high-light the importance of the extracellular loop for establishing the operatingvoltage range.

2351-Pos Board B367Quantum Calculations on a Voltage Sensing Domain of KV1.2: H

D Trans-fer and Gating CurrentAlisher M. Kariev, Michael E. Green.Chemistry, City College of the City Univ of NY, New York, NY, USA.Quantum calculations have been used to optimize the structure of a 904 atomsection of the Kv1.2 voltage sensing domain, with 24 water molecules added,starting from X-ray coordinates (pdb:3Lut), using HF/6-31G* for structureoptimization, and B3LYP/6-31G** for energy. The calculations show how pro-tons may traverse the section, and become the gating current. Proton transferwithin a triad of Y266, R297, and E283 is a key proton transfer. We have deter-mined the potential at which a proton switches from Y266 to R297; the R297-E283 salt bridge is not ionized; non-ionized salt bridges are not surprising (e.g.,Liao and Green Comput.Theor.Chem (2011), 963, 207); the switching poten-tial, approximately �15 mV across the membrane, is close to the potential atwhich these channels open. With the S4 segment free to move, and a �107

Vm�1 field applied (equivalent membrane potential: Vz-70 mV), the segmentbackbone moves<2 A, and not always in the intracellular direction. Five caseswith S4 free to move against the remainder of the VSD have been calculated,plus 24 cases with S4 end atoms frozen; these not only produce slightly betterresults by comparison with the X-ray structures, but arginine-phosphate com-plexing makes it likely to be more realistic physically; free/fixed differencesare small. The Y266-R283 proton transfer should contribute about 0.5-0.6charges to the gating current, so that several additional transfers would beneeded to account for the complete gating current. Charges on the atoms arefound not to be near integer values. The contributions of the quantum terms,tens of kT, are significant. See the accompanying abstract for extension tothe critical hydrophobic region near the conserved F233.

2352-Pos Board B368Quantum Calculations on an Interior Segment of the KV1.2 ChannelVoltage Sensing DomainAlisher M. Kariev, Michael E. Green.Chemistry and Biochemistry, City College of the City Univ of NY, NewYork, NY, USA.Quantum calculations on a 976 atom (includes 24 water molecules) VSD sec-tion, described in the accompanying abstract, show proton transfer in the VSD.The only hydrophobic section in the path from one end of the VSD to the otheris at the edge of this segment, centered on a phenylalanine (F233: 3Lut

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numbering). A similar phenylalanine appears in the Hv1 channel, known toconduct protons. F233 and its surrounding region must allow a proton topass, based on much evidence that the VSD conducts protons. In quantum cal-culations of this region, with 311 atoms, in 16 amino acids (including F233),there is a proton transfer with voltage, in the E226-R303 salt bridge; the saltbridge is ionized at zero field (neutralized: approximately 7 kT higher energy),while at�70 mV, the unionized form is about 2 kT lower in energy. Also, K306always transfers its extra proton to D259, so that that salt bridge is not ionizedwith or without voltage. This transfer occurs during the calculation, so there isno ionized form of this salt bridge, and thus no quantitative comparison to bemade. There is evidently only one energy minimum in this salt bridge; in theE226-R303 salt bridge, the H is in local minima so that both the ionized andunionized forms can be calculated. In this calculation, the F233 does not rotatewith voltage; possibly such a rotation might occur in a larger system, whichincluded more distant F233 neighbors. Calculations on a large system (1180atoms) are now in progress; these calculations extend the VSD, with someT1 interaction, almost to the gate; together with the 976 atom calculation,this will essentially complete the VSD. Some water may still require additionalstudy.

2353-Pos Board B369Does VSP Multimerize and Does It Matter?Vamseedhar Rayaprolu1, Perrine Royal2, Guillaume Sandoz2,Susy Kohout1.1Cell Biology and Neuroscience, Montana State University, Bozeman, MT,USA, 2Universit�e Cote d’Azur, CNRS, INSERM, iBV, Nice, France.Proteins form multimers for many reasons: to enhance function, to regulatefunction, to modulate degradation and sometimes to allow function at all.The original studies on the voltage sensing phosphatase (VSP) indicated a sin-gle subunit of VSP stays a monomer on the plasma membrane and functions asa monomer1. This conclusion was supported by PTEN (phosphatase and tensinhomologue deleted on chromosome 10), a closely related enzyme, also func-tioning as a monomer. However, in the last few years, researchers showedthat PTEN can homodimerize and an inactivating mutation in one subunit ofthe dimer can have a dominant negative effect on function2. Thus, we revisitedthe question of VSP multimerization in light of these PTEN studies. Using acombination of biochemical ensemble and single molecule measurements,we found compelling evidence for VSP multimerization. Because VSP ismade up of both a phosphatase domain and a voltage sensing domain, we arecurrently investigating which domain of the protein is involved and how func-tion is impacted by the multimers.

2354-Pos Board B370Detection of Voltage-Sensing Residues in Membrane ProteinsMarina A. Kasimova, Erik Lindahl, Lucie Delemotte.KTH, Stockholm, Sweden.Membrane proteins, which are able to transform electric energy into conforma-tional rearrangements, are called voltage-sensitive. They are responsible forelectric signaling and mediate a spectrum of key physiological processes atmultiple levels - from single cells to complex multicellular organisms. Forsome voltage-sensitive membrane proteins (VSMPs), such as voltage-gatedion channels, the mechanism of voltage sensitivity has been already character-ized, while for others even the voltage sensor itself remains unknown. Due tolarge diversity of VSPMs, the detection of a voltage sensor in each member ofthis group will require screening over tens or hundreds of residues. Here wesuggest a computational method to significantly reduce the number residuesto be tested experimentally. Our method relies on the estimation of a character-istic variable of voltage sensitivity: the gating charge transferred per distanceunit. The latter is calculated per residue of a given VSMP, and the residueswith the largest gating charge transfer are suggested to be voltage-sensitive.For voltage-gated ion channels, the method pinpoints positively charged resi-dues on the S4 helix and their negative countercharges on S2 and S3, whichare known to play fundamental role in voltage sensitivity. In TRPV1, it detectsthe residues of the S4-S5 linker, and in Cx26 - of the extracellular opening ofthe channel. Some of these residues were already shown to affect voltage sensi-tivity when mutated, while the others are a novel prediction of our method,which remains to be tested experimentally.

2355-Pos Board B371Key Residues in the Interface between Voltage Sensor and Pore Domain inShaker Potassium ChannelsJoao L. Carvalho-de-Souza, Francisco Bezanilla.Biochemistry and Molecular Biology, The University of Chicago, Chicago,IL, USA.Based on our recent demonstration of a non-canonical coupling between thevoltage-sensor-domain (VSD) and pore-domain (PD) in Shaker potassium

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channels, we sought key residues in the VSD-to-PD interface (VSD/PD) thatcould explain our findings. We used the Shaker-like Kv1.2 crystallographicstructure (PDB:3LUT) in open relaxed conformation, that shows the segmentsS4 (VSD) and S5 (PD) making contact, as a guide to important residues in S5that are part of the (VSD/PD). There are two very conserved residues (onlyfound in putatively domain-swapping potassium channels’ families Kv1-4):L409 and S412 which make contact with I371 and V369, respectively(numbering from Shaker). In Shaker-IR channel we replaced each of these res-idues in S5 into many different other amino acids (AA) using conventional site-directed mutagenesis. Robust voltage-activated potassium current were re-corded using cut-open oocyte voltage clamp and we estimated the voltage-dependence of mutants measuring G-V curves. Hydrophobic AAs replacingS412 (S412I, S412V and S412F) shifted G-Vs by þ67, þ44 and þ41 mV,respectively. Increasing side-chain size and keeping the overall hydrophilicityat the residue 412 (S412N, S412Q and S412T) shifted G-Vs by þ24, þ30 and33 mV respectively. At L409, hydrophilic substitutions in L409 (409Q and409S) shift G-V curve by þ52 and þ26 mV, respectively. Keeping 409 hydro-phobic but with different side-chain sizes (L409F, L409V and L409A) shiftedG-Vs by þ8.8, þ9.6 and þ30 mV, respectively. These data suggest that resi-dues 412 and 409 in S5 are key for VSD/PD interaction, setting voltage-dependence by their hydropathicity and size: at 412 the side chain is requiredto be hydrophilic and small and at 409 it needs to be hydrophobic, aliphatic andlong. Supported by NIH-R01GM030376.

2356-Pos Board B372Observing the Movement of Heterogeneous Voltage Sensing Domains viaIntermolecular FRETLee Min Leong1,2, Bok Eum Kang1,2, Bradley J. Baker1,2.1Center for Functional Connectomics, Korea Institute of Science andTechnology, Seoul, Republic of Korea, 2Division of Bio-Medical Science &Technology, KIST School, Korea University of Science and Technology,Seoul, Republic of Korea.Fusion of a voltage sensitive domain (VSD) to the fluorescent protein (FP), Su-per Ecliptic pHluorin A227D, allows for the optical measurement of changes inmembrane potential. However, the mechanism coupling fluorescence changesto changes in membrane potential is not fully understood. Primary studies ofthis mechanism suggest that dimerization via the cytoplasmic fluorescent pro-tein domain is required. To further explore the dimerization effect of the genet-ically encoded voltage indicator (GEVI) via intermolecular Forster ResonanceEnergy Transfer (FRET), novel constructs fusing FRET donors or FRET accep-tors to the VSD were constructed and co-transfected into HEK-293 cells. CFP-YFP FRET pairs showed a higher resting FRET than GFP-RFP FRET pairs pre-sumably due to the dimerization of the CFP-YFP pairs. Robust voltage-dependent FRET signals were observed which suggested the movement ofS4 transmembrane segment upon depolarization of the plasma membraneimproved the FRET efficiency between the chromophore dipoles of VSDstuned to different potentials. For instance, a FRET donor attached to a VSDtuned to extreme positive potentials can be paired with a FRET acceptorcoupled to a VSD that responds to physiologically relevant potentials. Thiscombination results in a GEVI that will only move one S4 transmembranesegment or both S4 transmembrane segments depending on the strength ofthe depolarization of the plasma membrane. Research reported in this publica-tion was supported by the National Institute Of Neurological Disorders AndStroke of the National Institutes of Health under Award NumberU01NS099691.

2357-Pos Board B373Tracking of the R1 of the S4 during Voltage Sensor Deactivation underImproved Optical ConditionsElizabeth E.L. Lee, Michael F. Priest, Francisco Bezanilla.University of Chicago, Chicago, IL, USA.Voltage sensor movement in voltage-gated channels remains debated becausetracking the motions of discrete gating charges in the voltage sensor is difficult.We previously employed monobromobimane (qBBr), a small positivelycharged fluorophore that is strongly quenched by tryptophans, to replace gatingcharges in S4 of Shaker. We found that depolarization leads to a rotation andtranslation of the first gating charge, R1. In the present work we study the deac-tivation pathway with this method. We generated several Shaker constructswhere R1 is mutated to a cysteine for qBBr conjugation and another residuein the putative path is changed to a tryptophan. These constructs were expressedin Xenopus laevis oocytes and labeled with qBBr. We simultaneously recordedelectrophysiological and fluorescence data to optically track the movement ofthe R1 by interrogating the interaction between R1 and a residue of interest. Alimitation of this technique is that the qBBr signal is often small due to its over-lap with the endogenous fluorescence of the oocyte when exciting at 405 nm.

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Through the application of a small molecule kinase inhibitor, we decreased theintrinsic background fluorescence of the oocyte. After this treatment, the oo-cytes produce qBBr fluorescence signals that are �3x larger. We found thatstarting at typical resting potential, R1 does not interact with residue F290W,the most intracellular residue of the hydrophobic plug. However, it doesshow a slow quenching when pulsing to extreme hyperpolarizations (�160mV), suggesting that R1 does not normally move past the hydrophobic plugand may provide the basis of the Cole-Moore shift. By looking at different du-rations of depolarizations, we have investigated the movement of R1 as thesensor evolves from the open to the relaxed state. Support NIH:R01GM030376.

2358-Pos Board B374The Role of Backbone Hydrogen Bonds in the Voltage Sensor of KD

ChannelsDaniel T. Infield1, Kimberly Matulef2, Jason D. Galpin1,Christopher A. Ahern1, Francis I. Valiyaveetil2.1Department of Physiology and Biophysics, University of Iowa, Iowa City,IA, USA, 2Department of Physiology and Pharmacology, Oregon Health andScience University, Portland, OR, USA.Voltage-gated potassium channels (Kv) activate in response to depolariza-tion via an outward movement of the S4 helix. The crystal structure ofthe Kv2.1 channel revealed that the S4 helix is unusual in that it includesboth alpha helical and 3-10 helical segments. Structures of VSDs in othervoltage gated channels now reveal that the S4 helix can include variousproportions of alpha and 3-10 helical segments. Based on molecular dy-namics simulations and functional studies, it has been proposed that duringchannel gating, a conversion between an alpha and a 3-10 helical structuretakes place in the S4 segment. The alpha and the 3-10 helical segment showdistinct hydrogen bonding properties and so this conversion will involve arearrangement of the backbone hydrogen bonds in the S4 segment. Wepostulated that disruption of specific backbone hydrogen bonds should altervoltage gating. In order to investigate the role of backbone hydrogen bonds,we used unnatural amino acid mutagenesis techniques. We used thenonsense suppression approach to introduce amide-to-ester backbone substi-tutions at several positions along the S4 of the Shaker potassium channel.We investigated the effects of the ester substitutions on the voltage depen-dence of activation and on gating charge movements. We observe dramaticfunctional effects with the disruption of specific hydrogen bonds in the S4helix and thereby identify a critical set of backbone hydrogen bonds in theS4 segment. The implication of our findings to the mechanism of voltagegating will be discussed.

2359-Pos Board B375Exploring Possible Conversion between Alpha- and 310-Helix in S4 ofShaker Potassium ChannelCarlos Alberto, Z. Bassetto Junior, Joao L. Carvalho-de-Souza,Francisco Bezanilla.Biochemistry and Mol Biology, University of Chicago, Chicago, IL, USA.The possible conversion between a-helix and 310-helix in S4 voltage sensordomain of voltage-gated ion channels has been under debate. Crystal struc-ture of KV1.2 shows the N-terminal of segment four (S4) in an a-helicalconformation whereas C-terminal is in a 310 conformation. The crystalstructures of Ci-VSP show S4 in the a-helical conformation while NaVAbis in 310 conformation. Here, we explored possible conversions betweena-helix and 310-helix conformations by evaluating the effects of interactionsof histidines replacing residues between charges in the S4 segment of con-ducting Shaker-IR using the cut-open oocyte voltage clamp technique. Wetested double mutants V363H-L366H and V363H-V367H in an attempt tofavor the S4 helix in 310-helix or a-helix conformations respectively. Addi-tionally, we evaluated the histidine effects on the single mutants (V363H,L366H, V367H). We analyzed the mid points (V1/2) of G-Vs as well asthe time constant for activation and deactivation. The construct V363Hhas G-V and time constants similar to WT. Remarkably, the mutants con-taining V367H are the slowest ones (V367H: 5 times slower than WT andV363H-V367H: 10 times slower than WT) and G-Vs are displaced to theright (V367H: �25mV and V363H-V367H: �5mV). On the other hand,L366H is 1.25 times slower and V363H-L366H is 2.5 times slowerthan WT while the G-Vs are displaced to the left (both �40mV). A p-pinteraction between histidines is possible but as all constructs are affectedby Ni2þand Zn2þ, the histidine metal bridge formation is uncertain. Apossible explanation for the left- and right-shifted G-Vs in single mutantsis the putative p-p stacking interaction between the histidine in questionand an aromatic residue nearby: F370 in S4 and F416 in S5. Support:NIH-R01GM030376.

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2360-Pos Board B376Determining the Target of Membrane Sterols on the Gating of Voltage-Gated Potassium Channels using Voltage-Clamp FluorometryZakany Florina, Ferenc Papp, Gyorgy Panyi, Zoltan Varga.Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary.Membrane lipids can affect the gating of voltage sensitive ion channels throughdifferent non-specific and specific mechanisms. It has been shown that choles-terol and 7-dehydrocholesterol (7DHC) have remarkable effects on the gatingof Kv1.3 (shift in voltage-dependency of activation and a slower rate of activa-tion) but it is not clear whether these effects are mediated by the actions throughthe voltage sensor domain (VSD) or the pore domain (PD). Our aim was toinvestigate whether the major target of the action of these membrane sterolsis the VSD, PD or the coupling between these two domains. To test the spec-ificity of the effect we carried out our measurements on channels with differingstructures and gating mechanisms: Kv1.3 and the non-domain-swappedKv10.1. Current recordings were performed with VCF technique using a Xen-opus laevis oocyte expression system. To monitor the movement of the VSDsof the channels the specific cysteine residues on the S3-S4 extracellular linkerwere labeled with MTS-TAMRA. By electrophysiological measurements wedetermined typical current parameters of the channels and F-V curves fromthe fluorescent signal in control and sterol-loaded cells. In the oocyte expres-sion system we were able to successfully reproduce the shift in voltage-dependency of activation and a slower rate of activation in the case of Kv1.3and as a novel finding we obtained similar results with Kv10.1. We found novoltage shifts in the F-V curves paralleling the G-V shift in either ion channel,only the slopes of these curves were decreased in both cases. These results sug-gest that cholesterol and 7-DHC exert their effects by acting directly on the PDand/or the coupling mechanism, instead of influencing the VSD. Support:KTIA_NAP_13-2-2015-0009

2361-Pos Board B377Functional Analysis of the Voltage Sensor Domain Present in the Mamma-lian Sperm-Specific NAD/HD Exchanger by Patch-Clamp Fluorometry ofChimeric Fluorescent Voltage SensorC�esar Arcos Hernandez1, Esteban Suarez2, Leon Islas2, Takuya Nishigaki1.1Gen�etica del desarrollo y fisiologıa molecular, Instituto de Biotecnologia,Cuernavaca, Mexico, 2Facultad de Medicina, Mexico City, Mexico.Several enzymes, transporters and ion channels accurately regulate spermmotility. Some of these proteins are expressed only in spermatozoa, but thefunctional relationship among them is not fully understood. Sperm-specificNaþ/Hþ exchanger (sNHE) is an essential protein for mouse sperm motilityregulation and is localized in the principal piece of mammalian spermatozoa.Differently from somatic NHEs, sNHE has two predicted regulatory domains,a voltage sensor domain (VSD) and a cyclic nucleotide binding domain(CNBD), although their functionality remains to be confirmed. In the case ofthe VSD, it has been proposed that hyperpolarization caused by Slo3 (a spermspecific Kþ channel) promotes an intracellular alkalization by the sNHEthrough this VSD. This signal cascade is thought to be involved in sperm hyper-activation. Interestingly, there are experiments that suggest the VSD of sNHE isfunctional in mice but not in humans. Correlating this, the fourth transmem-brane segment of the VSD of the human sNHE, but not the mouse sNHE, lacksan arginine in a critical position, which could cause the domain not to be func-tional. To demonstrate functional differences in the VSD of the sNHE betweenhuman and mouse, we will produce fluorescent voltage sensors (VSFPs) usingArclight, a popular VSFP, as a template and replace its VSD by that of sNHE.We will analyze the biophysical properties of the VSFPs expressed in HEK293cells by using patch clamp fluorometry and extrapolate the function of theVSDs of human and mouse sNHE. This project is supported by PAPIIT(DGAPA IN206116) and CONACyT-Mexico (Fronteras de la Ciencia 71).

2362-Pos Board B378Site-Specific Encoding of Citrulline, a Neutral Analog of Arginine, to Studythe Atomic Basis for Voltage-Dependent Gating In Ion ChannelsDaniel T. Infield, Jason D. Galpin, Grace D. Galles, Christopher A. Ahern.Physiology and Biophysics, University of Iowa, Iowa City, IA, USA.Voltage gated cation channels are gated by transient changes in transmembranepotential. Conventional mutagenesis studies have implicated conserved basicresidues, primarily arginines, within the S4 helix in the role of voltage sensing,but the unique structure of arginine makes it difficult to attribute functionalroles to specific chemical properties of the side chain. Glutamine, for instance,is a common chemical mimetic of arginine yet this substitution changes multi-ple features of the side-chain, in addition to neutralizing the charge. Citrulline,(CIT) in comparison, is an arginine analog wherein one of the distal aminegroups is changed to an ester, which neutralizes sidechain charge while main-taining the overall structure, H-bonding pattern and polarity of Arginine. Here,

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we have used a synthetic pyrrolysine orthogonal tRNA to site-specificallyencode citrulline in the place of basic residues within the voltage sensor ofShaker-IR potassium channels using nonsense suppression into Xenopus oo-cytes. The CIT substitution is well-tolerated producing Shaker channels withmacroscopic current expression and voltage-dependent gating parameters.However, at some positions, such as R1, CIT had an unexpectedly minimal ef-fect on voltage sensitivity (V1/2 of activation similar to WT), but at others likeR3, substitution with citrulline shifted the voltage sensitivity in the depolarizeddirection (approximately 25 mV for this position). In contrast, glutatmine sub-stitution at these same sites invariably produced large perturbations in the chan-nel equilibrium gating. These results highlight the utility of atomic mutagenesiswhen examining mechanistic details in ion channels. Further, these data repre-sent the first demonstration of CIT encoding in na ion channel via nonsensesupppression and pave the way for future mechanistic studies of conservedArg residues.

2363-Pos Board B379Using Unnatural Amino Acids to Probe the Interaction between TarantulaToxins and Voltage Sensing Domains in Kv ChannelsKanchan Gupta, Kenton J. Swartz.Molecular Physiology and Biophysics Section, NINDS, NIH, Bethesda, MD,USA.Peptide toxins folded into an inhibitor cystine knot (ICK) are found in thevenom of a variety of organisms (spider, scorpion, snakes, etc.), and many ofthese target the voltage-sensing domains in voltage-activated ion channels tomodulate their gating properties. Our previous studies suggest that tarantulatoxins partition into the lipid membrane and bind to the S3b-S4 paddle motifwithin the voltage-sensing domains to allosterically inhibit Kv channels.Alanine scanning mutagenesis studies have identified candidate residues inboth toxin and channel that may contribute to forming the protein-protein inter-face when in complex. To definitively identify protein interaction surfaces be-tween toxin and Kv channel, we applied site-specific crosslinking withunnatural amino acids and Strain - Promoted Azide Alkyne Click (SPAAC)chemistry. Azidohomoalanine (AHA), an analog of methionine, was incorpo-rated in Kv channels expressed in Xenopus laevis oocytes through endogenousmethionyl t-RNA synthetase by supplementing culture media with AHA.Simultaneously, guangxitoxin-1E (GxTx-1E) was engineered with spinstercysteine residues to incorporate an azide-reactive dibenzocyclooctyne(DBCO) group using a cysteine-maleimide conjugation method. We assayedfor formation of toxin-channel crosslinks in a functional manner by analyzingthe recovery of macroscopic ionic currents and kinetics of toxin dissociationusing the two-electrode voltage clamp (TEVC) technique. Our results indicatethat alkyne-conjugated GxTx-1E can be crosslinked with AHA incorporated atspecific positions in S3b helix within the voltage-sensing domain of Kv2.1.Currently, we are assaying the crosslinking behavior of other DBCO-conjugated GxTx-1E variants and Met mutants of Kv2.1 to probe into the inter-acting surfaces of toxin and channel in a membrane environment.

2364-Pos Board B380Orthogonality of a Synthetic Pyrrolysine tRNA in the Xenopus Oocyte andits use to Encode Unnatural Amino Acids into Ion ChannelsDaniel T. Infield, John D. Lueck, Jason D. Galpin, Grace D. Galles,Christopher A. Ahern.University of Iowa, Iowa City, IA, USA.The power of the chemical misacylation method of nonsense suppression stemsfrom the ability to attach a wide range of synthetic amino acids to tRNAwithout the need to evolve specific aminacyl-tRNA synthetases. This methodhas been powerfully paired with protein expression in the Xenopus oocyte,wherein injection of the target RNA and tRNA is facile. However, the fidelityof the method depends on the orthogonality of the tRNA species being used todeliver the amino acid; the tRNAmust be accepted by the ribosomal machineryof the cell but ignored by endogenous aminacyl-tRNA synthetases that may editthe tRNA with a natural amino acid. Tetrahymena thermophila -based tRNAsare orthogonal but have been shown to be re-edited under some conditions. Toimprove the method and expand its uses, we asked whether the PyrrolysinetRNA from Barkeri fusaro (PylT), which has already been shown to be highlyorthogonal in mammalian cells, could be used in nonsense suppression exper-iments in the Xenopus oocyte. Simultaneously, we also asked whether we coulduse fully synthetic (as opposed to in vitro transcribed) tRNA for this applica-tion. Functional and biochemical experiments indicate that PylT is orthogonalin the oocyte, and that it can be used to deliver diverse amino acids into mul-tiple targets (including voltage-gated sodium channels, voltage gated potassiumchannels and sodium transporters) to achieve high expression of engineeredproteins with limited evidence of ‘bleedthrough’ of the introduced TAG codonwithin the target cRNA. The demonstration that fully synthetic tRNA is useful

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in nonsense suppression eliminates the need for costly and tedious in vitro tran-scription and gel filtration in the manufacture of aminacyl-tRNA pellets, andshould expand access to this method.

2365-Pos Board B381Combining Electrical and Optical Measurements to Reveal the Structure-Function Relationship of Voltage-Gated Potassium ChannelsCorianne VandenAkker, Steven Boxer.Stanford University, Stanford, CA, USA.Although the structure of the voltage-gated potassium channel KvAP has beenresolved more than a decade ago, there is still controversy about their precisegating mechanism. The discrepancies among proposed models result in partfrom differences in experimental techniques: electrical measurements of chan-nels in lipid membranes to study their function versus determination of the struc-ture of channels purified using detergents. To be able to combine functional andstructural measurements, we developed a second-generation membrane interfer-ometer in which both electrophysiology measurements and high-resolution fluo-rescence microscopy imaging can be performed on ion channels reconstituted ina lipid membrane, a much advanced design over the original (Ganesan et al.,Proc. Natl. Acad. Sci. 106, 2008). In the membrane interferometer, a free-standing bilayer is formed over a micropore that is positioned above a reflectivemirror. The mirror allows the use of Fluorescence Interference Contrast (FLIC)and Variable Incidence Angle-FLIC (VIA-FLIC) microscopy, two surface char-acterization techniques that precisely locate the height of fluorescent objects rela-tive to the mirror with nanometer resolution. KvAP overexpressed in E. coli isdetergent-free extracted using polymer nanodiscs. We show that functionalKvAP can be reconstituted in black lipid membranes and freestanding lipid bi-layers on the membrane interferometer. No differences in gating propertieswere found between KvAP extracted by polymer nanodiscs versus detergents.We label the channels with a fluorescent tag at different positions at the S3 orS4 strand. Our goal is to correlate the change in position of the S3 and S4 strandsmeasured using FLIC microscopy with single-channel electrical measurementsto reveal the structure-function relationship of KvAP. Progress towards thisgoal will be described.

2366-Pos Board B382Reconstitution of Voltage-Activated Potassium Channel into PhospholipidBilayerChanhyung Bae, Swartz J. Kenton.NIH, Bethesda, MD, USA.Voltage-activated ion channels are expressed in the membranes of excitablecells, and open and close in response to changes in membrane voltage. Physi-ologically, they play diverse roles, from the generation of action potentials totriggering the release of neurotransmitters at synapses. To study the biochem-ical properties, these ion channels are usually purified and studied underdetergent-solubilized condition, but many ion channels behave differently indetergent when compared to their native membrane environment. One recentlyintroduced technique to overcome this issue is using lipid bilayer nanodiscs.Nanodiscs are a few nanometer-sized hockey-puck-shaped lipid bilayers solu-bilized by engineered apolipoprotein, membrane scaffold protein (MSP). MSPis comprised of short repeats of amphipathic alpha helies that surround the hy-drophobic acyl chains of phospholipids. To investigate the structure andbiochemical properties of voltage-activated potassium (Kv) channels in a mem-brane environment, we reconstituted a Kv channel into phospholipid bilayernanodiscs using phospholipid and MSP. We first attempted to assemble emptynandiscs (nanodisc without Kv) to determine the optimal ratio between lipidand MSP that provides the best yields. Using these conditions, we reconstitutedKv channels into nanodiscs, and show that they elute as a single peak in gel-filtration chromatography. SDS-PAGE result indicates that the preparation con-tains each subunit corresponding to the Kv and MSP, confirming successfulreconstitution of the Kv into nanodiscs. We also show that a voltage-sensortoxin can bind to the Kv channel in nanodiscs, confirming that the architectureof the Kv channel is well maintained in nanodiscs.

2367-Pos Board B383Imaging Voltage Gating of Endogenous Neuronal Ion Channels withFluorescent Tarantula ToxinParashar Thapa1, Rebecka Sepela1, Robert Stewart1, Mark Lillya1,Oscar Vivas1,2, Laxmi Parajuli2, Sebastian Fletcher-Taylor1, Karen Zito2,Bruce E. Cohen3, Jon Sack1.1Physiology andMembrane Biology, UC Davis, Davis, CA, USA, 2Center forNeuroscience, UC Davis, Davis, CA, USA, 3Biological NanostructureFacility, Lawrence Berkley National Labratory, Berkley, CA, USA.Diverse ion channel subtypes activate symphonically to orchestrate electricalsignals. Though the neural output is seemingly harmonious, the individualpatterns of channel activation are complex, biochemically regulated,

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location-dependent, and overlapping, making dissection of this cacophonousphenomenon difficult. Pinpointing the physiological role of solo ion channelsis further impeded by a lack in existing technologies capable of determiningwhen and where ion channel types are activated in native tissue. We havegenerated an endogenous channel activity probe (ECAP) that optically tracksvoltage activation of Kv2 type Kþ channels. Guided by a conformation- selec-tive, peptide toxin, the fluorescently tagged ECAP binds to resting voltagesensing domain of Kv2 and dissociates from activated voltage sensors. Wedemonstrate that loss of fluorescent ECAP signal from CA1 neurons in rat hip-pocampal slices coincides with voltage activation of endogenous Kv2 channels.Confirmatory tests show that the ECAP colocalizes with Kv2-GFP channels inneurons and in cultured cells with or without the presence of the channel’sauxiliary subunit AMIGO-1, but not with other channel subtypes. ECAP signaldynamics were both frequency and voltage-stimuli dependent, setting method-ical precedence for current and future explorations tracking channel activationor protein conformational changes during physiological signaling.

2368-Pos Board B384Lipid-Dependent Activation and Desensitization Mechanism of MthKNattakan Sukomon, Crina Nimigean.Anesthesiology, Weill Cornell Medical College, New York, NY, USA.MthK is a prokaryotic Ca2þ-activated Kþ channel with several high-resolutionstructures, and serves as a model for investigating gating mechanisms of Kþ

channels. Previous investigations using proteoliposome-based Tlþ fluxassay indicated that MthK underwent desensitization in seconds after Ca2þ acti-vation. In contrast,MthK studiedwith electrophysiology in a planar lipid bilayersystem show no evidence of desensitization.We hypothesized that this is causedby the different bilayer properties between the two preparations. The lipidcomposition of the bilayers in both preparations is the same (DOPC:POPG,3:1). However, the differences in curvatures (liposomes vs. planar bilayers)and thicknesses (decane, used for planar bilayer formation, leads to increasedbilayer thickness) may affect channel gating. Since MthK does not appear todesensitize in the thicker, decane-containing planar bilayers, we expect thatincreasing the bilayer thickness in liposomes will cause slower or no desensiti-zation in the flux assays. Conversely, decreasing the bilayer thickness in theplanar bilayers will lead to desensitization in the electrophysiology assays.We performed planar bilayer and Tlþ flux experiments using MthK reconsti-tuted in liposomes made with lipids with different lipid acyl chain lengths(PC, from C16:1 to C22:1) while maintaining the POPG mole fraction. Chang-ing the acyl chain length did not affect the voltage-dependent gating behavior ofMthK when tested in planar bilayers. In addition, MthK did not undergo desen-sitization in any lipid compositions tested. On the contrary, varying the acylchain length affected both MthK activation and inactivation in flux assays.We found that MthK inactivates more slowly in liposomes with longer acylchain lengths (C20:1 and C22:1), and faster in liposomes with shorter acyl chainlengths (C16:1). Additionally, MthK activation was markedly slower in lipo-somes with longer acyl-chain lipids. The mechanism for lipid-dependent activa-tion and desensitization of MthK is under investigation.

2369-Pos Board B385Alpha-B Helix of the RCK1 Domain is a Shared Structural Element forBoth Voltage and Calcium Activation of BK ChannelsYanyanGeng1, Zengqin Deng2, Guohui Zhang2, Alice Butler2, Jianmin Cui2,Peng Yuan2, Lawrence Salkoff2, Karl L. Magleby1.1University of Miami, Miami, FL, USA, 2Washington University, St. Louis,MO, USA.BK channels are comprised of an integral-membrane core, that includes fourvoltage sensors and a central pore-gate domain (PGD), and also a large cyto-solic domain (CTD), termed the gating ring, which is attached to the corethrough four S6-RCK1 peptide linkers. Structural studies (Yuan et al. 2011;Hite, Tao, MacKinnon, 2016) indicate that Ca2þ binding to the CTD rotatesthe N-lobe of each RCK1 outwards, which moves the a-B helices outwardsand upwards to push on the core near the S4-S5 linkers, while simultaneouslypulling the C-terminus of the S6-RCK1 linkers outwards and downwards. Thisgives a push-pull action between gating ring and core. We now further examinethe contribution of the a-B helix to channel activation by introducing point pro-line mutations along the a-B helix. Such mutations right shifted V0.5 for voltageactivation by þ50 to þ180 mV, while having negligible effects on the gatingcurrents, indicating that the a-B helix is involved in voltage activation. The mu-tations also decreased the delta leftward shift in V0.5 due to Ca

2þ activation, andthis was also the case for the Ca2þ bowl and RCK1 sites acting separately, indi-cating that both high affinity Ca2þ binding sites act, at least in part, through thea-B helix. Higher Ca2þ did not restore the delta Ca2þ shift to that of WT. Thecrystal structure of the CTD with L390P indicated one less helical turn in the a-B helix than WT without other notable differences in the CTD. These findings

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indicate that an intact a-B helix is required for effective coupling of both Ca2þ

binding and voltage depolarization to pore opening, and that a shared pathwayinvolving the a-B helix may be involved. NIIH GM114694.

2370-Pos Board B386Role of an Intrasubunit Ca2D Bridge Dependent Activation of BKChannelsAlberto J. Gonzalez-Hernandez1,2, Aravind Kshatri1,2, Teresa Giraldez1,2.1Biomedical Sciences (Physiology), University of La Laguna, San Cristobalde La Laguna, Spain, 2Biomedical Technologies Institute (ITB) andBiomedical Research Center of Canary Islands (CIBICAN), San Cristobal deLa Laguna, Spain.BK channels are high conductance potassium channels that are synergisticallygated by voltage and intracellular Ca2þ. Numerous studies have identified theCa2þ binding sites within an intracellular structure known as the gating ring.Recent crystal structures of the full BK channel from Aplysia californica(Tao et al., 2017; Hite et al., 2017) are consistent with previous findings butalso identified additional residues involved in Ca2þ coordination that had notbeen studied in the human BK channel (hBK). We have now validated theseresidues using alanine scanning mutagenesis. Consistent with the crystal struc-ture, our functional data indicate that the conserved residue hR514 participatesin Ca2þ coordination in the RCK1 site. Furthermore, our data support the exis-tence of an interaction between the side-chain of this residue with residueshY902 and hY904 from the Ca2þ bowl region of RCK2 site. The strongercation- p interaction between hR514 and hY904 residues may constitute astructural correlate underlying the cooperative interactions between the twohigh-affinity Ca2þ binding sites (Kshatri et al., 2017, Tao et al., 2017; Hiteet al., 2017; Guan et al., 2017). We have investigated the role of this cation-p interaction in BK Ca2þ dependent gating by altering its strength throughvarious amino acid substitutions. Patch-clamp fluorometry studies are currentlyunderway to unveil the structural mechanism underlying this cooperativity.This project has received funding from European Research Council (ERC) un-der the European Union’s Horizon 2020 research and innovation programme648936.

2371-Pos Board B387Coupling between Sensors and the Activation Gate in BKChannels Probedby a Chemical CompoundGuohui Zhang1, Xianjin Xu2, Hongwu Liang3, Jingyi Shi3, Kelli McFarland3,Xiaoqin Zou2, Jianmin Cui4.1Washington University, Ballwin, MO, USA, 2University of Missouri-Columbia, Columbia, MO, USA, 3Washington University, St. Louis, MO,USA, 4Department of Biomedical Engineering, Washington University, St.Louis, MO, USA.Large conductance Ca2þ-activated Kþ channels (BK channels) activate inresponse to both membrane voltage and intracellular Ca2þ. Although thevoltage sensor and Ca2þ binding sites have been identified, the coupling ofthe voltage sensor activation or Ca2þ binding to the opening of the activationgate is not clear. In a screening of compounds for BK channel modulators,we found BC1-5 as a candidate that alters BK channel function. Patch-clampstudies of mSlo1 BK channels expressed in the Xenopus oocyte membraneshow that BC1-5 activates the channel at 0 [Ca2þ]i by shifting theconductance-voltage (GV) relation to more negative voltages for up to 75mV with a concentration at half-maximal response (EC50) of 4 mM. The shiftof GV relation reduces when the channel is activated by increasing [Ca2þ]i,In 100 mM [Ca2þ]i, which is a saturating concentration for Ca

2þ dependent acti-vation of the channel, 100 mMBC1-5 cannot shift GV. This result suggests thatBC1-5 promotes channel opening by interacting with the pathway for Ca2þ

dependent activation. Although the BC1-5 effect depends on [Ca2þ]i the com-pound can activate the mSlo1 Core-MT channel, in which the cytosolic domaincontaining Ca2þ binding sites is deleted. Molecular docking analysis and muta-genesis studies suggest that BC1-5 interacts with the cytosolic face of thevoltage sensor domain to activate the channel. These results suggest thatBC1-5 modulates the coupling between Ca2þ binding and channel openingand the voltage sensor domain is involved in this coupling mechanism.

2372-Pos Board B388Quantitative Analysis of Subcellular Nanodomains Formed by BK andVoltage-Gated Calcium ChannelsAlejandro Cerrada1,2, Aravind Kshatri1,2, Roger Gimeno1,2,Teresa Giraldez1,2.1Department of Medical Sciences-Physiology, Universidad de La Laguna, LaLaguna, Spain, 2Center for Biomedical Research of the Canary Islands(CIBICAN), Institute of Biomedical Technologies (ITB), La Laguna, Spain.A plethora of diverse physiological responses initiated in the cell are finelyregulated by intracellular Ca2þ, which requires a strict control at both temporal

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and spatial levels. In different neuronal types and other excitable cells, the tightcoupling of Ca2þ influx and Ca2þ effectors results in the formation of nanoscalecalcium domains. One type of neuronal ‘‘nanodomains’’ is that constituted byvoltage-dependent Ca2þ (Cav) channels and the large-conductance Ca2þ- andvoltage-gated Kþ channels (BK). BK channels are allosterically activated bymembrane depolarization and elevation of cytosolic Ca2þ. In subcellular cal-cium nanodomains, Cav have been proposed to locate within 20-50 nm ofBK, permitting its rapid activation by Ca2þ influx and shifting the depolariza-tion required for channel opening into the physiological voltage range. In thiswork we have used Stochastic Optical Reconstruction Microscopy (STORM)as well as the Proximity Ligation Assay (PLA) technique to quantitativelystudy the structural organization of reconstituted nanodomains formed by het-erologously expressed BK-Cav complexes. These techniques have allowed usto quantify the number of clusters and the distance between molecules withinclusters (Nearest Neighbor Distance analysis). We have analyzed differentcombinations of various members of the Cav family, BK and their relevant reg-ulatory subunits. Our results show that Cav2.1, Cav2.2 and Cav1.2 channelsform nanodomains in our experimental conditions. However, Cav2.3 channelsshowed lower number of clusters with a significantly broader NND distribution,suggesting that they may not form clusters in physiological conditions. Theseresults are consistent with previous biochemical and functional studies (Berke-feld et al 2006 J Cell Sci, 120:985, Loane et al 2007 Science 314:615). Usingthese powerful techniques, we are currently performing additional experimentsto test the effect of different factors proposed to alter the clustering of BK andCav.

2373-Pos Board B389Novel Topology of BK Channel Beta1 Subunit Predicted on the Basis of the‘‘Positive-Inside’’ Rule Points at an Intracellular Location of the LoopMaria Simakova1, Shivantika Bisen1, Kelsey Cleland1, Avia Rosenhouse-Dantsker2, Alex Dopico1, Anna Bukiya1.1University of Tennessee HSC, Memphis, TN, USA, 2University of Illinois atChicago, Chicago, IL, USA.The most effective computational methods aimed at investigating the structureof transmembrane proteins (TMPs) are based on the prediction of their topol-ogy. Herein, topology predictions were performed by combining a new modi-fication of the sliding window method for topography prediction of helicalTMPs (Simakova and Simakov, 2014), and the empirical ‘‘positive-inside’’rule of the biased distribution of Arg/Lys between intracellular and extracel-lular domains of TMPs (von Heijne, 1986). We predicted the topologies ofKCNMB1-coded proteins of rat (UniProtKB P97678), mouse (Q8CAE3), andhuman (Q16558) that conform accessory beta1 subunits of the calcium-/voltage-gated potassium channel of large conductance (BK). BK betas consistof N-/C- termini, and two transmembrane regions connected by a loop. Accord-ing to our topology predictions, the beta1 loop resides intracellularly as thenumber of Arg/Lys in the loop exceeds the number in the termini (12 versus8 in rat/mouse, 14 versus 7 in human). Immunofluorescence labeling ofbeta1 in rat and mouse cerebral arteries against the loop epitope showed inten-sive staining only of the vasculature in permeabilized specimens. In non-permeabilized arteries, the beta1-associated signal was indistinguishablefrom those in cerebral arteries stained in the presence of an immunogenic pep-tide or in the absence of a primary antibody. Non-permeabilized arteries of wildtype (beta1-containing) mice lacked beta1-associated fluorescence to the sameextent as permeabilized arteries of KCNMB1 knock-out mice. The difference influorescence intensity between the permeabilized and non-permeabilized spec-imens disappeared when KCNMB1 knock-out arteries were electroporated witha beta1-mutant containing six additional Arg in the termini that likely causedthe beta1 subunit to adopt an inverted topology. In conclusion, our topologypredictions and our experimental data place the beta1 subunit loop inside thecell.

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2374-Pos Board B390Eliminating Warrantless Assumptions Facilitates Consideration of anElectrostatic Model of Ion-Channel ActivationH. Richard Leuchtag.Retired, Kerrville, TX, USA.Models of voltage-sensitive ion channels (VSICs) that don’t specify the wayreduction in electric field at threshold activation leads to observed outwardS4 motions don’t provide satisfactory explanations of voltage sensing [Beza-nilla F (2008) Neuron50:456-468]. Current models depend on unrealistic as-sumptions: screening of charges by a putative water-filled pore, and simplemechanical devices. A gate, screw, paddle or other everyday device cannotphysically explain activation of the highly energetic resting channel, a far-from-equilibrium macromolecular system: The a-helix’s hydrogen bondsare too weak to support such rigid structures. The energy required tolengthen the H-bonds is provided when the capacitive energy lost in thevoltage reduction to threshold does mechanical work on the channel. Simi-larities exist between excitable membranes and ferroelectrics [Leuchtag HR(1987) J Theor Biol 127:321-340; 127:341-359; (2008) Voltage-Sensitive IonChannels, Springer]. The ferroelectricity of excitable membranes must orig-inate in the channels, since VSICs are their active components. In ferroelec-trics, the dielectric coefficient ε is a function of the electric field.Electrostatic forces are inversely proportional to ε. Evidence from moleculeswith the sidechains of the branched-chain amino acids isoleucine, leucineand valine shows that their ε becomes remarkably large in a high electricfield [Yoshino K, Sakurai T (1991) in: Goodby JW et al. FerroelectricLiquid Crystals:317-363], comparable to that in a resting channel. Accordingto the Channel Activation by Electrostatic Repulsion (CAbER) model, ε ishigh at rest potential and drops on depolarization. The consequentlyincreased electrostatic repulsion between positively charged arginine andlysine residues at activation expands the S4 segments. This outward motionreconfigures the VSIC into an ‘‘open’’ structure with pore-domain sites wideenough to accommodate unhydrated permeant ions that hop stochasticallyacross the channel [Leuchtag HR (2016) Biophys. J. 112(3):544a;112(3):543a-544a].

2375-Pos Board B391Multicomponent Conduction and Selectivity of Biological ChannelsDmitry G. Luchinsky1, Will A.T. Gibby1, Igor Kh Kaufman1,Miroslav Barabash1, Dogan A. Timucin2, Peter V.E. McClintock1.1Physics, Lancaster University, Lancaster, United Kingdom, 2IntelligentSystems Division, Ames Research System, Moffett Field, CA, USA.We consider multispecies conduction and selectivity of narrow biological fil-ters such as KcsA [1] and NaChBac [2]. We use equilibrium statistical theoryto find filter occupation and conductivities when coupled to the bath solutionswith mixed types of conducting ions. The analysis of the filter conduction andselectivity conduction and selectivity is thus reduced to the analysis of theeffective grand canonical distribution of ions within the filter [3], energyspectra, and density of the states within the filter [4]. The effects of energyquantization due to discrete number of ions and due to dehydration are takeninto account. To account for the interaction between fluxes of different specieswithin the channel we introduce Maxwell-Stefan theory [5] of the ion channelconduction. The fluxes of various species through the filter are obtained analyt-ically in the limit of weak coupling. The theory is applied to derive Eisenmanrelations of the filter selectivity from the condition of nearly diffusion limitedconduction in KscA channel.The model predictions are compared with experimental results using self-consistent kinetic equations. The transition rates in kinetic model are derivedusing effective grand canonical ensemble. A good agreement with the experi-ment is demonstrated. An application of the theory to analysis of the channelmutation experiments are discussed.

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Posters: TRP Channels I

2376-Pos Board B392Decrypting the Heat Activation Mechanism of TRPV1 Channel by Molec-ular Dynamics SimulationWenjun Zheng, Han Wen.SUNY at Buffalo, Buffalo, NY, USA.As a prototype cellular sensor, the TRPV1 cation channel undergoes a closed-to-open gating transition in response to various physical and chemical stimuliincluding noxious heat. Despite recent progress, the molecular mechanism ofheat activation of TRPV1 gating remains enigmatic. Toward decrypting thestructural basis of TRPV1 heat activation, we performed extensive moleculardynamics (MD) simulations (with cumulative simulation time of �11 ms)for the wildtype (WT) channel and a constitutively active double mutantat different temperatures (30�C, 60�C, and 72�C), starting from a high-resolution closed-channel structure of TRPV1 solved by cryo-electronmicroscopy (cryo-EM). In the WT simulations, we observed heat-activatedconformational changes (e.g., expansion or contraction) in various key domainsof TRPV1 (e.g., the S2-S3 and S4-S5 linkers) to prime the channel for gating.These conformational changes involve a number of dynamic hydrogen-bondinteractions which were validated with previous mutational studies. Next, ourmutant simulations observed channel opening following a series of conforma-tional changes that propagate from the channel periphery to the channel porevia key intermediate domains (including the S2-S3 and S4-S5 linkers). Thegating transition is accompanied by a large increase in the protein-water elec-trostatic interaction energy which supports the contribution of de-solvation ofpolar/charged residues to the temperature-sensitive TRPV1 gating. Takentogether, our MD simulations and analyses offered new structural, dynamic,and energetic information to guide future mutagenesis and functional studiesof TRPV1 channel and development of TRPV1-targeting drugs.

2377-Pos Board B393Heat Sensitive Gating Mechanism of TRPV1 Channel Revealed by Molec-ular Dynamics SimulationSoon Woo Park, Soojin Jo, Moon Ki Kim.School of Mechanical Engineering, Sungkyunkwan University, Suwon,Republic of Korea.TRPV1, known as capsaicin receptor, is a cation channel formed by tetramer. Itresponds to various stimuli including capsaicin, DkTx/RTX, and heat. TRPV1channel is a tetramer protein and its selectivity filter is formed by GLY643 andMET644 of each monomer. Conformational change of the pore regionincluding selectivity filter plays an important role in determining ion perme-ability. It is also reported that ions can be transmitted to cytoplasmic regionwhen TRPV1 channel is opened over 43�C. However, there is no clear expla-nation about how ion permeability is related with conformational change ofspecific residues as temperature increases. In this study, conformational changeof TRPV1 gates was investigated by molecular dynamics simulation withCharmm36 force field. The pore radius was calculated using HOLE programin case of 30, 40, 50, and 60�C. As a result, we confirm that ions can be trans-mitted through the TRPV1 pore that is spatially distorted without any change ofpore radius as temperature increases. Consequently, this conformational changeplays a crucial role in ion permeability of TRPV1 channel.

2378-Pos Board B394Thermal Diffusion Pathways of TRPV1Fernando D. Gonzalez-Nilo1, Ignacio Diaz-Franulic2,Romina V. Sepulveda1, Felipe A. Gomez-Becerra1, German Mino-Galaz1,Ramon Latorre2.1Center for Bioinformatics and Integrative Biology, Universidad AndresBello, Santiago, Chile, 2Centro Interdisciplinario de Neurociencia deValparaıso, Facultad de Ciencias., Universidad de Valparaıso, Valparaıso,Chile.TRPV1 is a polymodal receptor gated by vanilloid ligands, heat, voltage, lipids,protons, and peptides from venomous animals (Diaz-Franulic et al., 2017). Thestructure of TRPV1 in the closed and open configurations (Gao et al., 2016) hasbeen resolved and may provide a frame work to identify the unknown struc-ture(s) responsible of heat sensing in TRPV1 channels. Here, taking advantageof the known TRPV1 structures and using molecular simulations and the aniso-tropic thermal diffusion method (ATD), we evaluated the heat transfer path-ways which could be responsible to guide the thermal energy from theenvironment to the channels gates. ATD is a non-equilibrium molecular dy-namics simulation method that consists in introducing vibrational energy ineach amino acid of the channel-forming protein and then to measure its impactin the kinetic energy of the remaining residues (Ota, N. and D.A. Agard, 2005).Thus, the ATD technique allows us to map the heat diffusion pathways in the

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TRPV1 channel and identify the amino acid involved in the allosteric commu-nication between the TRPV1 channel structures absorbing energy from the sur-roundings and the channel gates. Here, we present a detailed analysis of theamino acids involved in well-defined thermal diffusion pathways, and weshow that most of the thermal pathways identified converge into the hydropho-bic gating ring formed by Ile679.

2379-Pos Board B395Role of Hydrophobic Solvation in TRPV1 Temperature SensitivityMarina Kasimova1, Aysenur Yazici2, Yevgen Yudin2, Tibor Rohacs2,Vincenzo Carnevale1.1Temple University, Philadelphia, PA, USA, 2Rutgers–New Jersey MedicalSchool, Newark, NJ, USA.TRPV1, a channel permeable to Ca2þ, plays crucial role in pain sensation. Themolecular details of TRPV1 gating, including its temperature dependence,remain largely unknown. Here we use a combined computational and experi-mental approach to shed light on the TRPV1 closed-to-open transition. Wefind that gating relies on the rotation of N676, an evolutionarily conserved res-idue on the S6 helix: N676 can either face the channel pore or the S4-S5 linker.Only in the former case, the channel pore is hydrated. When N676 rotates to-ward the linker, we observe hydration of four so far unreported cavities. Basedon our findings, we propose a model for TRPV1 gating involving dynamic hy-dration of these cavities. Free energy calculations indicate that this gatingmechanism is markedly temperature dependent. On the basis of this model,which rationalizes seemingly conflicting experimental observations, we predictand experimentally confirm the behavior of two mutants.

2380-Pos Board B396Rotational Brownian Motion of TRPV1 Channel Observed by Synchro-tron Diffracted X-Ray Tracking and Laboratory X-Ray Blinking AnalysisKazuhiro Mio1, Masahiro Kuramochi2, Ken Matsubara2, Keigo Ikezaki2,Muneyo Mio1, Hiroshi Sekiguchi3, Tai Kubo1, Yuji C. Sasaki2.1National Institute of Advanced Industrial Science and Technology, Tokyo,Japan, 2Graduate School of Frontier Sciences, The Univ. Tokyo, Chiba,Japan, 3Japan Synchrotron Radiation Research Institute, Hyogo, Japan.The TRPV1 is a nonselective cation channel that responds to various signalsincluding capsaicin, heat, and low pH condition. Recent progress in the cryoe-lectron microscopy depicted a structure of TRPV1 at near atomic resolution.However gating mechanisms of TRPV1 is not clearly understood because in-formation about state-to-state transition is still missing. To understand the dy-namics of TRPV1, we adopted the Diffracted X-ray Tracking (DXT) technique,in which individual protein was labeled with gold nanocrystals and the motionof X-ray diffraction spots from the gold crystal were investigated as intramo-lecular movement of TRPV1 in real time. We introduced ‘‘Met tag’’ for label-ing nanocrystal and ‘‘His tag’’ for substrate absorption. Purified protein wasimmobilized on the Ni-NTA coated polyimide substrates. Trajectories ofdiffraction spots from gold nanocrystal were analyzed in two axis views, tiltingand rotation directions. Capsaicin enhanced Brownian motion of outer helicesin dose dependent manner, as anisotropic rotation movement around the parti-cle. Heating with acidified condition also induce the movement. These motionswell reflect the gating properties of TRPV1 reported in electrophysiologicalstudies. We further succeeded motion detection of TRPV1 in living cells.The HEK293 cells stably expressing TRPV1 were cultured on Kapton sheetand the cell surface TRPV1 was labeled with gold nanocrystal. Usingin-house X-ray generator, diffraction spots from the gold-nanocrystal were re-corded. Autocorrelation of diffraction intensity in each pixel was calculated asan indicator reflecting molecular dynamics. We succeeded to detect the capsa-icin induced TRPV1 movement on the cell surface. Lag t of capsaicin treatedcells was shortened to that of untreated cells, suggesting capsaicin also en-hances rotational Brownian motion of TRPV1 in living cells.

2381-Pos Board B397A Bimodal Activation Mechanism Underlies Scorpion Toxin-Induced PainShilong Yang1, Fan Yang2,3, Bei Zhang4, Bo Hyun Lee5, Bowen Li1,Lei Luo1, Jie Zheng3, Ren Lai1.1Key Laboratory of Animal Models and Human Disease Mechanisms ofChinese Academy of Sciences/Key Laboratory of Bioactive Peptides ofYunnan Province, Kunming Institute of Zoology, Kunming, China,2Department of Pharmacology, School of Medicine, Zhejiang University,Hangzhou, China, 3Department of Physiology and Membrane Biology,University of California, Davis, Davis, CA, USA, 4School of PharmaceuticalSciences, Southern Medical University, Guangzhou, China, 5Department ofPhysiology and Membrane Biology, University of California, Davis,Hangzhou, China.Venomous animals use peptide toxins for hunting and self-defense. To achievethese goals, toxins need to bind to their targets with high affinity due to the

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small amount a single bite or sting can deliver. The scorpion toxin BmP01 islinked to sting-induced excruciating pain; however, the reported concentrationsfor activating TRPV1 channel or inhibiting Kv channels (both in mM range)appear too high to be biologically relevant. Here we show that the effectiveconcentration of BmP01 is highly pH-dependent—it increases by about 10-fold in inhibiting Kv channels upon a one-unit drop in pH, but decreasesover 100-fold in activating TRPV1. Mechanistic investigation revealed thatBmP01 binds to one of the two proton-binding sites on TRPV1 and, togetherwith proton, uses a one-two punch approach to strongly activate the nociceptivechannel. As most animal venoms are acidic, proton-facilitated synergistic ac-tion may represent a general strategy for intoxication.

2382-Pos Board B398The Gq-GPCR Pathway Evokes Tightly Controlled TRPV1 ActivationAdina T. Hazan.Pharmacology, Hebrew University of Jerusalem, Ein Kerem, Jerusalem,Israel.The detection and propagation of a pain signal must be coupled with the regu-lation and control of such a signal. TRPV1 is well established as a major painreceptor, capable of detecting a multitude of stimuli, including endogenous in-flammatory mediators such as arachidonic acid and anandamide, in addition toexogenous molecules such as the spicy chemical capsaicin. However, endoge-nous inflammatory mediators evoke noticeably weaker channel activation incomparison to exogenous molecules. Here, we employed pharmacologicaland molecular biology tools to dissect the regulated activation of TRPV1 inthe inflammatory response via the Gq/GPCR pathway. By employing specifictechniques such as perforated-patch recordings, the DREADD system, and cal-cium imaging of both neurons and a heterologous system, we worked to pre-serve the native intracellular cascades which are essential for Gq-mediatedTRPV1 activation. By doing so, we found that two downstream pathwaysconverge on TRPV1 to evoke a tightly regulated response. The first, the produc-tion of endo-vanilloids by lipoxygenases, are essential in producing the ligandthat activates TRPV1 through the intracellularly located vanilloid binding site.The second, channel sensitization through PKC phosphorylation, is necessaryfor the metabolites to successfully activate the channel. We propose thatthrough this dual cascade, a tightly regulated channel activation is achieved.Functionally, this regulation prevents the depolarization block associatedwith exogenous activation of TRPV1 by capsaicin, and ensures prolongedneuronal firing, as is found during nociceptor activation by inflammatory me-diators. Overall, we propose that the requirement of multiple signalling eventsmaintains a subdued TRPV1 activation in order to evoke regulated neuronalresponse during inflammation.

2383-Pos Board B399Activation of TRPV1 by Capsaicin Analogs with a Shorter or Longer NeckSimon Vu1, Vikrant Singh2, Fan Yang3,1, Heike Wulff2, Jie Zheng1.1Department of Physiology and Membrane Biology, University of California,Davis, Davis, CA, USA, 2Department of Pharmacology, University ofCalifornia, Davis, Davis, CA, USA, 3Department of Pharmacology, ZhejiangUniversity, Hangzhou, China.The TRPV1 ion channel, also known as the capsaicin receptor, is a polymodalnociceptor known for its activation by vanilloids. Recent advances in cryo-electron microscopy revealed the vanilloid-binding pocket in high resolution,allowing atomic interactions mediating capsaicin binding to be proposed. Inthe current model, capsaicin adopts a ‘‘tail-up, head-down’’ configuration,with key interactions allowing capsaicin to tether the S4-S5 linker to S4.Guided by this model, the vanilloid-insensitive TRPV2 channel was success-fully converted to be vanilloid-sensitive with minimal changes. A key questionconcerning the current model is whether an optimal distance between thecapsaicin-interacting residues on the S4-S5 linker and S4 (E571 and T551,respectively) is required to activate the channel. To directly address this ques-tion, we generated two capsaicin analogs, which differ in ‘‘neck’’ lengths:CAPþ1 refers to the addition of one CH2 moiety, and CAP-1 denotes theremoval of one CH2 moiety from the chain. These capsaicin analogs are foundto still activate TRPV1 at nM-to-uM concentrations. Activation by these com-pounds reveals new aspects of the channel which shed new light on its ligand-gating mechanism.

2384-Pos Board B400Expression and Purification of the Pain Receptor TRPV1 for Spectro-scopic AnalysisPhanindra Velisetty1, Richard A. Stein2, Francisco J. Sierra Valdez1,Valeria Vasquez1, Julio F. Cordero-Morales1.1Physiology, Health Science Center, University of Tennessee, Memphis, TN,USA, 2Molecular Physiology and Biophysics, Vanderbilt University MedicalCenter, Nashville, TN, USA.

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The transient receptor potential vanilloid 1 (TRPV1) channel is an essentialcomponent of the cellular mechanism through which noxious stimuli evokepain. Functional and structural characterizations of TRPV1 shed light on vanil-loid activation, yet the mechanisms for temperature and proton gating remainlargely unknown. Spectroscopic approaches are needed to understand themechanisms by which TRPV1 translates diverse stimuli into channel opening.Here, we have engineered a minimal cysteine-less rat TRPV1 construct(eTRPV1) that can be stably purified and reconstituted for spectroscopicstudies. Biophysical analyses of TRPV1 constructs reveal that the S5-pore helixloop influences protein stability and vanilloid and proton responses, but notthermal sensitivity. Cysteine mutants retain function and stability for doubleelectron-electron resonance (DEER) and electron paramagnetic resonance(EPR) spectroscopies. DEER measurements in the closed state demonstratethat eTRPV1 reports distances in the extracellular vestibule, equivalent to thoseobserved in the apo TRPV1 structure. EPR measurements show a distinctpattern of mobilities and spectral features, in detergent and liposomes, for res-idues at the pore domain that agree with their location in the TRPV1 structure.Our results set the stage for a systematic characterization of TRPV1 using spec-troscopic approaches to reveal conformational changes compatible with ther-mal- and ligand-dependent gating.

2385-Pos Board B401Cell Unroofing to Study the PI3K-TRPV1 InteractionGabriela Bergollo Drouyn, Anastasiia Stratiievska, Sharona Gordon.Physiology and Biophysics, University of Washington, Seattle, WA, USA.Transient receptor potential vanilloid 1 (TRPV1) channels play an essentialrole in the sensitized response to painful stimuli that occurs in response to in-flammatory mediators including nerve growth factor (NGF). Sensitization in-volves NGF activation of phosphoinositide 3-kinase (PI3K), which increasesthe concentration of the signaling lipid phosphatidylinositol (3,4,5)-trisphos-phate (PIP3). PIP3 then triggers exocytosis of TRPV1-containing vesicles,increasing the number of TRPV1 channels on the plasma membrane. Wehave previously shown that TRPV1 and PI3K interact directly through thechannel’s N-terminus and PI3K’s p85 subunit. However, little is known aboutthe dynamics behind the interaction and its consequences. Cell unroofing is aneffective method for accessing biological membranes from the inside. In cellunroofing, a probe sonicator is mounted on the microscope stage and positionedwithin about 1 mm of adherent cells. A single pulse of the sonicator shears offthe top of the cells, causing loss of organelles and cytosolic components, andleaving a plasma membrane sheet with its proteins and lipids intact. Becauseof the near-instantaneous disruption of cellular contents, this system is idealfor measuring dissociation rate of membrane-associated proteins. By labelingproteins of interest with fluorescent tags, we can measure the time course ofthe decay in fluorescence that occurs upon unroofing. We used PI3K fused toYFP and compared the dissociation rates in cells without TRPV1 to thosethat were transiently transfected with TRPV1. We quantified the fluorescentsignals and obtained the dissociation rates (koff) by fitting the data with an expo-nential decay model. These studies revealed that PI3K dissociation from theplasma membrane was slower in TRPV1-expressing cells than in those not ex-pressing TRPV1. We conclude that PI3K interacts with TRPV1 in resting cells.Whether the interaction is altered in NGF-stimulated cells is still an openquestion.

2386-Pos Board B402Reciprocal Regulation of PI3K by TRPV1 during InflammationAnastasiia Stratiievska1, Gabriela Bergollo1, Sara Nelson1,Sharona Gordon2.1University of Washington, Seattle, WA, USA, 2Physiology and Biophysics,University of Washington, Seattle, WA, USA.Inflammatory hyperalgesia is caused by sensitization of a pain-sensing ionchannel TRPV1, via PI 3-Kinase (PI3K)-dependent fusion of TRPV1-containing vesicles with the plasma membrane of sensory neurons. The aimof this study was to understand the signaling pathway leading to TRPV1-sensitization by the inflammatory mediator nerve growth factor (NGF). NGF,through its receptor TrkA, activates PI3K, which generates the signaling lipidphosphatidylinositol (3,4,5)-trisphosphate (PIP3). We used total internal reflec-tion fluorescence (TIRF) microscopy to measure changes in PIP3 via plasmamembrane translocation of the PIP3-sensitive probe Akt-PH, as an indicatorof PI3K activity. F11 cells were transfected with TrkA/p75 neurotrophin recep-tors and fluorescently-labeled Akt-PH and TRPV1. Upon application of NGF,we observed a rapid increase in membrane-associated Akt-PH fluorescence,followed by an increase in TRPV1-associated fluorescence. Interestingly,NGF-induced PI3K activity was significantly greater in TRPV1-expressingcells compared to cells not expressing TRPV1. This suggests that TRPV1potentiates NGF-induced PI3K activity. We have previously shown that the

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N-terminus of TRPV1, containing the Ankyrin repeat domain (ARD), physi-cally interacts with PI3K. This interaction could underlie potentiation by pre-associating PI3K with the membrane and/or via an allosteric effect on catalyticactivity. To test whether an allosteric mechanism could be involved, we ex-pressed the soluble N-terminal domain of TRPV1. We found that the N-termi-nal domain partially potentiated NGF-induced PI3K, suggesting allostericregulation is involved. Other TRPV, TRPA1, and TRPC channels also containan N-terminal ARD. To test whether the potentiation of NGF-induced PI3K ac-tivity generalized to other ARD-containing channels, we examined the effect ofNGF in TRPV2- and TRPV4-expressing cells. We found that TRPV2 andTRPV4 channels also potentiated NGF-induced PI3K activity, whereas non-ARD-containing channels TRPM8 and TRPM4 did not. Together, these datademonstrate a novel reciprocal regulation of PI3K signaling by the ARD ofTRPV channels.

2387-Pos Board B403Binding of Capsaicin Slows Down Proton-Induced TRPV1 ActivationBo Hyun Lee, Jie Zheng.Physiology & Membrane Biology, University of California, Davis, Davis,CA, USA.The capsaicin receptor TRPV1 is a well-known nociceptor; its activationby protons contributes to inflammation pain. Capsaicin binds to a transmem-brane pocket near the S4-S5 linker. Current working model of capsaicinbinding indicates that capsaicin stabilizes the S4-S5 linker towards S4,causing an outward movement of S6 to open the activation gate. Extracellularprotons bind to outer pore acidic residues to promote activation, however,the underlying molecular mechanism has yet to be fully understood. Usinga rapid solution-switching method, we measured the ON and OFF rates ofmTRPV1 currents induced by low pH solutions in the absence or presenceof a low concentration of capsaicin (30 nM, about four times lower thanEC50). Interestingly, while the proton-induced current exhibited an antici-pated increase in the presence of capsaicin, the current ON rate was substan-tially decreased. This unexpected phenomenon cannot be explained by asimple allosteric coupling of these two activators. Instead, it points to anintriguing possibility that protons and capsaicin open TRPV1 into distinctconformations; furthermore, it suggests that the conformational rearrange-ments induced by capsaicin are not limited to the lower part of transmembranedomain.

2388-Pos Board B404Functional Analysis of TRPV1 Channels with a Genetically EncodedCross-LinkerDeny Cabezas-Bratesco1,2, Christopher A. Ahern2, Sebastian Brauchi1.1Physiology Department, Austral University of Chile, Valdivia, Chile,2Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA,USA.TRP channels constitute a family of non-selective, polymodal cation channels.They can sense different nature stimulus, i.e. chemical, electric, thermic andmechanic. The members of this family are homologous to the voltage-gatedcation channels superfamily. Consequently, TRP channels have a tetramericstructure, composed by four monomers with six transmembrane domains(TMD) each, and substantial N- and C-terminal intracellular domains. TheS1-S4 TMD region is considered the ligand binding domain, and the poreof the channel is comprised by the S5-S6 TMD and a pore loop betweenthem. High-resolution structures are now available from multiple membersof the TRPV subfamily. These structures reveal rich interaction networks be-tween the N- and C-terminal domains, the intracellular loops and the poreforming helixes. For instance, the TRP domain, which is formed by anextended alpha-helix from the sixth TMD, is localized in the core of thisinteraction network, thus serving as a putative signaling integrator betweenmodulatory signals and the channels gating mechanism(s). In order gathersite- and conformation-specific information on TRPV1 function, we appliednonsense suppression to encode the non-canonical amino acid p-Benzoylphe-nylalanine (BPA) into conserved aromatic sites into TRPV1 in HEK cells.This aromatic amino acid is useful for functional perturbation analysis studiesand in voltage-clamp UV dependent crosslinking assays. Specific encoding ofBPA was achieved with an evolved E. coli. tyrosine synthetase and tRNA.Full length channel rescue at introduced TAG sites in the presence of BPAwas first demonstrated by western blot of cell lysates. Functional expressionwas confirmed with whole-cell voltage clamp of TRPV1 channels inHEK293 cells, demonstrating that BPA containing channels could be gatedby temperature and/or capsaicin induced activation. These data set the stagefor site-specific cross-linking and the potential capture of transient gatingstates.

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2389-Pos Board B405Regulation of TRPV1 Expression in Non-Neuronal Tissues by BDNF, Sp1,and Sp4Rebecca Brenner, Padmamalini Baskaran, Baskaran Thyagarajan.University of Wyoming, Laramie, WY, USA.Transient receptor potential vanilloid subfamily 1 (TRPV1), popularly knownas the capsaicin receptor is an emerging target for treating pain, degenerativeand metabolic diseases. The expression of TRPV1 in non-neuronal tissueshas been recognized in the recent years. Although the regulation of its expres-sion by transcription factors like Sp1 and Sp4 and neurotrophic factors likeBDNF have been extensively studied in neuronal tissues, the current knowl-edge on the expression regulation of TRPV1 in non-neuronal tissues is verylimited. Here, we present novel data suggesting the role of adipose tissuederived neurotrophic factors (ATDNF) and Sp1 and Sp4 in the regulation ofTRPV1. TRPV1 protein is endogenously expressed in inguinal and brown fatpads of wild type mice. High fat diet (HFD) suppressed the expression and ac-tivity of TRPV1 in these tissues. However, feeding HFD þ capsaicin (TRPV1agonist) countered this. Analyses of mechanisms underlying the inhibition ofTRPV1 expression by HFD revealed that adipose tissues express transcriptionfactors Sp1 and Sp4 and brain derived neurotrophic factor (BDNF), whichpotentially regulate TRPV1 expression. HFD suppressed the expression ofSp1, Sp4 and BDNF to inhibit TRPV1 expression, while capsaicin reversedthis. We present a novel regulatory role of ATDNF in the regulation ofTRPV1 expression in adipocytes.

2390-Pos Board B406Chalcones Derivatives as Potent Inhibitors of TRPV1 ActivityBruna Benso1, Carolyne Lespay-Rebolledo2, Lisandra Flores1,Miguel Zarraga2, Sebastian Brauchi1.1Physiology Department, Universidad Austral de Chile, Valdivia, Chile,2Department of Organic Chemistry, University of Concepcion, Concepcion,Chile.TRPV1 receptors are allosterically gated, calcium permeable ion channels, sup-porting the detection of noxious and nociceptive input. We have synthesizedchalcones derivatives and studied their effect on TRPV1 receptors expressedin HEK-293T cells. The potency of inhibitor compounds was examined by theirability to capsaicin response in a calcium-imaging assay and outward currentsby whole-cell patch clamp technique. For a set of derivatives, we observed astrong antagonistic activity at picomolar concentrations and in a lower IC50levels compared to the BCTC control. Moreover, we found that chalcones de-rivatives are able to inhibit the temperature-dependent activation of thecapsaicin-insensitive cTRPV1, suggesting a different mechanism of interactionwhen compared with capsaicin competitive inhibitors. By performing an elec-trostatic field-based pharmacophore model, we inferred the channel-drug inter-action. Our study suggest that the antagonistic potency of chalcones is definedby the stabilization of an hydrogen bond network, together with the conjugationof the alpha, beta unsaturated carbonyl.

2391-Pos Board B407Mechanisms by which Botulinum Neurotoxin A Suppresses PainBaskaran Thyagarajan1, Louis Premkumar2, Padmamalini Baskaran1.1School of Pharmacy, University of Wyoming, Laramie, WY, USA, 2Dept. ofPharmacology, Southern Illinois University, Springfield, IL, USA.Botulinum neurotoxin A (BTX---A) is used for the treatment of several neuro-logical disorders and pain. BTX---A injections have been shown to be highlybeneficial for the treatment of neuralgia, joint pain, etc. in clinics. Nonetheless,the mechanism by which BTX---A inhibits pain still remains to be elusive.Here, we show that nociceptive transient receptor potential proteins may serveas potential targets for BTX---A as these proteins are integrators of noxiousstimuli and are regarded as crossroads for pain signaling. BTX---A (1 nM) in-jection significantly decreased formalin---mediated flinches, licking and pawlifting frequencies and suppressed carrageenan-mediated inflammation andpaw edema compared to controls. Further, acute treatment of BTX---A, in vitro,inhibited capsaicin (1 mM)--- stimulated Ca2þ influx and currents into HEK293cells that stably express TRPV1 (HEKTRPV1). Acute 90 min. treatment orchronic overnight treatment of BTX---A decreased TRPV1 expression inHEKTRPV1 cells. This was associated with a decrease in PKC---alpha expres-sion. These data suggesting the involvement of PKC in BTX---A mediated ef-fects. BTX-A also inhibited phorbol myristate acetate induced sensitization ofTRPV1. BTX-A pretreatment also inhibited formaldehyde-induced Ca2þ influxvia TRPA1 in vitro. Also, exposure to intracellular surface of the plasma mem-brane of commercially available botox stongly inhibits voltage-gated sodiumchannels (VGSC) and voltage-gated calcium channels (VGCC) in dissociatedrat DRG neurons. Collectively, our data show that BTX---A inhibits pain bysuppressing TRP expression/activity and by blocking VGSC and VGCC.

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2392-Pos Board B408Sub-Chronic Oral Safety Analysis of MetabocinTM

Padmamalini Baskaran, Laurel Markert, Liesl Zimmerman, Jane Bennis,Baskaran Thyagarajan.School of Pharmacy, University of Wyoming, Laramie, WY, USA.MetabocinTM (also known as capsaicin) activates TRPV1 expressed on themembranes of white adipocytes to cause browning of white adipocytes.Although, our sub-chronic MetabocinTM -feeding neither altered the energyintake nor any adverse reactions in mice, it is essential to demonstrate the safetyof MetabocinTM in preclinical toxicological studies in mice. Keeping this goalin mind, we conducted a dose response for MetabocinTM (0.133, 0.399, 0.665,1.33 or 3.99 mg/kg body weight of mouse corresponding to 0.001, 0.003, 0.005,0.01 and 0.03% of MetabocinTM in diet) to counter obesity in high fat diet-fedwild type mice. We conducted histopathological studies and analyzed theplasma levels of markers metabolic, liver and kidney functions. To ensurethat MetabocinTM does not cause adverse reactions when in normal chowdiet (NCD), we fed wild type mice a diet containing 0.01% MetabocinTM inNCD. Our data show that MetabocinTM inhibited diet-induced obesity at con-centration above 0.001% (0.133 mg/kg body weight) and the theoretical EC50of MetabocinTM is determined to 0.00157% (0.2881 mg/kg body weight). Me-tabocinTM did not alter food/water intake in mice in any of the concentrationsused. HFD feeding increased fasting plasma glucose, triglycerides and choles-terol levels and significantly elevated serum alanine aminotransferase andcreatinine levels, and MetabocinTM antagonized this. MetabocinTM counteredhypertension associated with obesity at all concentrations except 0.003%.Feeding MetabocinTM in NCD did not cause weight loss in mice. Analysesof histopathological sections revealed no pathological findings when micewere fed MetabocinTM either with NCD or HFD. Collectively, our study pro-vides compelling preclinical data that suggest that sub-chronic oral Metabo-cinTM feeding is safe and does not cause any adverse effects in mice. Thesedata are valuable for advancing the clinical uses of MetabocinTM P to counterobesity in humans.

2393-Pos Board B409TRPV2 is Crucial for the Development of Excitation-ContractionCoupling in Neonatal CardiomyocytesYuki Katanosaka1, Yoshihiro Ujihara2, Yumiko Chiba1, Satoshi Mohri2,Keiji Naruse1.1Cardiovasc. Physiol., Okayama Univ, Okayama, Japan, 2Physiology,Kawasaki Medical school, Kurashiki, Japan.We have previously reported that TRP vanilloid family type 2 channel(TRPV2) is a key molecule in stretch-induced Ca2þ response of cardiomyo-cytes. In this study, we aimed to understand the roles of TRPV2 in the devel-opment of excitation-contraction (E-C) coupling of cardiomyocytes andstructural organization of intercalated discs between neighboring myocytes.We examined the mechanical stimulation-induced Ca2þ response in neonatalcardiomyocytes isolated from normal (control) and TRPV2-deficient mice. Inneonatal cardiomyocytes isolated from control mice, hypotonicity- andstretch-induced Ca2þ responses were observed within 12 hours of culture afterenzymatic isolation. These responses were decreased by tranilast, an inhibitorof TRPV2, suggesting involvement of TRPV2 channel. At this stage of thecultured cardiomyocytes, robust expression of TRPV2 was observed. After48 hours, formation of intercalated discs and reorganization of myofibrils grad-ually progressed. After 72 hours, autogenic Ca2þ oscillation and beating wereobserved, indicating appropriate Ca2þ-handling for E-C coupling. In contrast,TRPV2-deficient myocytes showed no Ca2þ response to the hypotonicity-and stretch-stimulation. These myocytes neither formed intercalated discs be-tween neighbouring cells nor highly organized sarcomeric assembly of actin.These myocytes showed aberrant intracytoplasmic accumulation of connexin43, internally defused N-cadherin, and reduced expression of Na2þ/Ca2þ

exchanger 1, NCX1. Even after 72 hours, Ca2þ handling for E-C couplingwas not observed in TRPV2-deficient myocytes. Their beating was spontane-ously elicited, but weak and asynchronous. Taken together, TRPV2 has apivotal role in mechanical stimulation-induced Ca2þ response, and in structuraland functional development of cardiomyocytes that enable E-C coupling andsynchronous contraction with neighboring cells.

2394-Pos Board B410Molecular Mechanism of TRPV2 Channel Pore Dynamics during LigandActivationAmrita Samanta1, Yuhang Liu2, Franklin Mayca Pozo3, George R. Dubyak1,Taylor E.T. Hughes4, Seungil Han5, David T. Lodowski6,Vera Y. Moiseenkova-Bell3.1Physiology and Biophysics, Case Western Reserve University, Cleveland,OH, USA, 2Pfizer Research and Development, Groton, CT, USA,

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3Department of Pharmacology, Case Western Reserve University, Cleveland,OH, USA, 4Department of Pharmacology, Case Western Reserve University,Cleveland, OH, USA, 5Pfizer Research and Development, Case WesternReserve University, Cleveland, OH, USA, 6Department of Nutrition, CaseWestern Reserve University, Cleveland, OH, USA.The transient receptor potential (TRP) superfamily, one of the largest familiesof the non-selective cation channels, is subdivided into six major branches:TRPV, TRPC, TRPM, TRPA, TRPP, and TRPML. The TRPV subfamily con-tains six unique members, four non-selective cation channels (TRPV1-TRPV4)and two highly Ca2þ selective channels (TRPV5 and TRPV6).A hallmark of these polymodal TRP channels is their exceptionally high perme-ability to large organic cations in response to prolonged ligand stimulation. Thepore dilation phenomenon has been proposed to play a role in this process.Although cryo-electron microscopy structures of various TRP channels havebeen resolved in both apo and ligand bound states, the structural basis of thisuptake is unknown due to unavailability of the full-length TRP structures inligand-activated states.Recent cryo-electron microscopy (cryo-EM) structure of full-length rat TRPV2in the apo state revealed details about TRPV2 channel architecture, but did notelucidate aspects of channel activation and gating. We have determined aligand-activated TRPV2 structure, which displayed a distinct channel gatingmechanism that involves rearrangement of the S5 helix, pore turret and selec-tivity filter domains. Together these results provide foundation to further under-standing TRP channels large organic cations’ permeation, their gatingproperties and divergent physiological functions.

2395-Pos Board B411Evolutionary Variations in HLH Domain Modulate the Fast InactivationPhase in Calcium Selective TRP ChannelsLisandra Flores Aldama1,2, Daniel Bustos3, Juan G. Opazo1,Wendy Gonzalez3, Sebastian Brauchi1,2.1Universidad Austral de Chile, Valdivia, Chile, 2CISNE, Valdivia, Chile,3Universidad de Talca, Talca, Chile.TRPV5 and TRPV6 are highly calcium-selective channels from the Tran-sient Receptor Potential (TRP) family. These channels are considered gate-keepers of epithelial calcium transport, essential for calcium homeostasis.At negative potentials, the channels exhibit a two-phase calcium-dependentinactivation where the slow component is shared and determined by thebinding of Ca2þ-Calmodulin complex to the C-terminal region of the chan-nel. In contrast, the rapid phase of inactivation depends on the binding ofcalcium ions and allows differentiating both channels from a functionalpoint of view; while TRPV6 shows a very robust inactivation, at the samecalcium concentrations, the inactivation of TRPV5 conductance is modest.The intracellular loop S2-S3 and residues downstream the transmembranesegment S6 has been associated to the differences observed in the kineticsof the rapid phase of inactivation. However, the exact location of the puta-tive calcium-binding site and the molecular mechanism governing this pro-cess are not known. A thorough phylogenetic reconstruction in vertebratessuggest that the genes encoding for these channels duplicates more thanonce during evolution, naturally introducing the same set of mutationswithin a HLH domain located at the N terminal region. Further sequenceanalysis unveiled that the HLH domain acts as a fingerprinting in both chan-nels. Molecular dynamics simulations, allowed to identify a putativecalcium-binding site that put together three different portions of the foldedchannel. By means of site-directed mutagenesis and patch clamp electro-physiology we reversed the phenotype of inactivation in these channels,confirming that the HLH sequence serve as modulator for the calcium-induced inactivation process. We conclude that subtle evolutionary-relatedvariations within the binding region affect the phenotype of the fast inacti-vation phase.

2396-Pos Board B412Selective Inhibition of Thermosensitive TRPV3 Channel by NaturalCoumarin Osthole for Itch ReliefXiaoying Sun, Lilan Sun, Ningning Wei, Kewei Wang.Qingdao University, Qingdao, China.Natural coumarin osthole is a dominant bioactive ingredient of Cnidium mon-nieri plant that is widely used as traditional Chinese medicine for remedies suchas antipruritus and antidermatitis. However, the underlying molecular mecha-nism of action for osthole remains unknown. In this study we show that ostholeexhibits antipruritic effect by selective inhibition of thermosensitive and Ca2þ-permeable TRPV3 channels primarily expressed in the keratinocytes of theskin. We utilized the calcium fluorescent assay in FlexStation 3 96-well formatand identified coumarin osthole as a selective inhibitor of TRPV3 channelsexpressed in HEK293 cells or HaCaT keratinocytes. Whole-cell patch clamp

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recordings further confirmed that osthole selectively inhibited TRPV3 currentactivated by TRP channel agonist 2-APB (50 mM) in dose-dependent mannerwith an IC50 value of 34.1 5 0.7 mM. Behavioral evaluations revealed thatinhibition of TRPV3 by osthole or knockout of TRPV3 gene significantlyattenuated mouse scratching induced by either acetone-ether-water (AEW)or histamine in localized rostral neck skin. Taken together, our findingsidentify osthole as a relatively selective blocker of TRPV3, demonstrating acritical role of TRPV3 in chronic itch and also providing a molecular basisfor use of coumarin osthole from C. monnieri in anti-pruritic therapy or skincare.

2397-Pos Board B413Targeting Inflammation in Post-Operative Atrial Fibrillation: the Role ofTRPV4 in Augment Neutrophil Infiltration via Up-Regulation IL6 andCXCL2Shao-Shao Zhang1, Qiong-Feng Wu1, Chen Qian1, Huixia Liu2,Bin-bin Wang1, Jie Liao1, Lei Chen3, Yi-Mei Du1.1Department of Cardiology, Union Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan, China; ResearchCenter for IonChannelopathy, Union Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan, China; Key Labfor Biological Targeted Therapy of Education Ministry and Hubei Province,Union Hospital, Tongji Medical College, Huazhong University of Scienceand Technology, Wuhan, China, 2Medical College of Xiamen University,Xiamen, Fujian, China, 3Department of Physiology, Nanjing MedicalUniversity, Nanjing, China.Post-operative atrial fibrillation (AF) is a common complication occurringafter cardiac surgery and is close related with inflammation and fibrosis.Transient receptor potential vanilloid 4 (TRPV4) is a Ca2þ-permeablecation channel, highly expressed in heart and its activation promotes inflam-mation and fibrosis in a variety tissues. The current study is to investigatewhether TRPV4 activation contributes to POAF via aggravating inflamma-tion. Sterile pericarditis (SP) was induced in mice by the epicardial applica-tion of sterile talc. AF was induced by transesophageal burst pacing. Themice with SP showed a higher susceptibility to developing AF comparedto the sham-operated mice, with an increased duration and probability ofAF, as well as marked neutrophil infiltration and fibrosis in atria. Further-more, all above effects were significant reduced after treatment with aspecific TRPV4 antagonist GSK2193874 or TRPV4 knockout, but furtherenhanced by a specific TRPV4 agonist GSK1016790A. In vitro, functionalexpression of TRPV4 was detected in isolated mouse neutrophils. However,isolated neutrophils from sham, SP, treatment with GSK2193874 orGSK1016790A did not show any different in migration potency. Moreover,an enhanced neutrophil migration was observed in the presence of atrialhomogenate from SP mice, which was reversed by GSK2193874 but wasfurther aggravated by GSK1016790A. In addition, the mRNA expressionof inflammatory cytokines and all CXCL chemokines in atria were screenedand the results showed that IL-6 and CXCL-2 play obvious roles. Inconclusion, our results suggested that TRPV4 activation increasesneutrophil infiltration via up-regulation of IL-6 and CXCL-2 and maycontribute to post-operative AF in SP mice, but further studies are requiredto investigate the molecular pathways and a major source of IL-6 andCXCL-2.

2398-Pos Board B414Blockade of Transient Receptor Potential Vanilloid 4 Enhances Antioxida-tive Activity and Attenuates Hypoxia/Reoxygenation Injury in Cardiomyo-cytes: Involvement of Akt/Nrf2/AREQiong-Feng Wu1, Qian Dong1, Shao-Shao Zhang1, Bin-Bin Wang1,Jie Liao1, Lei Chen2, Yi-Mei Du1.1Department of Cardiology, Research Center of Ion Channelopathy, Instituteof cardiology, Key lab for biological targeted therapy of Education Ministryand Hubei Province, Union Hospital, Tongji Medical College, HuazhongUniversity of Science and Technology, Wuhan, Hubei, Wuhan, China,2Department of Physiology, Nanjing Medical University, the LaboratoryCenter for Basic Medical Sciences, Nanjing, China.Antioxidative stress provided the cardioprotective effects in myocardialischemia/reperfusion (I/R). Transient receptor potential vanilloid 4(TRPV4) is a Ca2þ-permeable nonselective cation channel and widely ex-pressed in the cardiovascular system. We previously demonstratedblockage of TRPV4 attenuates hypoxia/reoxygenation (H/R) inducesCa2þ influx and decreases reactive oxygen species (ROS) release, andfinally alleviates injury in cardiomyocytes. The current study focused onwhether blockade of TRPV4 reduced the injury via the antioxidative activ-ity during H/R and explored the molecular mechanisms in cardiomyocytes.

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Our results reveal that H/R induced cell injury as evidenced by the cellviability, the release of lactate dehydrogenase (LDH) and apoptosis, whichwere obviously alleviated by a selective TRPV4 blocker HC-067047 orspecific TRPV4-siRNA. Moreover, H/R also increased the contents ofROS and malondialdehyde (MDA) and decreased the activity of superoxidedismutase (SOD) and glutathione peroxidase (GSH-Px), which werereversed by HC-067047 or specific TRPV4-siRNA. Furthermore,HC-067047 treatment increased the expression of P-Akt, and the transloca-tion of nuclear factor E2-related factor 2 (Nrf2) and related antioxidantresponse element (ARE) mainly including SOD, GSH-Px andhemeoxygenase-1(HO-1) after H/R. In addition, the Akt inhibitor,LY294002, absolutely hampered these cardioprotective effects of HC-067047. Finally, we confirmed the antioxidative stress roles of blockadeof TRPV4 in myocardial I/R or application of exogenous H2O2. In conclu-sion, inhibition of TRPV4 exerts protective effects against oxidative stressinduced cardiomyocyte injury possibly related to the up-regulated Akt/Nrf2/ARE pathway.

2399-Pos Board B415TRPV4 Channel is an Osmosensor and Mechanosensor in ProximalTubule CellsRoberta Gualdani, Francois Seghers, Xavier Yerna, Philippe Gailly.Institute of Neuroscience, Laboratory of Cell Physiology, Universit�eCatholique de Louvain, Brussels, Belgium.The proximal tubule (PT) is the first segment of the nephron and accounts forreabsorption of water and solutes filtered by the glomerulus. In addition, theproximal tubule plays a pivotal role in the endocytosis to reabsorb andmetabolize proteins and other substances in glomerular filtrates. Recentstudies showed that the PT functions can be modulated by mechanical forces,namely the shear stress on the apical surface and the radial stretch on tubularlumen. Since TRPV4 channel is mainly expressed at the basolateral side ofthe tubule, we hypothesized that TRPV4 could play a role as stretch-activated channel rather than a shear stress sensor. Patch clamp and Ca2þ im-aging experiments showed that TRPV4 channel is expressed in culturedproximal tubule cells and can be activated by the specific agonistGSK1016790A and by hypotonic solution. Moreover, mechanical stretchinduced by uniaxial strain in silicone stretchable chambers activated a largeCa2þ influx that was significantly reduced in Trpv4-/- PT cells. Notably, noTRPV4-dependent Ca2þ entry was observed in response to flow-inducedcell deformation. Furthermore, we observed that the uptake of FITC-labeled albumin in PT cells, treated with GSK1016790A or mechanicallystimulated in stretchable chambers, was significantly reduced in Trpv4-/-

PT cells. Finally, we injected angiotensin II in mice, using osmotic mini-pumps, in order to challenge PT endocytotic activity and increase theproteinuria of mice. Our data showed a massive increase in diuresis ofmice. Interestingly, we observed an increase in albuminuria in wild-typemice, and this effect was significantly higher in Trpv4-/- mice. In contrast,glucose and phosphate urinary levels, which undergo PT reabsorption aswell, were not significantly affected. Taken together, these results supportthe view that TRPV4 channel is a mechanosensor in PT cells and modulatestheir endocytotic activity.

2400-Pos Board B416Molecular Dynamics Insights into TRPV5 Channel Inhibition by SmallMoleculesAbhijeet Kapoor1, Taylor E.T. Hughes2, David T. Lodowski3,Vera Y. Moiseenkova-Bell2, Marta Filizola1.1Department of Pharmacological Sciences, Icahn School of Medicine atMount Sinai, New York, NY, USA, 2Department of Systems Pharmacologyand Translational Therapeutics, Perelman School of Medicine, University ofPennsylvania, Philadelphia, PA, USA, 3Department of Pharmacology andDepartment of Nutrition, School of Medicine, Case Western ReserveUniversity, Cleveland, OH, USA.The transient receptor potential vanilloid 5 (TRPV5) channel is a calcium-selective renal protein that is directly involved in calcium reabsorption, andis therefore a potential target for human disorders with altered calcium homeo-stasis such as nephrolithiasis or kidney stone disease. An atomic-level under-standing of the molecular mechanisms underlying TRPV5 gating byexogenous or endogenous modulators has thus far been hampered by thelack of TRPV5 experimental structures. Here, we use a recently determinedcryo-electron microscopy (cryo-EM) structure of the TRPV5 tetramer boundto the small molecule antifungal inhibitor econazole as input to all-atommolec-ular dynamics (MD) simulations in a membrane-mimetic environment to gainfirst dynamics insights into TRPV5 channel inhibition by small molecules. Ourresults suggest high flexibility of the small-molecule econazole, which adopts

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different poses within the four binding pockets in the TRPV5 tetramer. Thesepockets are formed by residues of the S3 helix, the S4 helix, and the S4-S5linker region on one protomer, and the S6 helix on an adjacent one. As alsoseen in published simulations of a related channel, specifically TRPV1 boundto either antagonists or agonists, the four protomers of the TRPV5 tetramershow different dynamic behavior. This is particularly reflected in the differentcalcium coordination by selectivity filter residues on each protomer, as well asdifferent inter-residue distances within the lower gate region of econazole-bound TRPV5. Taken together, our preliminary molecular dynamics data pro-vide support to the putative role of specific residues in TRPV5 channel inhibi-tion inferred by the static cryo-EM structure.

Posters: Ion Channels, Pharmacology, andDisease II

2401-Pos Board B417Structural Modeling of hERG Channel Interactions with Drugs usingRosettaAiyana M. Emigh1,2, Kevin R. DeMarco1,2, Kazuharu Furutani2,Slava Bekker2,3, Jon T. Sack2, Colleen E. Clancy2,4, Igor Vorobyov2,4,Vladimir Yarov-Yarovoy2.1Biophysics Graduate Group, University of California Davis, Davis, CA,USA, 2Department of Physiology and Membrane Biology, University ofCalifornia Davis, Davis, CA, USA, 3Hartnell College, Salinas, CA, USA,4Department of Pharmacology, University of California Davis, Davis, CA,USA.The potassium-selective channel Kv11.1, encoded by the human Ether-a-go-go-Related Gene (hERG), mediates a major repolarization current (IKr) of thecardiac action potential. A major problem in the development of new pharma-ceuticals is that blockade of the channel by drugs can cause fatal cardiac ar-rhythmias. However, not all hERG blockers are pro-arrhythmic, and wehypothesize that the state-dependence of drug block may impact cardiac safety.In this study, a panel of 11 drugs was selected as a representative set based ontheir cardiac risk, inhibitory potency, and other characteristics of hERG block.We used Rosetta computational modeling software to build structural modelsof hERG in open and closed states based on cryo-EM structures of hERG(PDB ID: 5VA2) and the related EAG1 channel (PDB ID: 5K7L), respectively.Models were also built with mutations of the pore inner vestibule, F656A andY652A, which influence the efficacy of many hERG blockers. The selectedpanel of drugs was docked to these models using RosettaLigand. Our resultsprovide structural hypotheses concerning the state dependence of drug bindingto hERG. These results could help elucidate molecular interactions that play akey role in differentiating pro-arrhythmic from cardiac-safe hERG blockers.

2402-Pos Board B418Subunit Organization of K2P ChannelsGuillaume Sandoz.iBV, Nice, France.Heteromultimerization is a mechanism commonly used to increase the func-tional diversity of protein complexes. For example, with 15 genes, the K2P

channel family can potentially generate 120 combinations and, in theory,each of them would show different functional properties. Members of theTREK channel family and, more generally, K2P channels are colocalized inthe same brain region which raises the question: do these channels heteromerizeand what are the functions of the different heteromers? Despite the fact that K2P

channels show low sequence identity between members of the same subfamily(which is even lower between subfamilies) heteromerization of different K2P

channels have been postulated. The question of heteromerization in this chan-nel family has been explored for more than 10 years with the first description ofa K2P heteromer coming in 2002 (TASK1 and TASK3). We recently showedthat TREK1, TREK2 and TRAAK can functionally heterodimerize. Here, usingthe recently-developed single-molecule pull-down (‘‘SiMPull’’) assay com-bined with optogenetic, we have determined the ability of several K2P subunitsto physically and functionally heteromerize with each other and study the phys-iological and physiopathological impact of these heteromerizations.

2403-Pos Board B419Inhibition of NPC1 Increases Neuronal Excitability by Changing Phos-phoinositide LevelsOscar Vivas, Scott Tiscione, Eamonn Dickson.Physiology and Membrane Biology, University of California, Davis, CA,USA.NPC1 is a transmembrane protein localized to the lysosomal membrane. Itsmain role is to transport cholesterol from the lysosome to the ER atlysosome-ER contact sites. Mutations in this protein lead to accumulation of

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cholesterol in lysosomes and to the onset of a fatal neurodegenerative diseaseknown as Niemann-Pick. The mechanisms by which alterations in cholesterolmetabolism cause neuronal dysfunction are still not known. We tested the hy-pothesis that inhibition of NPC1 increases neuronal excitability by altering ionchannel activity. Using super-resolution microscopy, we found that overnightincubation with U18666A, a NPC1 inhibitor, caused the accumulation ofcholesterol in lysosomes of both sympathetic and hippocampal neurons. Thesame treatment rendered these neurons more excitable: i) the resting membranepotential was more depolarized, ii) the amount of current necessary to elicit ac-tion potentials was lower, and iii) the frequency of action potentials when neu-rons were stimulated with a sustained pulse was higher. These three hallmarksof increased excitability led us to hypothesize that the inhibition of NPC1 re-duces the activity of KV7.2/7.3 (KCNQ2/3) channel current. Using voltageclamp, we found a reduced current density of KV7.2/7.3, in agreement withour hypothesis. The mechanism underlying the decrease in KV7.2/7.3 channelcurrents involves the concurrent reduction in plasma membrane phosphatidyli-nositol 4,5-bisphosphate (PI(4,5)P2), a signaling lipid essential for the functionof KV7.2/7.3 and many other ion channels. Mass spectrometry and live cell im-aging of phosphoinositide levels revealed that PI and PI(4)P, precursors ofPI(4,5)P2, are also dysregulated. We propose that, in addition to its known ef-fects on cholesterol metabolism, mutations in or inhibition of NPC1 alterplasma membrane phosphoinositide levels. Thus, the regulated exchange ofcholesterol at lysosome-ER contact sites, indirectly controls plasma membranePI(4,5)P2, and as a consequence ion channel function.

2404-Pos Board B420Molecular Determinants of Steroid Hormone and Drug Induced Arrhyth-mogenesis via hERG Channel BlockIgor Vorobyov1,2, Brandon M. Brown2, Kevin R. DeMarco1,3,Sergei Y. Noskov4, Vladimir Yarov-Yarovoy1, Heike Wulff2,Colleen E. Clancy1,2.1Department of Physiology and Membrane Biology, University of California,Davis, Davis, CA, USA, 2Department of Pharmacology, University ofCalifornia, Davis, Davis, CA, USA, 3Biophysics Graduate Group, Universityof California, Davis, Davis, CA, USA, 4Centre for Molecular Simulations,Department of Biological Sciences, University of Calgary, Calgary, AB,Canada.Heart rhythm disturbances, such as long QT syndrome (LQTS), have beenlinked to mutations in cardiac ions channels, as well as unintended drug inter-actions with these channels. Female sex has been shown to be an independentrisk factor for both inherited and acquired LQTS. Previous experimental studiesand our recent multi-scale modeling (P.-C. Yang et al J. Physiol. 2017, 595:4695) demonstrated that this phenomenon is likely related to differential levelsof sex hormones (estradiol, progesterone and testosterone) playing oppositeroles in pro-arrhythmia proclivities, exacerbating or mitigating effects of ionchannel mutations or drug-induced blockade. In this study, we are focusingon hormone interactions with the human ether-a-go-go (hERG or Kv11.1) po-tassium channel, a major contributor to cardiac action potential repolarizationand an anti-target for diverse drug molecules. Our preliminary electrophysio-logical measurements indicate channel-specific 15% Kv11.1 current blockadeat physiological, low nM estradiol concentrations, and its combined additive ef-fect with a potent hERG blocker, E-4031. We will extend these experiments toseveral combinations of steroid hormones and drugs with proclivity for sex-dependent arrhythmogenesis. We will use a recently published hERG structure(PDB id: 5VA1) and perform RosettaLigand molecular docking and atomisticmolecular dynamics simulations to correlate experimental findings with spe-cific ligand - channel interactions. Also, we will test if hormone and drug bind-ing in the hERG cavity can synergistically increase their channel blockingaffinities, as was suggested by our previous study. This methodology can beused for in silico assessment of drug cardiotoxicities and development of car-diac safe pharmaceuticals. Supported by National Institutes of Health NHLBIR01HL128537-02 and U01HL126273-02 grants as well as American Heart As-sociation Pre-doctoral Fellowship (16PRE27260295), Western States Affiliate(to KRD).

2405-Pos Board B421Increased Pannexin 1 Expression and Activity in Ventricle of MdxDystrophic HeartsFrank J. Raucci, Jr.1, Kyunsoo Kim2, Sabine Huke3, Bjorn C. Knollmann2.1Pediatric Cardiology, Vanderbilt University Medical Center, Nashville, TN,USA, 2Clinical Pharmacology, Vanderbilt University Medical Center,Nashville, TN, USA, 3Cardiovascular Disease, University of AlabamaBirmingham, Birmingham, AL, USA.Duchenne muscular dystrophy is an X-linked disorder of sarcolemmaldystrophin, characterized by weakness of skeletal muscle. Progressive

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cardiomyopathy characterized by myocardial fibrosis, heart failure, andarrhythmia is the most common cause of mortality in these patients.Pannexins (Pxs) form large-conductance channels that have been implicatedas key mediators in signaling cascades involved with fibroblast recruitmentand activation as well as in the generation of pro-arrhythmogenic delayed afterdepolarizations (DADs). Pxs are large enough for passage of ATP moleculesand ATP-triggered beats have previously been demonstrated in isolated murineventricular myocytes. We have demonstrated for the first time increasedexpression of the Px1 isoform in mdx ventricular tissue by real time PCR[8.0 5 3.4 fold mRNA increase compared to wild type (WT), p = 0.03) aswell as by Western blot. In isolated Langendorff perfused WT and mdx mousehearts, adding ATP (30 mM) to the perfusate triggered both atrial and ventric-ular premature beats. The baseline total ectopy was increased in mdx heartscompared to WT (28.5 5 19.4 bpm vs 3.9 5 3.0 bpm, p = 0.006) withincreased fibrosis noted. In the presence of ATP, both ventricular and total ec-topy was increased in WT (3.8 5 3.0 bpm and 51.8 5 9.8 bpm, p = 0.02 andp< 0.001, respectively) and mdx hearts (24.6 5 16.4 bpm and 64.7 515.9 bpm, p = 0.009 and p< 0.001, respectively), although the increase in ven-tricular ectopy was larger in mdx hearts (p = 0.04). Blockade of Px1 with car-benoxolone (30 mM) demonstrated a decrease in ventricular and total ectopy(p = 0.03). We conclude that extracellular ATP activates an inward current suf-ficient to generate DADs and spontaneous action potentials via a mechanisminvolving Px1.

2406-Pos Board B422Mechanism of Selective Action of a Small Molecule Activator ofPhosphoinositide-Dependent GIRK ChannelsYu Xu, Lucas Noah Cantwell, Yuchen Yang, Sumanta Garai,Abhijit Kulkarni, Takeharu Kawano, Ganesh Thakur, Diomedes Logothetis.The Department of Pharmaceutical Sciences, Northeastern University,Boston, MA, USA.G protein-sensitive inwardly rectifying potassium channels (GIRK) areimportant integrators of G protein-coupled receptor (GPCR) signals.GIRK1/4 heteromers expressed in the heart hyperpolarize atrial myocytes,shorten the action potential, and slow heart rate. Meanwhile GIRK1,GIRK2and GIRK3 are coexpressed in multipule neuronal populations and are noveltargets for therapeutic intervention in multiple human nervous systemdisorders. GIRK4 expression is not prominent in the brain. Thus, the devel-opment of subunit-selective GIRK modulators could result in selectivemanipulation of GIRK channels to treat a variety of debilitating afflictions.For the first potent small activator of GIRK1-containing heteromersML297, Phe137 (F, in pore helix) and Asp173 (D, in TM2) of GIRK1have been identified to be necessary and sufficient for its activation effects.Our modeling studies have suggested the ML297 binding site is between theM1 and M2 helices which may perturb the pore region. Moreover, activationby ML297 requires PIP2 to bind to the non-FD subunits but competes withPIP2 in the FD-containing subunits. Chemical optimization based on ML297identified several new compounds such as GAT1508, that could changethe selectivity for the GIRK1/2 and GIRK1/4 heteromers. We performed aglobal search for the GAT1508 binding sites in the GIRK/GIRKFD channel,and probed its effects by molecular Dynamics (MD) simulations. Further-more, mutagenesis/voltage-clamp experiments were performed to test thesepredictions.Results and Conclusion: Our MD simulations suggest the binding region ofGAT1508 is differentiated in GIRK1/4 versus GIRK1/2 channels due to key in-teractions forming with two GIRK4 residues Val92 and Thr94 in the M1 helix.In oocyte expression experiments, GAT1508 slightly stimulated GIRK1/4channels but strongly activated GIRK1/2 channels. In cells expressingGIRK1 and the GIRK4 mutant with the two GIRK2 residues we identified,GAT1508-induced sensitivity is restored.

2407-Pos Board B423Engineered Transfer RNA Suppression of CFTR Nonsense MutationsJohn D. Lueck1, Adam L. Mackey1, Daniel T. Infield1, Marshall R. Pope2,Paul B. McCray3, Christopher A. Ahern1.1Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA,USA, 2Proteomics Facility, University of Iowa, Iowa City, IA, USA,3Pediatrics, University of Iowa, Iowa City, IA, USA.Ten percent of cystic fibrosis cases are caused by ‘‘nonsense’’ mutations thatlead to premature truncation of the cystic fibrosis transmembrane conductanceregulator (CFTR) protein. Examples of this ‘‘class 1’’ mutation are Gly542Xand Trp1282X, two common premature termination codons (PTC) whichcause a loss of CFTR function and severe cystic fibrosis phenotypes. Com-pounds that promote read-through of disease producing nonsense mutationsare only modestly successful as therapeutics due to a number of caveats

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with the approach, including poor specificity of the replaced amino acidreplacement, and the low efficiency of codon skipping in vivo. However,the widespread use of these compounds and the discovery that endogenousstop-codon read-through is common suggests that assisted suppression couldbe viable if delivered to a subset of cell types, i.e airway epithelium. Yet,when therapeutically assisted stop-codon read-through is successful, non-selective incorporation of amino acids at the location of the nonsense codonhas the potential to affect protein folding, trafficking and function (as is thecase with CFTR W1282X); and thus requires additional therapeutic interven-tion. We have developed a high-throughput platform for identifying codon-edited expressed tRNA, termed ACE-tRNAs, that suppress stop codons withthe cognate amino acid. This approach is theoretically ‘‘tuneable’’ to any dis-ease causing stop codon. Mass spectrometry of rescued proteins with thesetRNA indicate high-fidelity of rescue as only the cognate amino acid is en-coded at the stop site. Electrophysiological data obtained from mammaliancells and oocytes with either CFTR Trp1282X or Gly542X channels co-expressed with identified novel ACE-tRNA resulted in robust near wild-type CFTR channel function. The data describe the use of an edited tRNAto repair a CF causative mutation and serves as proof of principle forthe eventual use of ACE-tRNAs for the therapeutic rescue of PTC diseasecodons.

2408-Pos Board B424iPSC-Derived Neurons Harboring a Known Epilepsy Mutation Provide a‘Disease-in-a-Dish’ Capability that Displays Established and NovelEpileptic PhenotypesKile P. Mangan1, Imran Quraishi2, Yalan Zhang2, Michael McLachlan1,Benjamin Meline1, Chris McMahon1, Elisabeth Enghofer1,Christian Kannemeier1, Eugenia Jones1, Leonard Kaczmarek2.1Cellular Dynamics International, Madison, WI, USA, 2Yale University, NewHaven, CT, USA.Epilepsy is a disturbance in the electrical activity of the brain that effects 65million individuals, one-third of whom live with intractable epilepsy. A portionof this population is accounted for by single-gene epilepsy disorders resultingfrom mutations within sodium, potassium or inhibitory channels. Here weinvestigate the Slack gene (KCNT1), which encodes a sodium-activated potas-sium channel that is very widely expressed in the brain. Mutations in theKCNT1 gene in humans presents with autosomal-dominant nocturnal frontallobe epilepsy (ADNFLE), a disease marked by brief but violent seizures duringsleep and results in devastating effects on intellectual function. Advances inpersonalized medicine are crucial to combating these types of disablingdisorders.Central to the vision of personalized medicine is the technology of inducedpluripotent stem cells (iPSCs). iPSC technology provides a platform to expandour understanding of how single-gene mutations manifest disease states. Thisability provides unprecedented access to in vitro models of all-types of neuro-logical disorders. The approach presented here illustrates how the ‘‘disease-in-a-dish’’ iPSC-technology can be leveraged for phenotypic characterization andscreening, and how to navigate these types of investigations into the drug devel-opment space.Human cortical neurons harboring the KCNT1 {P924L} single-gene mutationwerer engineered and generated, as well as where the isogenic wild-type con-trol match. Using protein (Western Blots) and functional evaluations (patch-clamp and multi-electrode array), we illustrate iPSC-derived cortical neuronsharboring the KCNT1 {P924L} mutation recapitulate a well characterized‘gain-of-function’ ionotropic cellular-level fingerprint without altered cellularSlack-protein levels. We further present novel neural-network level, hyper-active phenotypes associated with this KCNT1mutation and use selective phar-macology to reverse the aberrant phenotypes. Collective results illustrate howhuman iPSC-derived neurons can successfully be harnessed within the person-alized medicine space.

2409-Pos Board B425Identification of a Modulatory Site of Action for the Volatile AnestheticIsoflurane in TREK1 Tandem Pore Potassium ChannelsPaul M. Riegelhaupt1, Kellie A. Woll2, Thomas T. Joseph2,Kiran A. Vaidya1, Crina M. Nimigean1, Roderic G. Eckenhoff2.1Weill Cornell Medicine, New York, NY, USA, 2University of Pennsylvania,Philadelphia, PA, USA.Many members of the K2P family of potassium channels are modulated by vol-atile anesthetic (VA) agents. While individual residues within a subset of K2Pchannels are known to be important for anesthetic modulation, those actuallycontributing to the VA binding site have not yet been revealed. Using a vali-dated photoaffinity analog of the haloether VA isoflurane (azi-isoflurane), weidentified a VA binding site within the TREK1 K2P channel. Azi-isoflurane

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was reacted with recombinantly expressed, purified and reconstituted TREK1protein, and modified residues were detected by liquid chromatography-tandem mass spectrometry. Two TREK1 residues are modified by Azi-isoflurane: a glycine in the inner helix bundle crossing region of TM2 and amembrane exposed residue in the TM2/TM3 loop. Protection from azi-isoflurane modification by photolabeling in the presence of 100-fold excess iso-flurane was only observed for the inner helix bundle glycine, suggesting that theparent VA isoflurane specifically binds in this region. Two electrode voltageclamp studies on TREK1-expressing Xenopus leavis oocytes demonstratethat mutagenesis of the identified inner helix glycine to tryptophan causes amarked increase in channel activity and eliminates TREK1 anesthetic sensi-tivity. In molecular dynamics simulations, an azi-isoflurane molecule placedin the TREK1 inner helix bundle region makes stable contacts with this glycineas well as a TM3 residue known to modulate anesthetic responsiveness ofTASK K2P channels. Mutating the glycine to tryptophan substantially de-creases azi-isoflurane binding affinity, as calculated using free energy perturba-tion. Future work combining photoaffinity labeling, functionalcharacterization, and MD simulation will aim to understand the mechanismby which anesthetic binding at this region leads to channel activation, whetherall VAs share this modulatory site, and whether the identified modulatory site isshared by all anesthetic sensitive K2P channels.

2410-Pos Board B426VX-770-Mediated Potentiation of Numerous Human CFTR DiseaseMutants is Influenced by Phosphorylation LevelGuiying Cui, Brandon Stauffer, Barry Imhoff, Andras Rab, Nael McCarty.Pediatrics, Emory University, Atlanta, GA, USA.VX-770 (Kalydeco) has been approved for clinical usage in CF patients withmultiple CFTR mutations, yet the binding site(s) and functional mechanismsare unclear. We previously found that the efficacy of potentiator P2 onD1152A-hCFTR depends on channel phosphorylation level. We hypothesizethat VX-770, like P2, potentiates multiple human CFTR disease mutants in aphosphorylation-dependent manner. We combined heterologous expressionin oocytes with more physiologically relevant Ussing chamber recordings uti-lizing polarized Fischer rat thyroid (FRT) and human bronchial epithelial cells(HBE) to study and characterize VX-770’s mechanism. Our results demon-strate that VX-770 potentiated WT-, E193K-, K1060T-, and N1303K-hCFTRin a manner-dependent upon phosphorylation level. VX-770 potentiation ofP67L-, F508del-, and G551D-hCFTR also exhibited phosphorylation leveldependence but was significantly more efficacious than the above mutations.We further established that P67L-, and F508del-hCFTR exhibit reduced theirPKA sensitivity to CFTR activation, contributes their severe gating abnormal-ities, and likely caused the alteration of VX-770 potentiation efficacy. Similarphenomena were also noted in N66S-, F508S-, and F508C-hCFTR. Further-more, G551D-hCFTR activation as well as inactivation was significantly de-layed compared to WT-hCFTR under the same experimental conditions. VX-770 not only strongly potentiated G551D but also greatly promoted G551Dactivation suggesting that VX-770 allosterically modulates CFTR in multipleways. Phosphorylation-dependent potentiation of WT-, F508del-, P67L-, andG551D-hCFTR was also observed in FRT and HBE cells studied by Ussingchamber recording. These results should be considered from the perspectivesof drug discovery and theratyping of new potentiators and lead to an expansionof ultra-orphan mutations for which VX-770 is approved.

2411-Pos Board B427Flos Magnoliae and its Chemical Constituents Modulate Cl- Secretion viaANO1 Cl- Channel Inhibition in Human Airway Epithelial CellsHyun Jong Kim1, Yu-Ran Nam1, Joo Hyun Nam1, Woo Kyung Kim2.1Physiology, Dongguk University, College of Medicine, Gyeongju-Si,Republic of Korea, 2Internal Medicine, Graduate School of Medicine,Dongguk University, Goyang-si, Republic of Korea.Flos Magnoliae (FM, Chinese name: Xin-yi) is a commonly used oriental me-dicinal herb for the relief of allergic rhinitis, sinusitis, and headache. FM alsohas been commonly used in traditional Chinese and Korean medicine formula-tions. FM has been reported to inhibit histamine release via mast cell and cyto-kine secretion from T cells; however, the mechanism of action of the ANO1 ionchannel, which is responsible for nasal hypersecretion in allergic rhinitis hasnot been elucidated. Therefore, we investigated whether FM and its chemicalconstituents can modulate the activity of anoctamin-1 (ANO1). A 30% etha-nolic extract of FM (FMEtOH) and its five major constituents in FMEtOH wereprepared. By using a conventional whole-cell patch clamp, we revealed thatthe ANO1 activity is inhibited by FMEtOH (30, 100, and 300 mg/ml) and itschemical constituent, tiliroside, in ANO1 was overexpressed in HEK293Tcells. In addition, we also reveal that the treatment of IL-4 in the airway epithe-lial cell line, Calu-3, significantly increases the ANO1 current (IANO1) but not

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the cystic fibrosis transmembrane conductance regulator (CFTR)-mediatedchloride current (ICFTR)and specifically modulated IANO1 by FMEtOH and tiliro-side. In this study, we found a new mechanism of action of FMEtOH for the alle-viation of allergic rhinitis. FMEtOH and its chemical constituent, tiliroside, maybe suitable candidates for the development of a potent agent for the preventionand treatment of allergic rhinitis.

2412-Pos Board B428Identification of Potent and Selective Inhibitors to Investigate the Role ofEpithelial Sodium Channels in NeurodegenerationVictoria Miller, John Atack, Martin Gosling.Sussex Drug Discovery Centre, University of Sussex, Brighton, UnitedKingdom.Members of the DEG/ENaC family have been recently implicated in the neuro-degeneration associated with a number of CNS disease states. More specificallythe neuronally-expressed ENaC d channel has been linked to the integration ofischemia-related signals in inflamed and hypoxic tissues. Although a recentstudy reported clinical efficacy of the prototypical ENaC inhibitor amiloridein multiple sclerosis, this drug was optimised for the non-CNS ENaC variant(abg). To date, the small number of compounds described in the literature asENaC blockers all share a structural similarities to amiloride, which lends toa collective poor selectivity between multiple ion channels and poor pharmaco-kinetic properties with respect to CNS penetration.As such our aim is to identify novel inhibitors of the ENaC d channel that arepotent and selective, and can be assessed for their potential as neuroprotectiveagents. Presently, we use an HEK293 stable cell line expressing ENaC b and gsubunits and a BacMam virus to transiently express the ENaC d subunit toreconstitute channel function. We have established a novel membranepotential-based fluorescence assay, in parallel to an IonWorks Quattro auto-mated electrophysiological assay, that have been used to screen for regulatorsof ENaC d channel function. Primary hits have been subsequently triaged usingconventional whole cell patch clamp electrophysiology, resulting in the identi-fication of novel chemotypes. This will be used to support an ongoing SAR-based approach to improve potency and selectivity in the development of atool compound to investigate the ENaC dbg channel.

2413-Pos Board B429Diverse Pharmacological Effects of Carbon Monoxide-Releasing Mole-cules on Mitochondrial BK ChannelDaria Rotko1, Piotr Bednarczyk2, Adam Szewczyk1.1Nencki Institute of Experimental Biology, Warsaw, Poland, 2WarsawUniversity of Life Sciences, Warsaw, Poland.Carbon monoxide (CO) is an endogenously produced gaseous transmitter,which is involved in maintenance of cellular homeostasis. A wide range ofCO signaling events is incited by a mitochondrial cascade of metabolic andROS signaling, which eventually contribute to cellular life-or-death decision.The mechanisms of its action are largely unknown, but one of the mitochondrialpathways might be realized by modulation of activity of large-conductance po-tassium channel in the inner membrane of mitochondria (mitoBK). MitoBKshares multiple biophysical properties with its plasma membrane counterpart,modulation of which by CO is emerging as a physiologically relevant phenom-enon. However, mitoBK resides in the environment of respiratory chain and hasbeen shown to be functionally and structurally coupled to cytochrome c oxi-dase. Since the latter constitutes a target for CO, such an interaction wouldintroduce another dimension to the CO regulation of mitoBK. To study modu-lation of mitoBK by CO, we chose several structurally different CO-releasingmolecules (CORMs), which are typically used for the experiments on plasmamembrane BK channels. Pharmacological action of CORMs was assessed inthe single-channel patch-clamp recordings of astrocytoma mitochondria.Apparent effects of CORMs were vastly disparate and varied by the class ofcompound applied. Therefore, our observations raise an issue of specificityof CORMs and their appropriateness in study of mitochondrial ion channelpharmacology.This work was supported by the Polish National Science Centre (grant no.2015/17/B/NZ1/02496) and MSCA-COFUND #665735 (Bio4Med).

2414-Pos Board B430Flexibility of a Transmembrane Helix Underlies Dramatic Reversal of NetAnesthetic Effects in a Pentameric Ligand-Gated Ion ChannelStephanie A. Heusser1, Marie Lycksell1, Xueqing Wang1,2,Rebecca J. Howard1, Erik Lindahl1,2.1Department of Biochemistry and Biophysics, SciLifeLab, StockholmUniversity, Stockholm, Sweden, 2Department for Theoretical Physics,SciLifeLab, KTH Royal Institute of Technology, Stockholm, Sweden.Mechanisms of action for general anesthetics have evolved substantially frompurely lipid-based theories to models involving specific protein interfaces, with

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the capacity to allosterically inhibit as well as potentiate human Cys-loop re-ceptors. However, we still lack a structurally detailed mechanism for thesebimodal allosteric effects. Recently, we mapped an allosteric model for anes-thetic modulation involving multiple state-dependent binding sites, based onnew co-crystal structures of the bacterial homolog GLIC and the general anes-thetic propofol. Notably, a point mutation (M205W) in the first transmembranehelix facilitated propofol binding in a membrane-accessible intrasubunit site,and conferred primarily potentiating rather than inhibitory net anesthetic ef-fects. Molecular dynamics simulations correlated the potentiated phenotypewith changes in backbone flexibility of the first transmembrane helix. Here,we used mutagenesis and two-electrode voltage-clamp electrophysiology toreveal that helix-perturbing residues at the same transmembrane positionlead to potent potentiation even by subclinical concentrations of propofol.Interestingly, these variants were so sensitive to propofol that they exhibitedpersistent potentiation for up to 40 minutes after exposure to moderate concen-trations of propofol, in contrast to an almost instantaneous washout in wild-typechannels. The extent of this effect depended on propofol exposure time, sug-gesting involvement of the membrane as a reservoir for a lipid-accessible bind-ing site. Persistent potentiation also appeared to be independent of an inhibitoryeffect observed at high acute doses, consistent with the presence of a discrete,water-accessible site for inhibition. The resulting mechanism for general anes-thetic modulation could bridge the lipid-based Meyer-Overton theory, postu-lated more than a century ago, with more complex receptor-based models,informing both our understanding of allostery and the development of newanesthetics.

2415-Pos Board B431Optogenetic Technologies Enable High Throughput Ion Channel DrugDiscovery and Toxicity ScreeningRiccardo Rizzetto1, Viviana Agus1, Sara Pizzi1, Jean-Francois Rolland1,Lia Scarabottolo1, Susanne Renhelt2, Daniela Malan2, Tobias Bruegmann2,Philipp Sasse2, Krisztina Juhasz3, Leo Doerr3, Matthias Beckler3,Michael George3, Andrea Br€uggemann3, Niels Fertig3.1Axxam SpA, Bresso, Italy, 2Institute of Physiology I, Medical Faculty,University of Bonn, Bonn, Germany, 3Nanion Technologies GmbH, Munich,Germany.The drug discovery process involving ion channels needs to rely on high-throughput screening (HTS) assays as well as fine-tuned characterization byelectrophysiological measurements. Combining optogenetic tools withinduced pluripotent stem-cells (iPSC)-derived cardiomyocytes can provide areliable, cost-effective and highly time-resolved approach to induce electro-genic proteins activation. The aim of the OPTEL project, which combinesthe expertise of two leading European companies and one academic labora-tory, was to develop integrated HTS-compatible platforms based on optoge-netic tools (Channelrhodopsin, ChR2) for effective drug discovery inheterologous expression systems and iPS-derived cardiomyocytes. We gener-ated HEK293 cell lines stably expressing ChR2 alone or with the cardiac so-dium channel, NaV1.5 which proper functional expression was validated byfluorescence and manual patch-clamp. The best clones were used to imple-ment light stimulation on an HTS automated patch-clamp platform (Synchro-Patch 384PE, Nanion Technologies). We recorded light-induced currents andaction potential-like responses in voltage and current-clamp, respectively,validating a new platform for high-throughput automated ‘‘opto-patch-clamp’’assays. For optogenetic drug toxicity screening, commercially available iPSC-derived cardiomyocytes were infected with adeno-associated viruses to ex-press ChR2 and analyzed by recording extracellular field potentials from 96wells with a modified CardioExcyte96 system (Nanion Technologies). Thiswas equipped with a custom designed 96 LED lid enabling pacing of cellsand frequency-dependent drug screening over the physiological heart rate(1-3 Hz). Thereby we characterized adverse side effects of known ion channelblockers and pro-arrhythmogenic drugs on Naþ, Ca2þ and Kþ channels. Theseresults validate new platforms that allow optogenetic control of electrical ac-tivity in HTS-compatible format with four different readouts: fluorescence,electrophysiology, impedance and extracellular field potentials. The use ofthese complementary readouts provides a new cost-effective and informativestrategy in early stages of ion channel drug discovery, especially in the cardiacfield.

2416-Pos Board B432Mechanisms Underlying Rate-Dependent Effects of State-Specific Bindingof Sodium Channel Blockers in Cardiac Tissue: Insights from IdealizedModelsSteffen S. Docken1, Timothy J. Lewis1, Colleen E. Clancy2.1Mathematics, UC Davis, Davis, CA, USA, 2Physiology and MembraneBiology, UC Davis, Davis, CA, USA.

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Increased propensity for arrhythmias is a well-documented but poorly under-stood side-effect of drugs. Many arrhythmogenic drugs bind to and blockion-channels in a conformational state-specific manner, making it difficult todiscern the fundamental mechanisms underlying pro-arrhythmic drug effects.We have developed mathematical models to examine the effects of state-specific Naþ channel blockers with various binding mechanisms on the rate-dependence of peak upstroke velocity, conduction velocity, and the vulnerablewindow of cardiac tissue. Specifically, we compared the effects of drugs thatbind via the guarded receptor and gate immobilization models, which are ideal-ized models consisting of motifs commonly found in modulated receptormodels of drug binding. For both the guarded receptor and gate immobilizationmodels, we considered drugs that bind to either inactivated or non-inactivatedNa-channels. Our results suggest that drugs that bind to inactivated Naþ chan-nels give rise to greater rate-dependent effects on peak upstroke velocity andconduction velocity than drugs that bind to non-inactivated Naþ channels.Additionally, we find drugs that bind to non-inactivated Naþ channels viathe gate immobilization motif induce a reversal of the rate-dependence ofpeak upstroke velocity. By exploiting the idealized nature of guarded receptorand gate immobilization models and utilizing singular perturbation analysis, weprovide an explanation of the fundamental mechanisms of rate-dependent drugeffects. Finally, we show that a gate immobilization model with inactivatedstate binding captures the rate-dependent effects of lidocaine, demonstratingthat idealized models can be useful models to study mechanisms of action ofreal drugs.

2417-Pos Board B433Highly Parallel All-Optical Real-Time Interrogation of Fast Voltage-Gated Ion Channels using Molecular Wire Voltage-Sensing CompoundsThomas Lila, Jay Trautman, Stephen Smith, Andrew Blatz.Photoswitch Biosciences, Inc., Menlo Park, CA, USA.Combining optogenetic control of cell membrane potential with real-time fluo-rescence-based voltage readouts has the potential to enable economical all-optical alternatives to patch clamp systems in screening large compound li-braries against ion channel targets. Rapidly-inactivating voltage-gated ionchannel targets are particularly challenging for high throughput opticalscreening because of the need for sub-millisecond scale temporal resolution,flexible voltage control, and highly parallel measurements.Here, we combine the instantaneous response of ‘‘molecular wire’’ voltagesensors with repetitive optical ion channel stimulation and simultaneousvoltage readouts of multiple wells at 10 kHz sampling rate. Using cells engi-neered to express a channelrhodopsin optical actuator plus either the Nav1.5or Nav1.7 sodium channel, induced action potentials were monitored at highS/N under regimens involving varying stimulation frequencies (0.5 – 8 Hz) ordurations of inactivating membrane voltage potentials. Appropriate isoformselectivity as well as state and use-dependent pharmacology are shown forlocal anesthetic and voltage sensor-binding test compounds. Of note are re-sults obtained with a ‘‘red-shifted’’ molecular wire probe at 660 nm excitationwavelength, allowing for completely independent voltage sensor monitoringand channelrhodopsin stimulation. The combination of optical sensors and ac-tuators offer a promising platform for truly high throughput interrogation ofion channel targets that are difficult to screen using preexisting opticalinstrumentation.

2418-Pos Board B434Bunyaviruses are Dependent on K2p Channels to Infect CellsSamantha Hover1, John N. Barr1, Steve AN Goldstein2, Jamel Mankouri1.1University of Leeds, Leeds, United Kingdom, 2Loyola University Chicago,Chicago, IL, USA.All viruses must infect cells in order to multiply and cause disease. Once in-side cells, viruses hijack normal cellular processes to create an environmentthat favours their own survival, typically at the expense of the host. Throughunderstanding how viruses do this, we can design new strategies to preventviruses from multiplying, and thus stop their ability to cause disease. TheBunyaviridae are the largest family of negative stranded RNA viruses. Theseare a group of over 350 different viruses spread throughout the globe. Theyare predominantly transmitted to humans by insects and are capable ofcausing fatal disease in humans, often as a result of devastating hemorrhagicfevers. Bunyaviruses are a serious and worrying threat to human healthbecause they have enormous capacity to mutate and evolve into new strains.In addition, the fact that bunyaviruses are mostly spread by biting insects thatare highly mobile means they can rapidly move into new geographic loca-tions, causing widespread disease. Despite this huge threat, no preventativeor therapeutic measures currently exist for any bunyavirus-mediated disease.Using model viruses within the Bunyaviridae family, predominantly Bunyam-wera virus (BUNV), we previously identified cellular potassium (Kþ)

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channels as an essential host cell factor required for bunyavirus infection. Us-ing a rationale panel of Kþ channel modulators, we identified the two-pore Kþ

(K2P) channel family as those required by BUNV. We have identified fourpossible K2P channels we believe are facilitating a cellular process requiredduring BUNV infection. Using genetic silencing we plan to determine the spe-cific channel(s) involved and from this, identify a new drug target for thedevelopment of novel anti-bunyavirus therapies.

2419-Pos Board B435Minocycline and Doxycycline Inhibit ASIC Currents in Dorsal Root Gan-glion NeuronsLaura C. Caba Sanchez1, Rosario Vega1, Audrey M. Ortega1,Ricardo F�elix2, Enrique Soto1.1Instituto de Fisiologıa, Benem�erita Universidad Autonoma de Puebla,Puebla, Mexico, 2Departamento de Biologıa Celular, Centro de Investigaciony Estudios Avanzado del Instituto Polit�ecnico Nacional (CINVESTAV-IPN),CDMX, Mexico.Acid Sensing Ion Channels (ASIC) are primary pH sensors in mammals andare expressed widely in neuronal and nonneuronal cells. ASIC are involved inlearning, fear behavior, neurodegeneration, nociception, mechanoreception,chemoreception, bone remodeling, ischemia, epilepsy, inflammation, amongothers. Thus, the discovery of pharmacological agents targeting ASICs hashigh therapeutic potential and can be used to elucidate their structural andfunctional properties. Minocycline and Doxycycline are semi-synthetic tetra-cyclines (Ts) of the second generation, which have neuroprotective properties,in processes in which ASICs have been involved (such as neurodegeneration,ischemia, nociception, and inflammation); which led us to postulate theASICs, as a minocycline and doxycycline target. Whole cell voltage clamprecordings from isolated dorsal root ganglion (DRG) neurons from the rat,shown that both Ts inhibit the peak amplitude of proton gated current witha IC50 in the mM range; depending the Ts perfused, the dose and the perfusionprotocol used (preapplication, sustained application or coapplication). Inter-estingly we observed a peak amplitude current increase with the ‘‘doxycyclinecoapplication’’ (in this perfusion protocol the Ts was applied only during the5s acid solution). Molecular anchor analysis (in-silico analyses) suggests the‘‘acid pocket’’ as a putative common binding site for both Ts in the chickenASIC1a channel. The importance of the acid pocket is that it participates inthe gating of ASICs and it is the binding site of several toxins, includingPcTx1 (specific inhibitor of ASIC1a) and mambalgin 2 (ASIC1a Inhibitorbut not ASIC2). The Ts may constitute a valuable tool for the modulationof ASICs, and may be a feasible starting point for the development of newstructurally molecules with a greater affinity, specificity, and selectivity forthe ASIC subunits.

2420-Pos Board B436An SCN1B Variant Found in a Child Diagnosed with Epilepsy andBrugada Syndrome Modifies Brain-Type (NaV1.1) and Cardiac-Type(NaV1.5) Sodium CurrentsRebecca Martinez-Moreno1,2, Helena Riuro1,2, Elisabeth Selga1,2,Michael F. Wangler3, Ramon Brugada1,2, Guillermo J. P�erez1,2,Fabiana S. Scornik1,2.1Medical Sciences, University of Girona, Girona, Spain, 2CardiovascularGenetics Center, Institute of Biomedical Research of Girona Dr. J. Trueta,Salt, Spain, 3Department of Molecular and Human Genetics, Baylor Collegeof Medicine, Houston, TX, USA.Generalized epilepsy with febrile seizures plus (GEFSþ) is an autosomal domi-nant syndrome linked to mutations in SCN1A,which encodes the brain voltage-gated sodium channel alpha-subunit (Nav1.1). Likewise, Brugada Syndrome(BrS) is a cardiac arrhythmogenic disease, associated with mutations inSCN5A, which encodes the heart sodium channel alpha subunit (Nav1.5). Inaddition, both syndromes have been linked to a small percentage of variantsin the sodium channel regulatory b1 and b1b subunits, encoded by theSCN1B gene. A missense mutation (c.308 A>T; p.D103V) in the SCN1Bgene was identified in a child diagnosed with concomitant GEFSþ and BrS.This mutation is located in the extracellular Ig-like domain of the b1 subunit,affecting both b1 and b1b isoforms. b1 subunits modulate sodium channelsin both brain and heart tissues. Thus, we investigated whether Nav1.1 and/orNav1.5 currents were affected by mutant b1 or b1b subunits.Sodium currents were studied using the whole cell patch-clamp technique intsA201 cells transfected with NaV1.1 or NaV1.5 and WT or D103Vb1/b1bsubunits.We observed a decrease in sodium current density in cells co-expressingNaV1.1 or NaV1.5 and b1D103V compared to b1WT subunit. Interestingly,when the isoform b1bD103V was co-transfected with NaV1.1, it did not affectINa density, but induced a positive shift in the voltage dependence of inactiva-

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tion and an accelerated recovery from inactivation, compared to b1bWT. How-ever, the isoform b1bD103V did not affect NaV1.5 current properties.Overall, our results show a loss of function in both, brain and cardiac sodiumchannels, caused by the mutation in SCN1B. This suggests that the D103V mu-tation is responsible for the combined GEFSþ and BrS phenotype of thepatient.

2421-Pos Board B437Novel CNG Channelopathy Model Generated using CRISPR/Cas9-Mediated Genome Editing in ZebrafishMichael D. Varnum1, Peter C. Meighan1, Samuel S. Hunter2,Lindsey M. Morey1, Tshering Sherpa1.1Integrative Physiology and Neuroscience, Washington State University,Pullman, WA, USA, 2Institute for Bioinformatics and Evolutionary Studies,University of Idaho, Moscow, ID, USA.Inherited defects in CNGA3 and CNGB3 genes encoding cone cyclicnucleotide-gated (CNG) channel subunits have been linked to complete andincomplete achromatopsia, progressive cone dystrophy and macular degener-ation in humans. Several disease-associated frameshift/truncation mutationsare located within the last exon of human CNGA3. To mimic these mutations,we used CRISPR/Cas9-mediated genome editing to target the two zebrafishorthologs of human CNGA3. We edited zebrafish cnga3a and cnga3b usingmicroinjection into single-cell embryos of Cas9 mRNA plus single guideRNAs targeting sites within the last exon of the genes (exon 9 and exon 5,respectively). Successful editing was confirmed via mismatch cleavage usingT7 endonuclease I, and DNA sequencing, demonstrating insertions/deletions(indels) at the edited sites. The functional effects of editing were determinedusing optomotor response (OMR) visual performance assays and electroreti-nogram (ERG) recordings with 6-8 dpf larvae. Retinal morphology was as-sessed by immunohistochemistry, while retinal cell death was investigatedvia TUNEL staining. Indels in cnga3a were associated with a profounddisruption of visual function in larvae, demonstrated by impaired OMR per-formance and attenuated ERG b-wave amplitudes, at a developmental stagewhen only cone-based vision is present. Compared to editing of cnga3a,CRISPR modification at homologous target sites in cnga3b produced amore modest effect on visual function in larvae, while editing of both cnga3aand cnga3b together completely disrupted visual performance. In addition,adult cnga3a-edited zebrafish exhibited disturbed retinal morphology withdisorganized cone photoreceptors and shortened cone outer segments, evi-dence of retinal cell death, and mislocalized cone CNG channels. Together,these results suggest that cnga3a exon 9 editing in zebrafish represents a novelchannelopathy model for human cone dysfunction and degeneration, which re-capitulates truncation mutations in human CNGA3 that are associated withblindness.

2422-Pos Board B438Acidosis Prolongs APD in Optically Mapped Adult Zebrafish WholeHearts as a Result of hERG Channel BlockYu P. Shi, Cherlene Chang, Marvin Gunawan, Eric Lin, Sanam Shafaattalab,Glen Tibbits, Tom Claydon.Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby,BC, Canada.During the early phase of myocardial ischemia, local extracellular acidosis (aslow as pHo 6.5) can play a part in the initiation of the ischemic cascade lead-ing to cardiac arrhythmia. Previously, we characterized the effect of acidosison hERG potassium channels in a heterologous expression system and showedthat external protons modify numerous gating properties that result in hERGchannel loss-of-function. In silico action potential simulations suggest thatthis could contribute to the development of arrhythmia. To explore thisfurther, we investigated the effects of acidosis on action potentials recordedusing optical mapping of isolated adult zebrafish whole hearts at 28oC. Zebra-fish hearts represent a good model for human cardiac electrophysiologicalstudies, since action potential morphology, intrinsic heart rate, and QT-interval are similar to those in human hearts, and repolarization is dependentupon hERG function, unlike in murine models. Measuring voltage transientsusing an optical dye (RH-237), we demonstrate that external acidosis (pHo

6.5) prolonged the rate-matched APD90 by 79 5 10 ms (n=4). This effectoccurred rapidly, within 1 min, and was readily reversed upon restorationof control pHo 7.4, indicative of a direct extracellular effect. Blockadeof hERG channels by dofetilide (250 nM) blunted the action potentialprolonging effects of acidosis by 55%, demonstrating a role for hERGchannels in the acidosis-induced APD90 prolongation. These data indicatethat hERG dysfunction during acute acidosis is partly responsible for prolon-gation of the action potential duration, which may contribute to arrhythmiageneration.

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2423-Pos Board B439Streptomycin Entry is Mediated by the Mechanosensitive Channel MscCGof Corynebacterium GlutamicumYoshitaka Nakayama1, Kosuke Komazawa2, Navid Bavi1,Kazuhiro Nobata2, Ken-ichi Hashimoto2, Hisashi Kawasaki2,Boris Martinac1.1Victor Chang Cardiac Research Institute, Sydney, Australia, 2Tokyo DenkiUniversity, Tokyo, Japan.Streptomycin is a widely used antibiotic for the treatment of a number of bac-terial infections, including tuberculosis. Bacterial cells exposed to streptomycinwere shown to leak potassium and glutamate; however it is not clear how strep-tomycin causes the efflux of these osmolytes although it has been proposed thatmechanosensitive (MS) channels serve as potential pathways for streptomycinentry into bacterial cells. To address this question, we evaluated the antibioticpotency on MS channels in the wild-type and knock-out strains of Corynebac-terium glutamicum, glutamate producing Gram-positive bacteria closelyrelated to mycobacteria. The cell viability against streptomycin is increasedwhen the mechanosensitive channel MscCG, a major glutamate exporter, isknocked-out. In order to characterize the gating of MscCG channel and itsinteraction with streptomycin in the native cell membrane, we developed anovel giant spheroplast preparation of Corynebacterium glutamicum as a modelof mycobacterial cells. Using the patch-clamp technique, we were able to re-cord the currents of two types of MS channels; MscCG and an MscL-like chan-nel, and examined their activation thresholds and gating kinetics. Furthermore,we were able to demonstrate that streptomycin increased the time that theMscCG channels spent in the open state. Our study thus suggests that strepto-mycin interferes with the gating kinetics of the MscCG channels by enteringinto the bacterial cells through these MS channels in C. glutamicum.

Posters: Other Channels II

2424-Pos Board B440Understanding the Structure and Function of the DcaP Channel fromAcinetobacter baumannii using MD SimulationsJigneshkumar D. Prajapati1, Satya Prathyusha Bhamidimarri2,Michael Zahn3, Dirk Bumann4, Mathias Winterhalter2, Bert van den Berg3,Ulrich Kleinekathofer1.1Department of Physics and Earth Sciences, Jacobs University BremengGmbH, Bremen, Germany, 2Department of Life Sciences and Chemistry,Jacobs University Bremen gGmbH, Bremen, Germany, 3Institute for Cell andMolecular Biosciences, Newcastle University, Newcastle, United Kingdom,4Focal Area Infection Biology, Biozentrum, University of Basel, Basel,Switzerland.DcaP is putative dicarboxylate specific channel, located in the outer membraneof the pathogen Acinetobacter baumannii. The X-ray crystal structure revealsthat DcaP is the first trimeric channel identified in Acinetobacter baumanniiand could play an important role in substrate and antibiotic permeation. Tocharacterize the permeation properties of this channel, we have carried outthe applied field MD simulations in the presence of ions (KCl), substrates(phthalic acid, succinic acid) and a b-lactam antibiotic (sulbactam). Addition-ally, free energy calculation have been carried out using metadynamics simu-lations to identify the lowest energy permeation path along the 2D free energysurfaces and the most prominent residues during translocation. These simula-tions clearly suggest that the DcaP channel is involved in the permeation ofthese solutes and results are complemented with electrophysiology experi-ments. Furthermore, the crystal structure reveals that DcaP have an extendedN-terminus domain in the periplasmic space, which is presumably known toform the coiled-coil structure (uniprot entry: A0A0B9X9I7). As the N-terminusdomain was not resolved in the crystal structure, we have predicted the struc-ture using an extensive modeling approach. Moreover, the simulations andelectrophysiology experiments suggest that the N-terminus might play animportant role in the formation of a stable trimer. Overall, we have built theso far unknown structure-function relationship for the DcaP channel whichcould help in designing next generation antibiotics efficiently permeatingthrough this channel.

2425-Pos Board B441Glial Channels and Transporters that Mediate Excretion of KD in theMicroenvironment between Glia and Neurons Shape Neuronal Output inC. elegansChristina Johnson, Ying Wang, Lu Han, Laura Bianchi.Physiology & Biophysics, University of Miami Miller School of Medicine,Miami, FL, USA.Isolated microenvironments, such as the tripartite synapse, where the concen-tration of ions is regulated independently from the surrounding tissues, exist

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throughout the nervous system. While the regulation of ions in these microen-vironments is known to be mainly mediated by glia, the molecular mechanismsof ion regulation and effects on neuronal output and animal behavior are poorlyunderstood. Using the model system C. elegans, our lab published that Naþchannels of the DEG/ENaC family expressed in glia control neuronal Ca2þ

transients and animal behavior in response to sensory stimuli. DEG/ENaCNaþ channels are known to establish a favorable driving force for Kþ excretion,which occurs via inward rectifier Kþ channels, in epithelial tissues across spe-cies. We hypothesized that a similar mechanism exists in the nervous system.Using molecular, genetic, in vivo imaging, and behavioral approaches, weshowed that expression in glia of inward rectifier Kþ channels and cationicchannels rescues the sensory deficits caused by knock-out of glial DEG/ENaCs,supporting our hypothesis. Based on this model, Naþ/Kþ-ATPases are alsoneeded to maintain ionic concentrations following influx of Naþ and excretionof Kþ. To test this prediction, we used RNAi to knock down each of the 5 a-subunits of the Naþ/Kþ-pump in glia and test the effect of the knock-downs onsensory behavior. We identified Naþ/Kþ-pump a-subunits eat-6 and catp-1 asspecifically required in glia for sensory perception, further supporting a modelin which glia shapes neuronal output and, consequently, animal behavior byexcreting Kþ in the microenvironment surrounding neurons. Given thatDEG/ENaCs, inward rectifier Kþ channels, and Naþ/Kþ-pumps are also ex-pressed in mammalian glia, we propose that this mode of regulation of neuronalfunction in conserved across species.

2426-Pos Board B442The Human Erythrocyte Mechano-Activated KD Channel a (HEMKCA):Effect of Ba2D on Burst ActivityYeimar Rortillo1, Alejandro Mata2, Jesus G. Romero1.1Escuela de Biologia, Universidad Central Venezuela, Caracas, Venezuela,Bolivarian Republic of, 2Institup de Biologia Experimental, UniversidadCentral Venezuela, Caracas, Venezuela, Bolivarian Republic of.Potassium channels conduct Kþ ions across the cell membrane down its elec-trochemical gradient. HEMKCA is a mechano-activated Kþ channel of humanerythrocytes, first recorded in our group. channel Selectivity is one of the mostexquisite processes studied in biology, and it is well known that some ions insolution may act as channel blockers. In this sense, the present work was aimedin study the effect of Ba2þ ions on the burst activity of the HEMKCA channel.Here, it was evaluated the effect of Ba2þ over the open probability (Po) of thechannel and the intraburst variables of the kinetic model of this channel. ABurst is a succession of channel openings being separated only by brief shutperiods, which length is shorter than a critical time (tc). It was found thatBa2þ affects either the Po as the intraburst variables, acting like a fast blockerof the ionic pathway of the channel. In presence of Ba2þ, the channel spendsmore time in the nonconductive state generating long periods of inactivity.The presence of 1 mM Ba2þ produces the occurrence of a set of shut eventswhich time constant is between the short close state (t1) and the long close state(t2). In this way, it was modified the kinetic model developed previously in ourlab, adding a fourth state, named as a blocking state. These results allow us todescribe a new kinetic state for the HEMKCA channel that arose by the fastinteraction of Ba2þ with the channel. The effects of this ion allow us to identifytwo types of blockages (fast and slow) and two binding sites (high and low af-finity) across the HEMKCA channel.

2427-Pos Board B443The Annexin V Transmembrane ChannelYichih Lin, Atsushi Miyagi, Simon Scheuring.Anesthesiology, Physiology and Biophysics, Weill Cornell Medicine, NewYork City, NY, USA.Annexins are a family of proteins that bind calcium. Upon calcium binding,annexins have high affinity for anionic phospholipids and bind to the plasmamembrane. They have been implicated in several cell biological processesincluding membrane stabilization and repair, and endocytosis and exocytosis.Annexins are also found in the extracellular space and have been shown tobe involved in coagulation and apoptosis. In the 1990s, based on functionalmeasurements, annexins have also been evidenced to form calcium ionchannels. However, the more than 80 annexin structures solved, all revealeda soluble protein, and the ion channel function remained an unexplainedoddity. Here, we report the direct monitoring of annexin-V (A5) interactionswith membranes in real time and native-like environments using high-speed atomic force microscopy (HS-AFM) imaging. We provide first compel-ling evidence of a transmembrane annexin ion channel structure that iscomposed of a central pore-forming trimer surrounded by a hexamer-of-A5-trimers. Our findings revive the proposal that Annexin can adopt a so far neverobserved transmembrane state and A5’s multiple functions in and outsidecells.

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2428-Pos Board B444Gain-of-Function of TMEM16E/ANO5 Scrambling Activity Caused by aMutation Associated with the Bone Genetic Disease GnathodiaphysealDysplasiaEleonora Di Zanni, Antonella Gradogna, Joachim Scholz-Starke,Anna Boccaccio.Institute of Biophysics, National Research Council, Genova, Italy.Members of the TMEM16 or anoctamin membrane protein family areinvolved in a variety of functions that include ion transport, phospholipidscrambling and regulation of other membrane proteins. In particular,TMEM16A, 16B and 16F have a clear plasma membrane localization butwhile 16A and 16B work as ‘pure’ ion channels, TMEM16F shows bothion channel and scramblase activities. Recent data suggest that alsoTMEM16E, the closest relative of TMEM16F, may work as a phospholipidscramblase, although a direct confirmation is missing. TMEM16E is highlyexpressed in bone tissue and skeletal muscle and different mutations in the hu-man TMEM16E (ANO5) gene are associated with the bone disease gnathodia-physeal dysplasia (GDD) or to different forms of muscular dystrophy(LGMD2L, limb-girdle muscular dystrophy-2l and distal MMD3, miyoshimuscular dystrophy-3). Our data show that human TMEM16E, when overex-pressed in mammalian cell lines, displays partial plasma membrane localiza-tion and give rise to phospholipid scrambling as well as non-selective ioniccurrents with slow time-dependent activation at highly depolarized membranepotentials and in presence of high intracellular Ca2þ. We address the effect ofthe GDD-causing mutation T513I, located in the second extracellular loop, onTMEM16E and we show phospholipid scrambling activity and large time-dependent ion currents even at low cytosolic Ca2þ concentrations. Our dataprovide the first direct demonstration of Ca2þ-dependent current and phospho-lipid scrambling activity for TMEM16E and suggest a gain-of-functionphenotype related to a GDD mutation.

2429-Pos Board B445Sphingosine-1-Phosphate-Induced ATP Secretion inMicroglia is Mediatedby LRRC8 Proteins of Volume-Regulated Anion ChannelsPhilipp Burow, Manuela Klapperst€uck, Fritz Markwardt.JB Institute for Physiology, Martin-Luther University Halle, Halle (Saale),Germany.Microglia cells are versatile players coordinating inflammatory and regenera-tive processes in the central nervous system. We investigated the activation ofion currents and ATP secretion by sphingosine-1-phosphate (S1P) and hypo-osmolarity in microglia BV-2 cells. S1P and extracellular hypoosmolar solu-tion evoked an anion conductance with almost linearly time-dependentincreasing amplitude. The currents were inhibited by intracellular hypoosmo-lar solution and by use of the anion channel antagonists NPPB and tamoxifenpointing to the activation of volume-regulated-anion channels (VRAC). Theanalysis by qPCR indicated the expression of S1P-receptor-subtypes 1, 2, 4and 5 in the BV-2 cells. The use of the S1PR1-antagonist W123 and theincubation with pertussis-toxin prevented the S1P-currents showing theinvolvement of the Gi-protein-coupled S1PR1 in the activation of VRAC byS1P. According to the progress in the molecular identification of VRAC-channels, we found a high inhibition of S1P- and hypoosmolariy-inducedVRAC-currents after LRRC8A-knockdown whereas knockdown of subtypesB-D did not significantly influence the currents. Subtype E was not presentin BV-2 cells as revealed by qPCR. Furthermore, S1P and hypoosmolarbuffer induced an increase of ATP-levels in the supernatants of BV-2cells being sensitive to treatment with NPPB, tamoxifen and W123. This in-dicates that in microglia cells sphingosine-1-phosphate or hypoosmolaritymediate an ATP-secretion via volume-dependent anion channel LRRC8proteins.

2430-Pos Board B446Zinc Inhibition of an Insect Voltage-Gated Proton ChannelGustavo Chaves1, Stefanie Bungert-Pl€umke2, Arne Franzen2, Boris Musset1.1Institut of Physiology, PMU Nuremberg, Nuremberg, Germany, 2ICS-4,Forschungszentrum Juelich, Juelich, Germany.Zinc is a physiological inhibitor of voltage-gated proton channels (HV1). Asubstantial number of reports have shown that inhibition of HV1 by zincchanges the physiology of several cell types; e.g. diminishing the oxidativeburst in phagocytes, limiting histamine secretion in basophils, reducing inva-siveness of cancer cells, and preventing the final maturation of human sperm.We investigated the zinc inhibition of a new member of the voltage-gated pro-ton channel family, NpHV1, from the insect Nicoletia phytophila. We foundthat NpHV1 is inhibited less by external zinc than the human channel hHV1at pHo 7. Both slowing of activation (tact) and the shift in voltage thresholdare diminished. Aligning the insect channel with the human channel reveals

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that NpHV1 possesses only one of the two externally accessible histidines.Instead of a histidine in the second extracellular loop NpHV1 expresses anaspartate (Asp145). Asp has a very low pKa value which leaves it deprotonatedin solution at pHo 5. The effects of zinc were abolished at pHo 5. This con-tradicts the hypothesis that Asp permits channel inhibition even at low pH.Substitution of Asp145 with His145 restores zinc sensitivity of NpHV1 compa-rable to hHV1. Substitutions of the His92 to Ala92 and His92/Asp145 to Ala92/Ala145 render the channel almost zinc insensitive. The data support the ideathat strong zinc inhibition of the proton channel is dependent on a histidinein the S3-S4 linker.

2431-Pos Board B447Optically Active, Self-Assembled Solid-State Nanopores for Single ParticleDetectionAndreas Schlegel1, Paul V. Gwozdz1, Christian Heyn1, August Dorn1,Andr�e Drews2, Wolfgang Hansen1, Robert H. Blick1.1Center for Hybrid Nanostructures (CHyN) and Institute of Nanostructureand Solid-State Physics, Universit€at Hamburg, Hamburg, Germany, 2Instituteof Applied Physics, Universit€at Hamburg, Hamburg, Germany.Nanopores (NPs) have critically impacted single molecule screening andbecame crucial tools to DNA sequencing research. So far, NPs are used inDC measurements, tracing ionic blockage due to translocating DNA. Biolog-ical NPs are currently used to bring about the potential of the Coulter principlefor sequencing implementations. Typically, those measurements lack speedand multiplexing, reducing their appeal for high throughput applications. Aim-ing to overcome these limitations we present an approach to use a solid-stateNP (SNP) system with optical readout which is based on self-assembly. Viamolecular beam epitaxy, GaAs membranes are grown on a wafer substratewith the SNPs being formed inside by local droplet etching and subsequentrelease of the membranes. The SNPs in our system show photoluminescenceat room temperature and are optically active due to the quantum confined Starkeffect. This we intend to employ in DNA sequencing.We introduce a procedureto transfer the membranes from its wafer substrate onto a transparent polymer.Suspending the membranes while sealing laser-ablated holes in the new sub-strates enables the use of the embedded SNPs in a combined setup of DCand optical readout. Due to the growth procedure, this SNP quantum well setupoffers tunability to vital properties such as pore diameter, emission wavelengthand membrane rigidity, expanding detection applications from quantum dotsand proteins to nucleic acids.

2432-Pos Board B448Exp2 in the Role of the Small Molecule Pore in the ParasitophorousVacuole Membrane of Plasmodium falciparumMatthias Garten1, Josh R. Beck2,3, Svetlana Glushakova1,Armiyaw S. Nasamu2, Jacquin C. Niles4, Daniel E. Goldberg2,Joshua Zimmerberg1.1NICHD, National Institutes of Health, Bethesda, MD, USA, 2Department ofMolecular Microbiology, Washington University School of Medicine, St.Louis, MO, USA, 3Iowa State University, Ames, IA, USA, 4Department ofBiological Engineering, Massachusetts Institute of Technology, Cambridge,MA, USA.Plasmodium falciparum (Pf), the causative agent of malaria, invades red bloodcells (RBCs) to multiply during its 2-day asexual cycle. In the process of inva-sion Pf surrounds itself with the parasitophorous vacuole membrane (PVM) inwhich it remains until the end of the cycle. A VDAC-like PVM pore had beenidentified as permeation pathway for small molecules, such as nutrients, yet itsmolecular identity is unknown. We asked if the pore of the Plasmodium trans-locon of exported proteins (PTEX), Exp2, serves a second role as the smallmolecule pore.PVM permeability was assessed by on-cell patch clamp onto parasites osmot-ically extruded out of the RBC, leaving the PVM intact. We found that grad-uated conditional knock-down and over-expression of Exp2 correlates withthe frequency of finding the PVM pore in patch clamp experiments, whileinterference with PTEX ATPase, HSP101, did not change the frequency bywhich the pore can be seen. Together, these results implicate a role ofExp2 in the PVM pore formation, independent of protein export. To test ifExp2 is part of the PVM pore, we hypothesized that if Exp2 were a part ofthe pore, a truncation of Exp2’s highly charged, but not well-conserved c-ter-minus would reduce the pores coupling to a transmembrane electric field.Indeed, we found that the voltage response of the PVM pore is significantlyreduced around 40 mV.We conclude that Exp2 serves a dual function in protein in protein export andsmall molecule translocation. It remains to be shown if Exp2 forms the porealone or in complex with other proteins.

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2433-Pos Board B449Unitary Water Permeability Measurements via Lipid Vesicle SystemsAndreas Horner, Thomas Barta, Christof Hannesschl€ager, Peter Pohl.Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria.Water transport across lipid membranes is fundamental to all forms of life andplays a major role in health and disease. It is becoming more and moreapparent that not only typical water channels such as aquaporins facilitate wa-ter flux. Transporters1, ion channels2 or receptors can also act as highlypermeable water channels. The efforts directed towards both a mechanisticunderstanding of water flow determinants and the development of waterflow inhibitors would profit from reliable and accurate ways of measuring uni-tary water channel permeabilities pf. A standard way of doing so is to subjectproteoliposomes to an osmotic gradient in a stopped-flow device: Fast record-ings of scattered light intensity are converted into the time course of vesiclevolume change. Some of the underlying empirical procedures misjudge pf byseveral hundred percent. The error may be amplified to reach orders of magni-tude by miscounting the amount of purified and reconstituted water channels.We developed a method that allows accurate determination of pf based on (a)a precise count of channels per lipid vesicle1-3 by fluorescence correlationspectroscopy and (b) an adaptation of the Rayleigh-Gans-Debey equation toaccurately acquire the water efflux from scattered light intensities. Here wedemonstrate possible flaws of widely used simplifications and their impacton published pf values.1. Horner, A. et al. J. Biol. Chem. 291, jbc.M115.706986 (2016).2. Hoomann et al. Proc. Natl. Acad. Sci. U. S. A. 110, 10842-7 (2013).3. Horner, A. et al.Sci. Adv. 1, 1-5 (2015).

2434-Pos Board B450Control of Membrane Permeability via Voltage Regulated LyseninChannelsPhilip Belzeski, Sheenah Bryant, Nisha Shrestha, Daniel Prather,Samuel Kosydar, Daniel Fologea.Boise State University, Boise, ID, USA.Lysenin channels inserted into planar lipid membranes containing asolectin,cholesterol, and sphingomyelin present voltage-induced gating which mani-fests as conformational changes that abrogate the channel’s conductance atpositive bias potentials. This remarkable feature enables controlled transmem-brane transport by modulation of the transmembrane voltage with diffusionpotentials created by using selective transporters. In this work we show thatthe macroscopic conductance of lysenin channels, thus the membrane perme-ability, can be adjusted with electrochemical gradients produced by the actionof specific transporters such as valinomycin. The lysenin channels close andthe macroscopic conductance of the membrane approaches zero as the trans-membrane potential becomes greater than 20 mV (membrane depolarization).After channel closure, the membrane conductance is reinstated by adjustingthe ionic concentrations such that the diffusion potentials drop under 20mV (membrane hyperpolarization). In addition, upon investigating the selec-tivity of lysenin channels to monovalent ions we found that their moderateselectivity may be used to produce diffusion potentials sufficient to elicitconformational changes and modulate the channel conductance. Accordingly,we succeeded to adjust the membrane permeability by sole adjustments of theionic concentrations and in the absence of any other selective transporter. Ourexperiments suggest that lysenin channels may be used to achieve controlledtransport across artificial and natural lipid membranes, with direct applicationsto drug delivery or the design of microbioreactors with controlledpermeability.

2435-Pos Board B451Access Resistance in Atomically Thin NanoporesSubin Sahu1, Michael P. Zwolak2.1IREAP; CNST, University of Maryland College Park; National Institute ofStandards and Technology, Gaithersburg, MD, USA, 2Center for NanoscaleScience and Technology, National Institute of Standards and Technology,Gaithersburg, MD, USA.Access resistance indicates how well current carriers from the bulk medium canconverge to a pore or opening, and it sets the upper limit of the current that canflow into ion channels. In classical electrical conduction, Maxwell - and laterHall for ionic conduction - predicted this access or convergence resistance tobe independent of the bulk dimensions and inversely dependent on the poreradius for a perfectly circular pore when the bulk dimensions are balancedand infinite. These conditions are often not valid in simulations of transportproperties due to the computational cost of large simulation cells, and caneven break down in micro- and nano-scale systems due to strong confinement.More generally, though, this resistance is contextual, it depends on the presence

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of functional groups/charges and fluctuations, as well as the effective constric-tion geometry/dimensions. Addressing the context generally requires all-atomsimulations, but this demands enormous resources due to the algebraically de-caying nature of convergence. We develop a finite-size scaling analysis - remi-niscent of the treatment of critical phenomenon - that makes the convergenceresistance accessible in such simulations. This analysis suggests that there isa ‘‘golden aspect ratio’’ for the simulations cell that yields the infinite systemresult with a finite system. We employ this approach to resolve the experi-mental and theoretical discrepancies in the radius-dependence of graphenenanopore resistance.

2436-Pos Board B452Backbone Amide Nitrogen Atoms are Key Determinants of Inter-AnionDiscrimination in CLCsLilia Leisle1, Eva Fortea1, Jason Galpin2, Christopher Ahern2,Alessio Accardi1.1Dept of Anesthesiology, Weill Cornell Medical School, New York, NY,USA, 2Dept of Mol Phys and Biophys, University of Iowa, Iowa City, IA,USA.Most known Cl- channels are more permeable to other anions, such as I-

or NO3-, than to their namesake Cl- ion. CLC channels, in contrast, are

more permeable to Cl- and have a unique selectivity sequence ofCl->Br->NO3

->I-, suggesting that the mechanism of ion selectivity in theCLCs is different from that of other Cl- channels. It has been proposedthat CLC selectivity is primarily determined by interactions of pore-liningside chains with the permeating ions. Specifically, a serine residue (Sercen)which participates in the central Cl- binding site was shown to control theCl- vs NO3

- selectivity of CLC channels and transporters. Surprisingly, inthe structure of the ClC-K channel, which follows the signature CLC selec-tivity sequence, Sercen points away from the pore. Here, we show that muta-tions at Sercen do not alter the selectivity sequence of the CLC-K channel,suggesting that this residue is not a general determinant of CLC selectivity.Since all CLCs share a common selectivity sequence, we hypothesize that thebackbone amides, which coordinate the ions in the pore, might be theconserved determinants of selectivity. To test this possibility directly, weused atomic-scale mutagenesis with non-canonical amino acids to replacepore-lining amide nitrogen atoms with oxygen atoms, by introducing a-hy-droxy acids in ClC-0 and ClC-K. This maneuver eliminates the hydrogenbonding capability of the backbone NH groups while leaving the side-chain composition unperturbed. We found that CIC-0 and CIC-K channelswith individual backbone substitutions displayed macroscopic currents inXenopus laevis oocytes with a strongly degraded inter-anionic selectivity.The data suggests that the pore-lining backbone amides are the general de-terminants of inter-anion selectivity in CLCs and that side chains contributeto selectivity in only a subset of CLCs.

2437-Pos Board B453Simulating the Permeation of Fosfomycin from the Extracellular Space tothe Site of Action in Gram-Negative BacteriaVinaya Kumar Golla, Karunakar Reddy Pothula, Ulrich Kleinekathofer.Department of Physics and Earth Sciences, Jacobs University Bremen,Campus Ring 1, 28759 Bremen, Germany.The research interest in the development of ‘old’ antibiotic fosfomycin was re-introduced by its activity against multidrug-resistant (MDR) Gram-negativebacteria and against extended spectrum b-lactamase (ESBL) producing patho-gens. Fosfomycin is a phosphonic acid antibiotic used in the treatment of uri-nary tract infections. It has a broad spectrum of antibiotic activity by itsirreversible inhibition of the MurA (UDP-N-acetylglucosamine-3-enolpyruvyltransferase) enzyme, which plays an essential role in the biosynthesis of thepeptidoglycan of the bacterial cell wall. In order to show its mechanism of ac-tion, the foremost important property to consider is the translocation of fosfo-mycin into the bacterial cell. Hence, we studied the translocation of fosfomycinthrough E. coli outer membrane and inner membrane proteins using all-atommolecular dynamics simulations. Our findings help in understanding the com-plete translocation path of fosfomycin from the extracellular space to the intra-cellular site of action.

2438-Pos Board B454Unitary Water Channel Permeability and Arrhenius Activation Energyare Intricately LinkedAndreas Horner, Peter Pohl.Biophysics, Johannes Kepler University, Linz, Austria.Measurements of the Gibbs activation energy barrier DGz

t constitute the tradi-tional way of showing the presence of an aqueous pore. The expected DGz

t

value of about 4 kcal/mol corresponds to the activation energy for the

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self-diffusion of water. However, structural investigations within the last twodecades have revealed radical differences in the water environment for mol-ecules in the narrow channel lumen, which casts doubt on the validity ofthe standard argumentation. Water molecules may lose up to two of theirfour hydrogen bonds when entering channels where they are lined up in singlefile. Moreover, the number of hydrogen bonds which permeating water mol-ecules may eventually form with pore-lining residues is known to determinethe unitary water permeability pf.

1 Here we present a different line of argu-mentation in favor of DGz

tz4 kcal/mol: high pf values indicate bulk-like wa-ter mobility. pf can be converted into the transport rate r. Transition statetheory intricately links r to DGz

t: r=noexp(-DGzt/kBT) where T, kB, and

noz1013s�1 are the absolute temperature, the Boltzmann constant, and theuniversal transition state theory attempt frequency. Calculating theoreticalpf values from DGz

t, we find a satisfactory match to experimentally obtainedpf values of aquaporins

1, potassium channels1, gramicidinA2 and other chan-nels that we have measured to bolster the experimental basis for the link be-tween r and DGz

t. Only channels in which water retains bulk mobility requireDGz

tz4 kcal/mol, while less permeable channels exhibit a higher barrier towater transport.1. Horner, A.; ...; Pohl, P., The mobility of single-file water molecules is gov-erned by the number of H-bonds they may form with channel-lining residues.Science Adv. 2015, 1, e1400083.2. Pohl, P.; Saparov, S. M., Solvent drag across gramicidin channels demon-strated by microelectrodes. Biophys. J. 2000, 78, 2426.

2439-Pos Board B455Rectifying Ionic Current in Conical Sub-Micropores Functionalized withPoly-L-LysineChih-Yuan Lin, Cody Combs, Zuzanna S. Siwy.Physics, University of California Irvine, Irvine, CA, USA.Inspired by biological ion channels, synthetic pores/channels functionalizedwith various chemical groups including DNA and proteins, attracted a lotof scientific interest in using them as platforms for rectifying ionic currentand sensing biomolecules. Such pores also provide a model system that allowsfundamental understanding of electrokinetic transport of ions and fluid in aconfined space. Conically shaped nanopores are often investigated due tothe ion current rectification that they exhibit: ion currents for one voltage po-larity are higher than currents for the opposite voltage polarity. Ion currentrectification is typically associated with nanoscale of pores. In this study weshow experimental and theoretical studies of ion current rectification in coni-cally shaped sub-micropores with opening diameter as large as 450 nm. Thepores rectify due to modification with high-molecular weight poly-lysine, sothat asymmetric current-voltages curves are observed at concentrations ashigh as 1 M, thus at conditions where the thickness of the electricaldouble-layer is hundreds time smaller than the pore opening. We show thattransport properties of the mesopores are determined by the space charges car-ried by poly-L-lysine so that at no voltage applied a fraction of the pore vol-ume is filled primarily with counterions. In addition, the currents approach aplateau at high voltages, especially pronounced at KCl concentrations below100 mM. This finding points to the importance of concentration polarizationcreated in the pore and at the pore entrance. For elucidating the underlyingmechanisms, a continuum-based model based on a set of Poisson-Nernst-Planck, Stokes, and Brinkman equations was adopted. The modelingconfirmed that when the pores were modified with poly-L-lysine, voltage-dependent accumulation and depletion of ions is observed. The results extendthe effect of ion current rectification to high concentrations of 1 M andmesoscale.

2440-Pos Board B456Phosphatidylinositol-(4,5)-Bisphosphate is a Necessary Cofactor forTMEM16F Ion Channel ActivitiesWenlei Ye, Tina W. Han, Layla M. Nassar, Mario Zubia, Yuh Nung Jan,Lily Y. Jan.Physiology, UCSF, San Francisco, CA, USA.Membrane scrambling, the collapse of phospholipid bilayer asymmetricity, isessential for important cellular functions including blood coagulation andcell apoptosis. TMEM16F, whose activation by elevation of intracellular cal-cium triggers membrane scrambling so as to cause exposure of phosphatidyl-serine to the cell surface, also mediates a small conductance calcium-activated cation current. How TMEM16F activation may be regulated is anopen question. We found that the TMEM16F response to Ca2þ is desensitizedby a brief exposure to high intracellular Ca2þ, which is caused by the depletionof phosphatidylinositol-(4,5)-bisphosphate, PIP2, from the inner leaflet of themembrane. The application of artificial or natural PIP2 restores TMEM16Fchannel activity. This TMEM16F modulation by PIP2 requires the presence

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of several positively charged amino acids at the cytoplasmic N-terminus. Ourstudy reveals the dependence of TMEM16F activity on phosphoinositide,and provides one mechanism for TMEM16F activation to be strictly regulatedin the cell membrane.

2441-Pos Board B457Cooperativity and Flexible Domains Participation in PIP AquaporinGatingKarina Alleva1, Florencia Scochera1, Agustina Canessa Fortuna1,Cintia Jozefkowicz2, Victoria Vitali1, Gerardo Zerbetto de Palma1,Gabriela Soto3, F. Luis Gonzalez Flecha1.1Universidad de Buenos Aires - Conicet / Facultad de Farmacia yBioquimica, Caba, Argentina, 2Conicet / Inta, Buenos Aires, Argentina,3Conicet / Inta, Bueonos Aires, Argentina.From structural data two distinct mechanisms have been proposed to describehow aquaporins are gated (Frick et al. FEBSLett. 2013). Gating of some aqua-porins -AQP0 or AQPZ- is mediated by the displacement of a single residueinside the pore (pinching), while for others -e.g. PIP- the blocking of the poreoccurs as the result of loopD rearrangement (capping). Regarding PIP cappingmechanism, it has been suggested that the open-closed transition not only in-volves loopD but also N and C-terminal movements. The PIP subfamily con-sists of two paralogs, PIP1 and PIP2, which can form heterotetramers.Heterotetramerization occurs with random stoichiometry and all formedheterotetramers have different water transport capability and pH0,5 thanPIP2 homotetramers (Jozefkowicz et al. Biophys J. 2016). Since there is nostructural information available for PIP1, many of the molecular events thatexplain changes in the pH sensing in heterotetramers are still unknown.We investigate the cooperative gating of homo and heterotetramers. Inparticular, we study the participation of flexible domains in this process.Our results show that all homo and heterotetrameric species share the samedegree of cooperativity for proton sensing. However the cooperativity isapparently lower when PIP1-PIP2 heterotetramers and PIP2 homotetramersare both present in the membrane due to their different pH0,5. In addition,we detected loopD participation in PIP2 gating is more efficient than C-term, since mutation of Leu202 keep the channel open even at low pH, whiletruncation of C-term do not modify pH sensing. The elucidation of the pecu-liarities of PIP gating sheds light on the regulatory mechanisms that controlwater transport trough membranes and extends the knowledge about theparticipation of flexible domains in the control of the biological activity ofoligomeric channels.

2442-Pos Board B458A New Method to Study the Lysomal Electrical Activity in Living CellsElla Matamala1,2, Cristian Castillo3, Kirill Kiselyov4, Sebastian Brauchi1,2.1Physiology Department, Faculty of Medicine., Universidad Austral de Chile,Valdivia, Chile, 2Millennium Nucleus of Ion Channels-associated Diseases(MiNICAD)., Valdivia, Chile, 3Department of Electronic Engineering,Universidad Austral de Chile, Valdivia, Chile, 4Department of BiologicalSciences, University of Pittsburgh, Pittsburgh, PA, USA.Lysosomes are acidic organelles responsible of the macromolecule digestion,nutrient recycle and cellular clearance. To perform these functions, they ex-press an array of degradative enzymes and several membrane proteinsincluding ion channels, pumps and transporters, that allow a regulated ions ex-change in and out of the lysosomal compartment, stablishing a voltage gradientthrough the membrane of the organelle. Lysosomes also are involved incoupling the cellular nutrition status to global cellular responses throughsignaling pathways under the control of the master regulator mTOR. It hasbeen described that nutrient deprivation or decrease in ATP levels inactivatemTOR, leading to an increase of sodium conductance in lysosomes (Lyso-NaATP) through a protein complex formed by TPC 1 and 2 channels. The acti-vation of the voltage-sensitive TPC1 channels, generates an important drop inthe lysosomal membrane potential, supposedly triggering the calcium efflux tothe cytosol via TRPML1. We wonder whether the activity of calcium-dependent Cl- channels is associated to lysosome electrical signaling. Toanswer this, we adapted a non-invasive method to evaluate changes in the mem-brane potential (DJ) of subcellular structures in intact cells. The method takesadvantage of a FRET pair consisting of a GFP-Lamp1 fusion acting as a donorand a colorless hydrophobic anion as an acceptor that rapidly moves across themembrane in response to changes in polarity, altering GFP emission. We testedthis approach by reporting DJ of lysosomal membranes in HEK-293T cells inresponse a pharmacologic inactivation of mTORC1/TRPML1 pathway withmTORC1 inhibitors (i.e. rapamacyn). Additionally, we evaluated changes inluminal pH, calcium and chloride permeability of lysosomes by fluorescencemicroscopy.

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2443-Pos Board B459Cryo Electron Tomography and Reaction-Diffusion Simulations Reveal aMolecular and Evolutionary Basis for Charged Archaeal Surface LayerProteinsPo-Nan Li1,2, Jonathan R. Herrmann2,3, Frederic PB Poitevin3,Rasika Ramdasi4, Bradley B. Tolar5, John Barger2, David Stahl6,Grant Jensen4, Soichi Wakatsuki2,3, Henry van den Bedem2.1Electrical Engineering, Stanford University, Stanford, CA, USA, 2SLACNational Accelerator Laboratory, Menlo Park, CA, USA, 3StructuralBiology, Stanford University, Stanford, CA, USA, 4Howard Hughes MedicalInstitute, Division of Biology and Biological Engineering, California Instituteof Technology, Pasadena, CA, USA, 5Earth Systems Science, StanfordUniversity, Stanford, CA, USA, 6Civil and Environmental Engineering,University of Washington, Seattle, WA, USA.Surface layers (S-layers) are 2D, proteinaceous lattices that form the outermostcell envelope component of manymicrobes. S-layers, which exhibit exceptionalsequence diversity, are found in nearly all archaea and numerous bacteria.Despite their variation, S-layer proteins display several unifying characteristicssuch as their ability to form crystalline sheets punctuated with nano-scale pores,and their propensity for charged amino acids. However, the precise role of thesecharged functional groups and how they relate to cellular function is unknown.Here, we offer a rationale for charged S-layer proteins in the context of the struc-tural evolution of S-layers.We have chosen ammonia-oxidizing archaea (AOA)as a model system for S-layer and used the cryo electron tomographic recon-struction of the AOA to develop a 2D electro-diffusion reaction computationalframework to simulate diffusion and consumption of the charged solute ammo-nium. The AOA create energy directly from electrons evolved during ammoniaoxidation by ammonia monooxygenase (AMO). While the specific location ofthe archaeal AMO active site is unknown, a bacterial homologue indicates alocation underneath the S-layer in pseudo periplasmic space (PPS). Our simula-tions suggest that charged S-layers and nanopores expedite diffusion of chargedsolutes into the PPS when the electro-diffusion-reaction system is driven awayfrom equilibrium, replenishing reacted NH4

þ in PPS. By contrast, a neutralS-layer would inhibit diffusion of charged molecules, while removing theS-layer altogether dramatically reduced ammonium concentration throughoutthe PPS. Strikingly, analysis of annotated S-layer amino acid sequences fromall known archaeal clades indicated a clear dearth of sequences in the neutralregime. Our simulations suggest that charged S-layers and nanopores impart apotential fitness advantage. Thus, S-layer charge may have emerged by conver-gent evolution to enhance metabolic function in diverse ecosystems.

2444-Pos Board B460Subunit Dependent Regulation of LRRC8 Mediated VRAC Currents byOxidationAntonella Gradogna, Paola Gavazzo, Anna Boccaccio, Michael Pusch.Istituto di Biofisica, Genoa, Italy.Volume-regulated anion channels (VRAC) are heteromers composed of theessential LRRC8A subunit and at least one among the LRRC8B-E subunits[1]. Reactive oxygen species (ROS) play physiological and pathophysiologicalroles and VRAC channels are known to be highly ROS sensitive. Using fluo-rescently tagged LRRC8 proteins that give rise to constitutive currents in Xen-opus oocytes [2] we tested direct effects of oxidation on different subunitcombinations of LRRC8 channels. 8A/8E heteromers were dramatically poten-tiated (more than 10-fold) by oxidation of intracellular cysteine residues bychloramine-T or tert-butyl hydroperoxide. Oxidation was not necessary for hy-potonicity induced activation. In contrast, 8A/8C and 8A/8D heteromers werestrongly inhibited by oxidation. In agreement with the non-inactivating currentcharacteristic of VRAC in T lymphocytes that is a typical feature of the 8C sub-unit, we found that VRAC currents in Jurkat cells are inhibited by oxidation andthat the 8E subunit is the least expressed in Jurkat cells. A reduction of VRACduring acute T cell activation associated with ROS production might be ofphysiological relevance in the immune response.References[1] Voss FK et al. 2014. Science 344, 634-8[2] Gaitan-Penas H et al. 2016. Biophys J 111, 1429-43

2445-Pos Board B461Subtle Modifications of the Pannexin-1 N-Terminus Results in AlteredChannel ActivityKevin Michalski, Toshi Kawate.Cornell University, Ithaca, NY, USA.Pannexins are a family of large oligomeric channels which form an ATPpermeable pore in a variety of cell types including neurons, macrophages,and epithelial cells. Released ATP can act as a signaling molecule, thus

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involving pannexins in various purinergic signaling pathways including neuro-transmission and vasoconstriction. Of the pannexin family, pannexin-1 is mostwidely studied due to its various activation mechanisms and readily observablecurrents when expressed in heterologous systems. However, currently, contra-dictory data from various labs suggest pannexin-1 activation can be achievedby mechanisms which appear to be mutually exclusive. Additionally, reportsalso suggest that mouse and human pannexin-1 have wildly different activationproperties despite being over 85% identical in primary sequence. Here, weshow that in HEK cells wild-type mouse and human pannexin-1 share similaractivation properties, with both channels presenting significant CBX-sensitivecurrents when stepped to voltage potentials greater than þ120 mV. Addition-ally, we report our findings that the channel properties of pannexin-1 can besignificantly altered by inserting amino acid into its N-terminus, in some casesresulting in stronger channel activity. Our data suggests a significant role for theN-terminus in pannexin-1 gating which may explain functional discrepanciesapparent in the literature.

Posters: Cardiac Muscle Mechanics andStructure II

2446-Pos Board B462Pathogenic Mechanisms of the Cardiomyopathy-Associated Alpha-Tropo-myosin Variant E192K as Revealed by Multiscale Modeling and Experi-mentsLorenzo R. Sewanan1, Jinkyu Park2, Michael J. Rynkiewicz3,Stephen M. Hollenberg4, Nikolaos Papoutsidakis2, Daniel L. Jacoby2,Jeffrey R. Moore5, William Lehman3, Yibing Qyang2, Stuart G. Campbell1.1Biomedical Engineering, Yale University, New Haven, CT, USA,2Cardiovascular Research Center, Yale University, New Haven, CT, USA,3Physiology & Biophysics, Boston University, Boston, MA, USA,4Biological Sciences, University of Massachusetts Lowell, Lowell, MA,USA, 5Department of Biological Sciences, University of MassachusettsLowell, Lowell, MA, USA.Missense mutations to alpha-tropomyosin (TPM1) are implicated in the devel-opment of hypertrophic cardiomyopathy (HCM). Linking HCM to mutation-induced molecular changes is challenging but critical for improved treatment.We have used modeling and experiments at the molecular, ensemble, and tissuelevels to reconstruct the molecular pathophysiology of the TPM1 E192Kvariant. Beginning at the atomic scale, molecular dynamics simulations pre-dicted a steep drop in tropomyosin (TM) stiffness after introduction ofE192K. Regulated in vitro motility assays showed increased motility ofTPM1 E192K-decorated actin filaments at low calcium. Fitting of these datawith a model of thin filament activation suggested that measured actin dysregu-lation was consistent with the predicted TM stiffness loss and a simultaneousalteration in TM affinity for the blocked (inactive) actin state. The modelfurther predicted that these molecular changes would lead to increased systolicand diastolic twitch force production. In order to test these predictions in thecontext of human tissue, we generated induced pluripotent stem cell cardio-myocytes (iPSC-CMs) from a young TPM1 E192K-positive HCM patient. En-gineered heart tissues (EHTs) were created from patient IPSC-CMs andcultured for 16 days under electrical pacing. Contractile characterization ofEHTs revealed that the peak force generated by mutant EHTs was several timeshigher than in two independent sets of non-mutant control EHTs. Contractionkinetics were also changed, exhibiting significantly slower time to peak andrelaxation time. Furthermore, the stiffness of the mutant EHTs during diastolesignificantly increased. Altogether, experimental observations and modelingindicate that E192K leads to lower TM stiffness and a consequent inabilityof the actin filament to inhibit myosin-based force production. We posit thatthis multiscale, multi-modal approach may be generally useful for predictingand understanding pathogenicity of TPM1 variants in cardiomyopathy.

2447-Pos Board B463Native Redox Posttranslational Modifications as Regulators of TitinMechanical PropertiesElıas Herrero-Galan1, Cristina Sanchez-Gonzalez1,Diana Velazquez-Carreras1, Elena Bonzon-Kulichenko2, Enrique Calvo3,Jesus Vazquez2, Jorge Alegre-Cebollada1.1Molecular Mechanics of the Cardiovascular System, Centro Nacional deInvestigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain,2Cardiovascular Proteomics, Centro Nacional de InvestigacionesCardiovasculares Carlos III (CNIC), Madrid, Spain, 3Proteomics TechnicalUnit, Centro Nacional de Investigaciones Cardiovasculares Carlos III(CNIC), Madrid, Spain.

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The relevant role of titin in the contractility of cardiac muscle has been recentlyevidenced by the discovery of mutations in the titin gene that lead topathologies involving changes in the elasticity of the heart. The elastic proper-ties of titin depend on the folding/unfolding equilibria of its immunoglobulin(Ig) domains. Strain-induced posttranslational modifications of buried cyste-ines have been recently shown to be key regulators of the folding dynamicsof titin Ig domains. However, the identity, extent and specific residues targetedby these modifications in vivo remain unexplored. We have optimized a methodfor in-gel detection of oxidized thiols by fluorescent labeling, which has al-lowed us to provide the first experimental evidence that a significant fractionof titin’s cysteines are oxidized. By mass spectrometry and high resolutionstructure modelling we study the redox state of specific cysteine residues andpredict the presence of disulfides in the different Ig domains of titin. Further-more, by means of single-molecule atomic force spectroscopy, we aim to deter-mine the effect of these modifications in the elasticity of the protein. Thisapproach may lead to a better understanding of how the contractility of theheart is modulated in physiology and disease.

2448-Pos Board B464High-Resolution Structural Basis of a Dual Titin/Obscurin Complex withTwo Well-Separated SitesPhilipp Hornburg1, Atsushi Fukuzawa2, Mathias Gautel2,Matthias Wilmanns1,3.1EMBL Hamburg, European Molecular Biology Laboratory, Hamburg,Germany, 2King’s College London, London, United Kingdom, 3UniversityMedical Centre Hamburg-Eppendorf, Hamburg, Germany.The sarcomere is a highly complex cellular network in striated and cardiacmuscles. The giant muscle protein titin guides numerous proteins to their spe-cific spatial location within this underlying structural network in the sarcomereusing specific protein/protein interaction sites. Previous studies revealed twoseparate interaction sites between titin and the dynamic filament protein ob-scurin: one is formed by the N-terminal obscurin domain Ig1 and the C-termi-nal titin domainM10 at the sarcomeric M-band and its high-resolution structurehas been determined; one is formed by the obscurin domains O58-O59 and titindomains Z8-Z9 at the sarcomeric Z-disk, however, without known molecularstructure.To provide insight into this interaction, we obtained the high-resolution crys-tal structure of the Z-disk titin Z8-Z9 / obscurin O58-O59 complex using X-ray crystallography. The structure reveals three distinct domain/domain inter-faces: between Z8/O58, Z9/O58, and Z9/O59. The binding affinity for com-plex assembly is about 2 mM, thus comparable to that of the M-band titin/obscurin assembly site. Targeted interface mutations at Z9/O59 led to a com-plete loss of complex formation whereas mutations introduced at either Z8/O58 or Z9/O58 still allowed assembly with reduced binding affinities. Thesefindings indicate that the Z9/O59 interface is crucial for complex formationwhile the Z8/O58 and Z9/O58 interfaces appear rather supportive. Based onthese observations, the nature of the Z-disk titin/obscurin site is considerablymore complex than the M-band site. Experiments validating these observa-tions in cell lines are ongoing and will be presented. Taking our data together,the observations on two distinct titin/obscurin sites lead to an attractive modelto consider the overall spatial roles of these two filaments in connection to theoverall architecture of the sarcomere and possible connections to neighbour-ing cell organelles.

2449-Pos Board B465Impact of Dilated Cardiomyopathy Mutation and Small Molecule Regu-lator on Human Beta-Cardiac MyosinWanjian Tang, William C. Unrath, Rohini Desetty, Christopher M. Yengo.Department of Cellular and Molecular Physiology, Penn State College ofMedicine, Hershey, PA, USA.Missense mutations in human b-cardiac myosin are associated with inheritedcardiomyopathies. The mechanisms for how mutations alter myosin’s motorperformance are still unclear. We expressed and purified human beta-cardiac myosin subfragment 1 (M2b-S1) containing a point mutation in theconverter domain (F764L) that is associated with dilated cardiomyopathy(DCM) and compared it to wild-type (WT) M2b-S1. We demonstrate thatthe F764L mutation slows down maximum actin-activated ATPase activity(20-30%). Direct measurements of ADP release in the presence of actin usingmant labeled ADP demonstrate the F764L mutant has a 20% slower ADPrelease rate constant. The in vitro motility assay demonstrates a 10-15%slower actin sliding velocity for F764L. Monitoring ATP binding and hydro-lysis with intrinsic tryptophan fluorescence demonstrates that the F764L mu-tation causes a 2-fold increase in K0.5 without changing the maximal rate. Wealso examined the impact of the heart failure drug Omecamtiv Mecarbil (OM)

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on the F764L mutant. The steady-state actin-activated ATPase results suggestthat the F764L mutant shares a similar OM binding affinity with WT. Inter-estingly, the drug does not reduce the maximum ATPase activity and actinsliding velocity as much as it does with WT. We observed no difference inthe ionic strength dependence or density dependence of in vitro motility inWT and F764L in the presence and absence of OM. Our previous results sug-gested OM dramatically slows down the ADP isomerization step in WT M2b-S1, making it the rate-limiting step. Our results demonstrate OM does notslow the ADP isomerization step as much in F764L, but this step is stillrate-limiting. Therefore, we propose that OM will increase the myosin dutyratio in the F764L mutant enough to enhance isomeric force while onlymoderately slowing shortening velocity.

2450-Pos Board B466The Giant Protein Titin Regulates the Length of the StriatedMuscle ThickFilament-Titin RulesHenk Granzier, Paola Tonino, Balazs Kiss, Joshua Strom, John Smith,Mei Methawasin, Justin Kolb.Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA.The contractile machinery of heart and skeletal muscles that powers life hasas its most essential component the thick filament, comprised of the molec-ular motor myosin. The thick filament is of a precisely controlled length,defining thereby the force level that muscles generate and how this forcevaries with muscle length. It has been speculated that the mechanism bywhich the length of the thick filament is so exquisitely controlled involvesthe giant protein titin, but no conclusive support for or against exists. Herewe show studies on a mouse model in which we deleted two of titin’sC-zone super-repeats, located in the A-band region of the sarcomere. Struc-tural studies using immunoelectron microscopy and super-resolution opticalmicroscopy in both cardiac and skeletal muscles reveal a thick filamentlength that is reduced by �170 nm; functional studies reveal reduced forcegeneration and a dilated cardiomyopathy (DCM) phenotype. Our studiesshow for the first time the important role of titin in regulating thick filamentlength, with each of titin’s C-zone repeats being responsible for a quantal43 nm thick filament length. We conclude that thick filament length regula-tion is titin-based and that this mechanism is crucial for maintaining musclehealth.

2451-Pos Board B467Similar Effects of Humoral or Mechanical Stress on Cell-Cell Contacts inCultured CardiomyocytesOliver Koldyka, Pragati Pandey, Thomas Iskratsch, Elisabeth Ehler.Randall Centre for Cell and Molecular Biophysics, King’s College London,London, United Kingdom.We have previously characterised the specific changes that occur at the cell-cellcontacts (intercalated discs) of cardiomyocytes in dilated cardiomyopathy.These include increased membrane convolution and increased presence ofactin-associated proteins as well as signalling molecules such as PKCalphaat these sites. Aim of the current project is to study these alterations as theyhappen during humoral (excessive beta-adrenergic stimulation) or mechanical(substrates that are mimicking a fibrotic environment) stress on neonatal rat car-diomyocytes in culture. After only three days exposure to stress, we alreadyobserved increased membrane convolution at the cell-cell contacts, assuggested by a broader signal for e.g. beta-catenin and also increased totalbeta-catenin expression levels as analysed by immunoblotting. The signal forphosphorylated FHOD1, the active form of a formin that is located at the inter-calated disc, was also increased at cell-cell contacts of stressed compared tocontrol cardiomyocytes. These changes in cytoarchitecture were accompaniedby increased targeting of phosphorylated PKCalpha to the cell-cell contacts.These data suggest that the cardiomyocytes respond very rapidly to humoralor mechanical stress in the 2D environment in vitro and that mainly the myofi-bril anchorage sites at the cell-cell contacts are reinforced following stressinduced by arrhythmic contractions. This may be a useful model system tostudy the time course of events that lead to changes in cytoarchitecture duringcardiomyopathy.

2452-Pos Board B468Mechanobiology of Myosin Mutations and Myofibril Remodeling in iPSC-CardiomyocytesAlison Schroer1, Kristina Kooiker2, Arjun Adhikari3, Kathleen Ruppel3,Daniel Bernstein2, James Spudich3, Beth Pruitt1.1Bioengineering, Stanford University, Stanford, CA, USA, 2Pediatrics,Stanford University, Stanford, CA, USA, 3Biochemistry, StanfordUniversity, Stanford, CA, USA.

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Hypertrophic cardiomyopathies are the most prevalent form of heritable cardio-vascular disease and are caused by a host of mutations that result in hypercon-tractility, though the largest number of known mutations are found in betacardiac myosin heavy chain (bMYH). Myosin is a key protein responsiblefor muscle contraction and is arranged into a dynamic, hierarchical structureof sarcomeres and myofibrils within cardiomyocytes. We have previouslymeasured differences in the kinetics of isolated myosin proteins with differenthypertrophic cardiomyopathy mutations in bMYH, but it has been difficult todetermine how these alterations manifest at the cellular level. Induced plurip-otent stem cell-derived cardiomyocytes provide a powerful tool for studyingspecific features of human cardiomyocyte biology including contractility, hy-pertrophic growth, and intracellular organization. We have developed a micro-patterned hydrogel platform that promotes myofibril alignment, cardiomyocyteforce generation, and other metrics of maturation. Using CRISPR-Cas9 gene-editing, we have created cell lines with specific bMYH mutations, includingD239N and P710R. We used traction force microscopy in a preliminary studyand measured increased forces in the mutant cells compared to isogenic con-trols, and we have observed larger cell size in one of the mutant lines in bothpatterned and unpatterned contexts. Infection with LifeAct adenovirus also al-lows for the visualization of actin structures including myofibrils and sarco-meres during cell spreading and the recovery of beating in cells recentlytransferred to patterned substrates. Immunostaining for bMYH revealed higherexpression and more regular organization of sarcomeres in patterned cells, andwe plan to use these techniques to quantify differences in myofibril and sarco-mere organization in cells with bMYH mutations. This work provides valuableinsight into the etiology of hypertrophic cardiomyopathy and the utility ofinduced pluripotent stem cell-derived cardiomyocytes as a model for humancardiac biology and disease.

2453-Pos Board B469The Role of cTnT Isoform Switching in Modulating Sarcomeric Cardio-myopathiesMelissa L. Lynn1, Teryn A. Holeman2, Grace Benitez3,Mark T. McConnell4, Lauren Tal-Grinspan5, Jil C. Tardiff1.1Medicine, University of Arizona, Tucson, AZ, USA, 2Medicine, Universityof Arizona, Marana, AZ, USA, 3University of Arizona, Tucson, AZ, USA,4Biomedical Engineering, University of Arizona, Tucson, AZ, USA,5Medicine, Columbia University Medical Center, New York, NY, USA.An oft-noted component of sarcomeric dilated (DCM) and hypertrophic (HCM)cardiomyopathies is that the same primary mutation can exhibit significantphenotypic variability. This lack of a distinct link between genotype and pheno-type suggests independent modifiers exist that differentially affect susceptibleindividuals. The tropomyosin (Tm) mutation Asp230Asn (D230N), exhibits a‘‘bimodal’’ distribution of severity primarily impacting children. Of note, sur-vivors often recover significant systolic function by adolescence. To explorethe molecular mechanism of this recovery (despite the persistence ofD230N-Tm) we hypothesized that the age-dependent remodeling is causedby the Tm binding partner, cardiac Troponin T, transitioning from its fetal(cTnT1) to adult (cTnT3) isoform. Divergent cardiac remodeling was observedin D230NþcTnT1 (DTg) mice, where DTg mice exhibited an additivelyreduced %FS compared to D230NþcTnT3 mice. We investigated these func-tional changes at a molecular level via differential scanning calorimetry(DSC). cTnT1 alone had little effect on the structure or function of the thin fila-ment (TF) resulting in a slight increase in flexibility of the overlap. However,D230NþcTnT1 filaments exhibited an additive decrease in flexibility of theTm-overlap. Coupled with this decreased flexibility was a cTnT1-induced ad-ditive reduction in the calcium sensitivity of sliding velocity likely due to dis-rupted flexibility in the overlap. In contrast, the HCM-linked cTnT mutationArg92Leu (R92L), also proximal to the Tm-overlap, resulted in an elevated%FS. This supra-normal systolic function decreased toward non-transgeniclevels in R92L-cTnT1 mice. Given these functional differences, we suspect a‘‘pinning’’ of the Tm-overlap in the presence of cTnT1 that lends stabilitywhen a mutation that perturbs the flexibility of the Tm-overlap is present. Thesedata suggest a potent modifier effect of fetal cTnT, capable of altering diseaseseverity in a mutation specific manner likely via effects at the crucial Tm-overlap.

2454-Pos Board B470Differences in Myofilament Interactions and Structural Dynamics betweenMouse and Human Cardiac Myosin-Binding Protein CThomas C. Bunch, Victoria C. Lepak, Brett A. Colson.Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA.Mutations in the gene encoding the sarcomeric protein cardiac myosin-bindingprotein C (cMyBP-C) are a leading cause of hypertrophic cardiomyopathy

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(HCM). Mouse models targeting cMyBP-C and use of recombinant proteinshave been effective to study its roles in normal contractile function and disease.However, it remains unclear how species-specific sequence differences influ-ence cMyBP-C structure and function in myocardium. We have used a newcompetition assay to resolve differences in myofilament binding betweenmouse and human cMyBP-C. Using chimeric fusion proteins, we investigatedeffects of species-specific sequences on cMyBP-C interactions with myosin oractin filaments. We have also performed time-resolved FRET (TR-FRET) toinvestigate the species-specific differences in protein structural dynamics be-tween domains C0 and C1. These Ig-like domains are separated by theproline-alanine-rich linker (P/A), a region with low sequence homology anddifferential effects on actomyosin reported between the mouse and human iso-forms. For TR-FRET structural measurements, we engineered pairs of cyste-ines in C0-C2 (cMyBP-C domains C0 through C2) for site-directed labelingwith fluorescent donor-acceptor probes. Fluorescence lifetime analysis oflabeled C0-C2 revealed profound species-dependent effects on structure anddynamics. With probes reporting distances and dynamics from C0 to C1, hu-man C0-C2 was much more compact and less disordered than mouse C0-C2.Our findings suggest that care should be taken with interpretation of mouse iso-form studies for extrapolation to cMyBP-C and HCM mechanisms in human.The importance of these uniquely evolved isoforms is underscored by the diver-gent functional properties, governed in part by species-specific P/A content andN-terminal structural dynamics, and thereby provides additional tuning of con-tractile performance. This work was supported by an NIH R00HL122397 andUA Sarver Heart Center ‘‘Novel Research Project Award in the Area of Cardio-vascular Disease and Medicine’’ to B.A.C.

2455-Pos Board B471Genetically Engineered Human Stem-Cell Derived Cardiomyocytes toInvestigate the Function of Cronos TitinRebecca Zaunbrecher1, Kevin Beussman2, Andrea Leonard2,Marion von Frieling-Salewsky3, Lil Pabon4, Hans Reinecke4, Xiulan Yang4,Wolgang A. Linke3, Nathan J. Sniadecki2, Charles E. Murry4,Michael Regnier1.1Bioengineering, University of Washington, Seattle, WA, USA, 2MechanicalEngineering, University of Washington, Seattle, WA, USA, 3Institute ofPhysiology II, University of Muenster, Muenster, Germany, 4Pathology,University of Washington, Seattle, WA, USA.Familial dilated cardiomyopathy (DCM) is most commonly caused by trun-cating mutations in the gene encoding for the sarcomere protein titin (TTN).Mutations in the A-band region of TTN are more commonly associated withdisease than those in the Z-disk or I-band domains. To investigate the functionof titin in human cardiomyocytes (CMs), we used CRISPR/Cas9 to generatehomozygous truncations in the Z-disk (TTN-Z-/-) and A-band (TTN-A-/-) re-gions of the TTN gene in human induced pluripotent stem cells. Following dif-ferentiation, we were surprised to find that TTN-Z-/- CMs had sarcomeresand visibly contracted despite carrying the more upstream truncation, whileTTN-A-/- CMs did not form functional myofibrils. Through the use of a customantibody we demonstrate that a novel isoform of titin, Cronos, is exclusivelyexpressed in TTN-Z-/- CMs, and is expressed in combination with full-lengthtitin in wild type cells. TTN-Z-/- CMs produce lower contractile force andhave perturbed myofibril morphology compared to controls, indicating thatCronos titin is able to partially replace full-length titin. Additionally, Cronostitin is highly expressed in early human fetal cardiac tissue, but is down regu-lated in later fetal and adult samples, suggesting it is predominantly a develop-mental isoform. We are currently investigating the function of Cronos titin inheart development using a CRISPR interference-based system to specificallyknockdown this isoform in stem cell-derived CMs. Future studies will investi-gate the role of Cronos titin in heart disease and whether Cronos titin expressionis able to partially rescue deficiencies in structure and function resulting fromtitin truncations located in the Z-disk and I-band, making mutations in these re-gions of TTN less pathogenic.

2456-Pos Board B472Ultrastructural Surveys of Cardiomyocytes Isolated from Failing HumanHeartsPatrick Robison, Christina Yingxian Chen, Matthew A. Caporizzo,Kenneth Bedi, Kenneth B. Margulies, Benjamin L. Prosser.Physiology, University of Pennsylvania, Philadelphia, PA, USA.Previously our laboratory has reported on the role of microtubules and theirviscoelastic properties in cardiac mechanics. Current work aims to extend thesefindings to myocytes derived from human patients, where biochemical assayssuggest robust alterations in the relative abundance of various cytoskeletal el-ements. This work has afforded access to a steadily increasing pool of clinical

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samples exhibiting a wide range of functional impairment, some of which wehave been able to mount for electron microscopy. The resulting images showclear ultrastructural detail including mitochondria, microtubules and the myo-fibrils. In order to produce unbiased surveys of these structures we turned tomachine learning through trainable image segmentation software (Weka).We find that the method identifies ultrastructural features faithfully and repro-ducibly in the heavily stereotyped organization of striated muscle in an unbi-ased fashion. Differences in the proportions, packing density andarrangement of sarcometic, membranous and other cytoskeletal elements arepresented with implications for how these changes may contribute to the pas-sive mechanical properties of failing myocytes.

2457-Pos Board B473Unique Structural and Functional Effects of Alpha-TropomyosinMutations in HCM and DCMTeryn A. Holeman, Melissa L. Lynn, Jil C. Tardiff.Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA.Hypertrophic (HCM) and dilated (DCM) cardiomyopathies are diseases of thecardiac sarcomere with complex phenotypical heterogeneity. Although the ge-netic basis of HCM and DCM is widely recognized, our understanding of theprecise mechanisms underlying these diseases remains unclear. Cardiaccontraction is regulated by the thin filament (TF) composed of actin, tropomy-osin (Tm), and the troponin complex (Tn). The ‘‘gate-keeper’’ protein, a-Tm, isa highly conserved a-helical, coiled-coil dimer that spans actin and regulatesmyosin-actin interactions. Two DCM causing mutations in Tm (D84N andD230N) were compared with two HCM causing mutations (E62Q, D219N)to study how primary alterations in protein structure can cause functional def-icits. We hypothesize that structural changes from point mutations propagatethrough Tm, and affect the regulatory function of the TF via distinct perturba-tions of structure and protein-protein interactions. Differential scanning calo-rimetry (DSC) was employed to determine mutational effects on thestructure of the Tm dimer and TF. All mutants in the Tm dimer system showedan increase in thermal stability at the proximal terminus, suggesting proximityhad a dominant effect on Tm stability. DSC of mutated TF revealed corre-sponding shifts in Tm C- and N-termini thermal stability. The second domain,however, reflected the TF interaction strength and cooperativity, consistentwith phenotype-concordant trends. DCM causative-mutations exhibitedincreases in interaction strength, while HCM mutations resulted in a decrease.These data suggest that the structural alterations of each mutation areunique, and pathogenesis of HCM and DCM is both mutation and location spe-cific. To characterize the resultant functional effects of these discrete changes,in-vitromotility is in progress to assess myofilament activation. Together, thesedata will provide a molecular level understanding of the primary deficits causedby these mutations and help elucidate the differential primary diseasemechanisms.

2458-Pos Board B474Neonatal Permeability Transition Pore Closure is Associated withIncreased Cardiac FunctionRyan E. Alanzalon, George A. Porter.Pediatrics, University of Rochester Medical Center, Rochester, NY, USA.Introduction: Recent studies have shown that embryonic myocytes have lessactive mitochondria and immature cellular structure. The neonatal heart isalso closer in structure and function to the fetal heart, but progressively dif-ferentiates into its mature form with time. Mitochondria appear to regulatethese changes during the neonatal transition. The closure of the mitochondrialpermeability transition pore (PTP), by pharmacologic or genetic inhibition ofCyclophilin D (CyPD), is associated with differentiation in cultured neonatalmyocytes. We evaluated CyPD inhibition’s effect on in vivo cardiac function.We hypothesized that closure of the mPTP in the neonatal mouse heart willincrease cardiac function by enhancing myocyte differentiation and matura-tion without longer-term deleterious effects. Methods: One-day-old WT orCyPD null mice were treated with daily intraperitoneal injections of vehicle(inert), Cyclosporin A (CsA), or NIM811 for five days. At six days old andweanling, the mice underwent echocardiography to evaluate ejection fraction(EF), a measure of cardiac function. Hearts were then harvested for furtherbiochemical analysis.Results: WT mice hearts treated with CsA or NIM811,as well as CyPD nulls, had significantly higher EF at six-days-old(P<0.0001) compared to WT untreated or vehicle-treated mice. At weanling,there was no significant difference in EF across all groups and all survived tothis time point. Work regarding assays of electron transport chain functionand structure remains ongoing. Conclusion: Our results demonstrate thatPTP closure in the neonatal mouse heart is associated with increased EF, ascompared to vehicle treated mice. Our most recent data suggests that there

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is no significant difference in EF at weanling across all groups. This suggeststhat PTP closure enhances the neonatal transition to a more differentiatedmyocardium leading to an early increase in ejection fraction usually observedin mature animals.

2459-Pos Board B475Effect of Truncated Mutations in the Titin Gene on Cardiac FunctionPetr G. Vikhorev1, Amy Li2, Sean Lal2, Cristobal G. dos Remedios2,Steven B. Marston1.1National Heart and Lung Institute, Imperial College, London, UnitedKingdom, 2Discipline of Anatomy and Histology, University of Sydney,Sydney, Australia.Dilated cardiomyopathy (DCM) is characterized by left ventricular dilationand impaired systolic function, where up to 40% of cases are associatedwith mutations. Truncating mutations in the TTN gene are the most commoncause of familial DCM. While it is known that titin is the major contributor tomyocardial stiffness and is an essential component of the Frank-Starlingmechanism of the heart, its function is not fully established. Titin mechanicalproperties are particularly important in assisting with myofibril viscoelasticityand consequently contribute to the work output characteristic of myocardium.In contrast, changes in myofilament Ca2þ-sensitivity and loss of its modula-tion by phosphorylation are involved in the proposed mechanism fordisease-causing mutations in contractile proteins but not necessary in theTTN gene.We studied mechanical properties and isometric contractility in single cardiacmyofibrils of DCM patients with and without TTNmutations. We found that allstudied DCM mutations in contractile protein genes (TNNC1, TNNI3, andMYH7) including truncations in the titin gene (TTN), result in lower passivestiffness compared to donor samples. Furthermore we showed that the myofil-ament Ca2þ-sensitivity was higher than donor and without any difference inmyofibril kinetics. In fact, heart function is highly coordinated and stronglydependent on spatiotemporal control during the cardiac cycle so data obtainedin isometric conditions may be inadequate to understand the effect of muta-tions. To further characterize mutations and the impact of myofibril lengthchanges on cardiac output, we now use cyclic length and Ca2þ concentrationchanges to imitate the changes in single myofibril similar to those that haveplace during the cardiac work-producing cycle.

2460-Pos Board B476Cardiac Light Chain Amyloidosis, Understanding the Implications ofCellular Toxicity in a 3D ModelKeely Redhage1, Chris Dick1, Yi Lin2, Marta Marin-Argany1,Angela Williams3, John S. Wall3, Marina Ramirez-Alvarado4.1Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA,2Hematology, Mayo Clinic, Rochester, MN, USA, 3Medicine and Radiology,University of Tennessee, Knoxville, TN, USA, 4Biochemistry and MolecularBiology, Immunology, Mayo Clinic, Rochester, MN, USA.Light chain (AL) amyloidosis is a fatal protein misfolding disease characterizedby extracellular deposition of immunoglobulin light chains as amyloid fibrils.In AL amyloidosis there is an abnormal proliferation of monoclonal plasmacells that secrete high amounts of monoclonal light chains into the bloodstream.These light chains misfold, aggregate, and deposit in vital organs ultimatelyleading to organ failure and death. Survival rates for individuals diagnosewith AL amyloidosis are often determined by the severity of cardiacinvolvement.There are two light chain subtypes (kappa and lambda). These subtypes arecomposed of variable (VL) and constant (CL) domains. Previous research inour laboratory demonstrated that different subtypes and species exert differentcytotoxic effects in human RFP-AC16 cardiomyocytes. Soluble kappa proteins(particularly full length) cause apoptosis while fibrillar proteins (particularlyVL) cause cell growth arrest. Mesenchymal stromal cells (MSCs) were shownto protect cardiomyocytes from cytotoxic effects of cell growth arrest exertedby fibrillar species. Currently, one major limitation in studying cardiac ALamyloidosis is the lack of an effective model that recapitulates the in vivo envi-ronment. In order to better understand the exact mechanisms behind apoptoticand cell growth arrest we have developed a 3Dmodel with cardiomyocytes sus-pended in a diluted matrigel network. In 3D cell culture, the entire surface ofthe cardiomyocyte is exposed to its external environment providing an in vitrocell model that mimics cell-to-cell contact of the in vivo environment observedin AL amyloidosis patients. Interestingly, similar effects of clustering and cellgrowth inhibition observed in 2D cell culture were observed in 3D cell culture.3D cell culture experiments in progress will determine the mechanisms behindfibrillar species cellular toxicity and the implications of MSCs as a potentialtherapeutic model.

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2461-Pos Board B477Solving for the Rate of Diffusion in Cardiac Transverse Tubules fromFluorescence Recovery after Photobleaching ExperimentsCherrie H.T. Kong, Clive H. Orchard, Mark B. Cannell.Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol,United Kingdom.Excitation-contraction (E-C) coupling in cardiac ventricular myocytes is criti-cally dependent on the transverse (t-) tubules, which are invaginations of thesurface sarcolemma. Many key proteins are preferentially located at the t-tu-bules, for example, the L-type Ca channel, Na-Ca exchange, and steady-stateK channel (1), and thus, the rate of passive solute transport into and out ofthe t-tubular system might be of functional significance. Use of FluorescenceRecovery After Photobleaching (FRAP) to assess solute mobility withincellular structures is commonplace. However, the unique structure of the car-diac myocyte and t-tubular system poses specific problems to the quantitativeinterpretation of the data. We have developed a new 3D computer model basedon measured cardiac ventricular myocyte and t-tubule system geometries toanalyse experimental FRAP data. Cells undergoing FRAP were immersed ina fluorescent tracker that illuminated the extracellular space and permeatedthe t-tubular system. Cell size was obtained from a 3D confocal image stackrecorded prior to performing FRAP, while t-tubule structure was quantified us-ing a separate cohort of cells stained with di-8-ANEPPS. We extended our un-derstanding of FRAP data by including measured geometry of the illuminating(bleaching) laser beam, which we show to have significant impact on the fluo-rescence recovery profile within the t-tubules. Our model is able to reproducethe time-course of recovery within the t-tubule system, as well as that in theadjacent extracellular space. These results show that extracellular accumula-tion/depletion problems affect the observed time-course of FRAP, however,these effects can be accounted for by detailed modelling that includes the ge-ometry of the illumination field and relationship of the cell to the coverslip.This work was supported by the British Heart Foundation and the MedicalResearch Council (UK).

2462-Pos Board B478Quantifying the Contribution of Cardiomyocyte Metabolic Dysfunction tothe Heart Mechanical FunctionRachel Lopez, Xin Gao, Francoise Van den Bergh, Santosh Dasika,Daniel Beard.University of Michigan, Ann Arbor, MI, USA.Hydrolysis of ATP provides the chemical free energy to drive the molecularprocesses underlying cardiac pumping. Under normal conditions the heart hy-drolyzes and resynthesizes the entire ATP pool several times per minute. In thefailing heart, cardiomyocyte metabolic dysfunction leads to a reduction in thechemical potential (free energy of ATP hydrolysis) at which ATP is synthe-sized and is made available to drive cellular processes. We hypothesize thatthis metabolic/energetic dysfunction causes contractile dysfunction of themyocardium in heart failure, contributing to the whole-body phenotype (venouscongestion, exercise intolerance) of heart failure in humans. To test this hypoth-esis, we used a rat transverse aortic constriction (TAC) model of cardiacdecompensation/heart failure to measure the relationships between functionalparameters (cardiac output, ejection fraction, etc.) and myocardial energeticstate, determined based on measurements of myocardial metabolite pools andmitochondrial oxidative capacity. We observed reductions in fatty acid and car-bohydrate oxidative capacities and adenine nucleotide pool levels that correlatewith impaired mechanical function. Furthermore, we used multi-scale compu-tational models of myocardial metabolisms and metabolic state-dependent con-tractile dynamics to quantify the contribution of reductions in oxidativecapacity and metabolite pools levels to heart mechanical function. Our resultssuggest that there is a causal link between a reduction in free energy of ATPhydrolysis and kinetic impairment of the myosin ATPase cross-bridge cyclein decompensated hypertrophy/heart failure.

2463-Pos Board B479Computational and Experimental Investigation of Tropomyosin D230Nand S215l Mutation Specific Correlates to DiseaseAndrea Deranek1, Anthony Baldo2, Melissa L. Lynn3, Mark T. McConnell1,Michael R. Williams2, Steven D. Schwartz2, Jil C. Tardiff3,4.

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1Biomedical Engineering, University of Arizona, Tucson, AZ, USA,2Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA,3College of Medicine, University of Arizona, Tucson, AZ, USA, 4Cellularand Molecular Medicine, University of Arizona, Tucson, AZ, USA.Mutations in alpha-tropomyosin (Tm), an integral component of the cardiacthin filament, are known to cause changes in protein structure and dynamicsleading to pathological cardiac remodeling observed in patients with hyper-trophic (HCM) and dilated (DCM) cardiomyopathies. In this complex sys-tem, mutations in Tm that are separated by only a few amino acids cancause divergent cardiomyopathies, thus highlighting the importance of theprimary structural and dynamic ‘‘triggers’’ that lead to disease. To beginto correlate genotype to phenotype, we investigated two cardiomyopathy-linked mutations in Tm: D230N, a DCM-associated mutation and S215L,an HCM-associated mutation. We coupled computational predictions within-vitro biophysical measurements to better understand the divergent diseasemechanisms. We propose that D230N and S215L cause differential structuralchanges in the thin filament leading to atomic and molecular level changesthat initiate pathogenic remodeling. Molecular dynamics (MD) simulationsof thin filaments containing the S215L mutation showed an increased dis-tance in the overlap between cardiac troponin T (cTnT) and Tm, but thosefor D230N containing filaments exhibited a decreased distance. These resultscorrelate well with the respective changes in cooperativity we hypothesizeaccompanies HCM and DCM -linked cardiac remodeling respectively. Dif-ferential scanning calorimetry (DSC) showed both mutations causedincreasing cooperativity (FWHM) in the azimuthal shifting of Tm:Tn onactin. However, while D230N increased thermal stability (Tm) of the Tmoverlap, S215L decreased the stability. Further clarification of these datawill be provided by ongoing Time-resolved Forster Resonance Energy Trans-fer (TR-FRET) studies to confirm structural distance changes in the overlapin fully reconstituted thin filaments. This iterative process will allow us tobuild predictive tools to test known and de-novo thin filament mutationsassociated with these cardiomyopathies and eventually to design and imple-ment more precise therapeutic interventions.

2464-Pos Board B480Quantifying Nuclear Remodeling in Heart FailureLogan Bailey1, Danny Smyl2, Sven Bossuyt3, Julie Bossuyt1.1Pharmacology, University of California, Davis School of Medicine, Davis,CA, USA, 2Mechanical Engineering, Aalto University, Espoo, Finland,3Engineering Design and Production, Aalto University, Espoo, Finland.Nuclear remodeling has been well-described in cancer as a source of genomicinstability and altered gene expression. Recent studies suggest that nuclear re-modeling is also a conserved feature of heart failure occurring before manifes-tation of cytosolic calcium handling dysfunction; however, detailedmeasurements of changes in nuclear organization and chromatin structurehave not yet been described in heart failure. Here, we use immunocytochem-istry, super-resolution stimulated emission depletion (STED) microscopy, andanalysis via Imaris and MATLAB to quantify three-dimensional nuclear struc-ture changes in a mouse pressure-overload model of heart failure (transverseaortic constriction, TAC) versus sham-operated control animals. Using Mito-tracker staining to track cell volume and DAPI staining to track nuclear vol-ume, in TAC vs. SHAM animals we find increased cardiomyocyte cellvolume (59000 5 3600 vs. 27000 5 1500 mm^3) and increased individualnuclear volume (440 5 35 vs. 300 5 17 mm^3), but a decreased nuclear vol-ume to cell volume ratio (1.8 5 0.16 vs. 2.6 5 0.22%). Of note, STED mi-croscopy and double-stranded DNA (dsDNA) staining with PICOGreenrevealed decreased dsDNA mean intensity (73 5 4.5 vs. 110 5 5.0) andincreased surface area to volume ratio of reconstructed dsDNA (2.0 50.25 vs. 0.96 5 0.029) in TAC vs. SHAM, suggesting altered physicalaccess to DNA in heart failure. The dynamic modification of chromatin archi-tecture was further assessed by staining for H3K4me3 and Brd4, while LaminA/C immunostaining captured nuclear envelope remodeling. Images wereanalyzed for changes in intensity, surface area, volume, distribution, and co-localization. Our results indicate this is a valid approach to quantify nuclearremodeling in heart failure and to delineate the role of specific signaling path-ways therein.

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Posters: Cardiac Muscle Regulation II

2465-Pos Board B481Defining a Unifying Mechanism for Select Cariomyopathy-Linked Vari-ants of DesmoplakinHeather R. Manring1, Ronald Ng2, Taylor Albertelli3, Prameela JyothiBobbili1, Olivia Carter1, Tyler Stevens1, Daniel Jacoby4, Paul M.L. Janssen1,Ahmet Kilic5, Nathan Wright3, Stuart Campbell2, Maegen A. Ackermann1.1Physiology and Cell Biology, The Ohio State University, Columbus, OH,USA, 2Biomedical Engineering, Yale University, New Haven, CT, USA,3Chemistry and Biochemistry, James Madison University, Harrisonburg, VA,USA, 4Internal Medicine, Yale School of Medicine, New Haven, CT, USA,5Surgery, The Ohio State University, Columbus, OH, USA.Arrhythmogenic cardiomyopathy (ACM) is a disease that affects 1 in 2000Americans every year and segregates with sudden cardiac death (SCD).ACM is linked to genetic variants in desmosomal genes, however the molecularmechanisms by which desmosomal variants result in ACM remain unclear. Ournovel results in human heart explants identify that desmoplakin (DSP), adesmosomal gene product, is cleaved via a calpain-dependent mechanism.To define a unifying mechanism for ACM-linked variants, we assessed the bio-molecular properties of variants localized to the NH2-terminus of DSP. Ourbiochemical and molecular dynamics (MD) assays indicate no overt structuralchanges compared to wildtype, however there is a decrease in stabilizing intra-molecular interactions in the presence of select variants (S299R, S442F andS507F). In addition, these same variants present with a significantly increasedexposure of an auto-inhibited canonical calpain cleavage site. Biochemicalassays support that these three variants are more susceptible to calpain-dependent cleavage. This led us to hypothesize that increased susceptibilityto calpain-dependent cleavage resulting in decreased functional DSP proteinmay be a unifying mechanism for select variants of DSP. To test this hypoth-esis, we evaluated a novel ACM-linked DSP variant, R451G, identified in aproband that suffered SCD. Heart autopsy tissue revealed a classic ACMhistological phenotype with pronounced fibrofatty scarring. Notably, immuno-fluorescent microscopy showed a significant decrease of DSP protein at theintercalated disc. MD and biochemical assays reveal that the R451G variant ex-hibits increased susceptibility to calpain-dependent cleavage due to decreasedintramolecular interactions and increased exposure of the auto-inhibited cal-pain cleavage site. Taken together we have created a workflow to screen novelvariants of unknown significance and identified a unifying mechanism forselect ACM-linked variants of DSP grounded in increased susceptibility tocalpain-dependent cleavage leading to decreased functional DSP protein.

2466-Pos Board B482TNNT2Restrictive and Hypertrophic CardiomyopathyMutations Depressthe Inhibitory Properties of the Troponin-T1 Fragment, In VitroAditi Madan1, Sineej Madathil2, William Schmidt1, Larry S. Tobacman2,Anthony Cammarato1.1Medicine, Johns Hopkins University, Baltimore, MD, USA, 2Medicine,University of Illinois at Chicago, Chicago, IL, USA.Restrictive (RCM) and hypertrophic (HCM) cardiomyopathies are heteroge-neous diseases of the heart, characterized by impaired relaxation and diastolicdysfunction, without/with ventricular wall thickening. The pathogenesis can beattributed to hyperdynamic contractile function of mutant sarcomeres, which isconsidered the most proximal effect of disease-causing mutations. �100 caus-ative thin filament mutations have been identified, of which 15% occur incardiac troponin-T (cTnT/TNNT2), the tropomyosin-binding subunit of the reg-ulatory troponin complex. Of these, 70% are clustered in the N-terminal cTnT1tail, which contains a highly conserved tropomyosin binding element spanningresidues 112-136. At low Ca2þ concentrations, troponin constrains tropomy-osin to the blocked/B-state, occluding acto-myosin interactions. cTnT1 contrib-utes to B-state formation and, thus, contractile inhibition. Here, we investigatedthree charge-altering mutations in invariant cTnT1 residues: E136K (RCM-causing), K124N, R130C (HCM-causing). We hypothesize that these mutationsweaken cTnT1’s inherent inhibitory properties via altered cTnT1-tropomyosinbinding, leading to destabilization of the blocked/B-state and excessive acto-myosin cycling. Human cTnT1 was cloned, mutagenized, expressed, purified,and reconstituted with vertebrate F-actin-tropomyosin to determine the effectsof the mutations on in vitro motility. We verified the inhibitory effect ofWT-TnT1 on F-actin-tropomyosin sliding speed. All 3 mutant-cTnT1-actin-tropomyosin filaments showed significantly higher sliding speeds versuscontrol: E136K (1.1550.22 vs. 1.0050.24mm/s), K124N (1.2150.21 vs.1.0050.18mm/s) and R130C (1.6350.51 vs. 1.0050.18mm/s). We also exam-ined sliding speeds as a function of myosin concentration, and observed differ-ences between WT and mutant filaments to be amplified at 50-75mg/ml.Additionally, there was a 6-12% increase in the number of moving versus

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non-moving mutant filaments. These data suggest a destabilized cTnT1-mediated B-state, which could contribute to the cardiac hypercontractilityand, importantly, impaired relaxation observed in patients. We are currentlyinvestigating the effects of these mutations on cTnT1-tropomyosin bindingaffinities.

2467-Pos Board B483Defining a Novel Mechanism Mediating the Rate of Heart FailureProgressionHeather R. Manring1, Prameela Jyothi Bobbili1, Abigail Beer1,Paul M.L. Janssen1, Ahmet Kilic2, Maegen A. Ackermann1.1Physiology and Cell Biology, The Ohio State University, Columbus, OH,USA, 2Surgery, The Ohio State University, Columbus, OH, USA.Heart failure accounts for 1 of every 4 deaths in the United States every year.Heart failure is a multifaceted disease involving numerous complex axes of theheart. Efforts over the last several decades have sought to define pathogenicmechanisms leading to a decline in heart function. However, to date manydisease pathways remain uncharacterized. Here we report a novel axis forthe progression of heart failure linked to both structural and signaling abnorm-ities and reliant on an imbalance of obscurin polypeptides. Our novel results inhuman heart explants identify a decrease in the levels of giant obscurin proteinsin conjunction with an increase in the levels of small obscurin peptides infailing hearts compared to donor controls. Furthermore we show that giant ob-scurins are cleaved via a calpain-dependent mechanism. Obscurin is a family ofmulti-functional adaptor proteins expressed in the heart. Giant obscurins func-tion as molecular scaffolds while the smaller obscurin-80 plays a role in modu-lating PI3K signaling. We utilize two specialized mouse models, a transgenicline lacking only giant obscurins and an AAV-induced overexpression modelof obscurin-80, to study the functional implications of an imbalance of obscurinpeptides. Mice lacking giant obscurins show disruption of internal membranestructures and altered phosphorylation of RyR indicative of Ca2þ mishandling.Increased levels of obscurin-80 in the myocytes result in increased size,decreased cellular adhesion and aberrant PI3K signaling along with hyperphos-phorylation of RyR. Taken together, our data suggests that an imbalance ofobscurin polypeptides provides a novel axis for the progression of heart failuregrounded in both structural and signaling phenotypes due to loss of giant ob-scurins and an increase in small obscurin peptides, respectively.

2468-Pos Board B484Influence of cTn Ca2D Binding Properties and Cooperative Mechanismson Cardiac Muscle Contractile DynamicsSrboljub M. Mijailovich1, Djordje Nedic2, Boban Stojanovic2,Joseph D. Powers3, Jennifer Davis4, Michael A. Geeves5, Michael Regnier3.1Biology, Illinois Institute of Technology, Chicago, IL, USA, 2Mathematicsand Informatics, University of Kragujevac, Kragujevac, Serbia,3Bioengineering, University of Washington, Seattle, WA, USA, 4Pathology,University of Washington, Seattle, WA, USA, 5School of Biosciences,University of Kent, Canterbury, United Kingdom.Mutations in cardiac muscle sarcomeric proteins are often associated with heartfailure. Relaxation abnormalities are rarely studied, though this can stronglyaffect early phase diastole and, perhaps, diastolic performance (filling) viaresting ventricular wall tension. Using MUSICO simulations, tightly coupledwith experiments, we quantitatively estimated the contributions of three mech-anisms to contraction and relaxation kinetics: (1) Tm azimuthal movement as acontinuous flexible chain (CFC); (2) reduction of cTn calcium affinity resultingfrom crossbridge detachment to create a negative feedback effect that reducesmyosin binding sites; and (3) inclusion of a super-relaxed myosin state (SRS) toreduce the pool of myosins that can rebind during relaxation. Simulationsprovided force-pCa relationships and force transient responses where nativecTnC was replaced with either cTnC L48Q (increased Ca2þ affinity) orcTnC I61Q (decreased Ca2þ affinity). Simulations demonstrated that force-pCa relationships with cTnC mutants require not only changes in Ca2þ affinity,but also CFC elasticity and its interaction with actin. Adjusting Ca2þ bindingand myosin affinity to actin accounted for the apparent loss of cooperativityof thin filament activation for both mutants. Intact cell twitches could not besimply related to force-pCa curves of skinned muscle, they also required a shiftto lower Ca2þ. To achieve the observed relaxation rates the reduced flux ofmyosin binding was achieved by moving detached states into SRS. Finally, pre-dictions of cTnC mutational penetrance with the present model suggests thatmodulation of cTnC Ca2þ binding only partially describes the phenomenaand more complex models including, for example, variable confined persis-tence lengths need to be developed. The predicted effects of varying mutantpenetrance on contractile properties suggests a tunable effect on cardiomyocyteperformance that may reflect the severity of contractile abnormality in cardiacmuscle disease.

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2469-Pos Board B485Cardiac Myosin Strutural Kinetics are Modulated by Myk461John Rohde1, David D. Thomas2, Joseph M. Muretta2.1University of Minnesota, Minneapolis, MN, USA, 2Biochemistry, MolecularBiology, and Biophysics, University of Minnesota, Minneapolis, MN, USA.The biochemical and structural kinetics of bovine cardiac myosin are perturbedby the putative heart failure therapeuticMyk461.Myosin’s lever arm undergoesstructural changes that are coupled to its biochemical state during ATPasecycling in muscle. Structural states of myosin’s lever arm can be detecteddirectly using the high-precision, high-throughput technique of transient time-resolved FRET revealing kinetics, thermodynamics, and allosteric coupling.We have previously used this technique to investigate mechanochemicalcoupling in skeletal myosin and cardiac myosin perturbed by the heart failuredrug omecamtiv mecarbil. Here we investigate the MyoKardia compoundMyk461, in clinical trials for treating hypertrophic cardiomyopathy. We findthat the small molecule therapeutic primarily inhibits actomyosin association,both in the absence and presence of nucleotide. This inhibition is also detectedin the coupled kinetic steps following actin activation of myosin: the force-producing structural isomerization known as the powerstroke, and the principalfree energy-driven biochemical step of Pi release. Other actin-dependent exper-iments reveal a potent inhibition by Myk461, steady-state ATPase cycling andsingle-turnover of fluorescent ATP. We also find that the drug’s affinity formyosin varies by two-orders of magnitude, dependent on the structural andbiochemical state of myosin: binding strongest to actomyosin-ATP and weakestto apo myosin. This work was supported by NIH AR032961 & AR057220(DDT), American Heart Association Scientist Development Grant (JMM), andGraduate Excellence Fellowship-University of MN (JAR).

2470-Pos Board B486Altered Signaling Pathways in Hearts of Ames Dwarf MiceEmily Eijansantos, Shuchita Tiwari, Aldrin Gomes.University of California, Davis, Davis, CA, USA.Much of the age-related decline in health is markedly associated with impairedcardiac function and increased vulnerability to cardiovascular disorders. Amesdwarf mice have been of considerable interest in studies of aging as they exhibitprolonged longevity and delayed symptoms of aging compared to normal mice.These mice possess an autosomal recessive mutation on chromosome 11 thatcauses severe dwarfism and produces a long-lived phenotype that allowsthem to live an average of 50% longer than their normal siblings. Previousstudies have shown that alterations in the cell’s molecular mechanisms thatrespond to stress are involved in the process of aging, and that differentialexpression of key metabolic pathway proteins also affects the longevity of an-imal models. To understand more about the mechanisms and pathwaysinvolved in their extended lifespan, hearts were collected from one-year-oldfemale Ames dwarf mice and one-year-old wild-type female mice and massspectrometry using tandem mass tag (TMT) labeling were carried out usingheart lysates. Data from mass spectrometry were analyzed with MaxQuantand PEAKS and several biological pathways were found to be altered in heartsfrom Ames dwarf mice relative to wild-type mice. These systems include path-ways associated with cytoskeletal regulation, energy production, and cellularstress. The expression of several chaperones and calcium-binding proteinswere altered in Ames dwarf mice. Components of the 19S proteasome werefound to be upregulated in Ames dwarf mice. The proteasome is part of theubiquitin-proteasome system (UPS), which is the main intracellular degrada-tion system in cells and is critical for intracellular protein homeostasis. Overall,the data suggest that the Ames dwarf mice have higher levels of chaperones andUPS components which are likely to be beneficial in reducing the levels ofoxidized and aggregated proteins that accumulate in aged hearts.

2471-Pos Board B487Tributytin Induces Negative Inotropic Effect, Reduces Cardiac SRCalcium Content and Increases Calcium Sparks Frequency in Cardiomyo-cytesIvanita Stefanon1,2, Cleydianne Luiza Vieira Pereira1, Bruno BarcellosJacobsen1,2, Rog�erio Faustino Ribeiro Junior1,2, Donald M. Bers2.1Ciencias Fisiologicas, Universidade Federal do Espırito Santo, Vitoria,Brazil, 2Pharmacology, University of California Davis, Davis, CA, USA.Tributyltin (TBT) is anorganotin environmental contaminant used in farming andantifouling ship paints. Its release directly into the water, from ships, shipyardsand ports caused environmental impacts on aquatic ecosystems. Organotin com-pounds produce neuro-, cyto- and geno-toxic effects but its toxic effect on thecardiovascular system has not yet been fully elucidated. We used Langendorff-perfused rat hearts (pH 7.4, 37�C) to assess left ventricular systolic pressure(LVSP) and LVdiastolic pressure (LVDP) via a latex balloon in the LV. Animalswere randomized into: Control (N = 7) and those exposed for 5 min to TBT solu-

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tion (50 mM) (TBT group N = 8) at different perfusate [Ca] and/or with 100 mMisoproterenol (ISO). TheFrank-Starling responsewas assessed by raising preloadfrom0 to30mmHg.TBTperfusion caused an acute negative inotropic effect, evi-denced by reduced contractile response to: Baseline 1.25 mM extracellular [Ca](LVDP = 1155 9 in control vs 665 4*mmHg in TBT, *p<0.05), ISO (133522 mmHg Control vs. 24 5 13* mmHg TBT, *p <0.05) and during Frank-Starling curve (88 5 6 vs = 45 5 2* mmHg, for 10 mmHg preload,*p <0.05). We also used isolated rat ventricular myocytes to test the effects ofTBT on spontaneous sarcoplasmic reticulum (SR) Ca release (Ca sparks) andSR content. In permeabilized rat myocytes 10-100 nM TBT acutely increasedCa spark frequency (CaSpF), which resulted in a reduction in Ca spark amplitudeandSRCa load.Weconclude thatTBTcauses acute andpotent negative inotropiceffects on the intact heart that may depend, at least in part, on increasing SR Caleak and reduction in calcium SR content.

2472-Pos Board B488Biophysics of SERCA2a/DWORF Complex and Implications for Thera-peutic DesignAng Li1, Daniel R. Stroik1, Tory M. Schaaf1, Benjamin D. Grant2,David D. Thomas1.1Biochemistry, Molecular Biology, and Biophysics, University of Minnesota,Minneapolis, MN, USA, 2Fluorescence Innovations Inc, Minneapolis, MN,USA.We have developed cell-based FRET biosensors and used time-resolved FRET(TR-FRET) to measure protein-protein interactions and structural changes of thecardiac sarcoplasmic reticulum Ca-ATPase 2a (SERCA2a) in complex withphospholamban (PLB) or dwarf open reading frame (DWORF). SERCA2a re-duces intracellular [Ca2þ] in cardiac myocytes, inducing relaxation. DWORF,a 35-residue transmembrane peptide, has been proposed to enhance SR Ca2þ up-take and myocyte contractility through its displacement of the inhibitory peptidePLB from SERCA. FRET between GFP-SERCA2a and RFP-DWORF indicatesbinding of DWORF to SERCA. To test the hypothesis that DWORF competeswith PLB for SERCA2a binding, we have transiently transfected unlabeledDWORF into a stable cell line expressing GFP-SERCA2a (donor) and RFP-PLB (acceptor). We observed a significant decrease of SERCA-PLB FRET ef-ficiency due to co-expression of DWORF. Detailed analysis of TR-FRET dataallows us to determine whether this is primarily due to a decrease in themole-fraction of SERCA2a molecules bound to PLB, or to a change in the struc-ture of SERCA2a-PLB complex. The DWORF concentration dependence ofFRET and SERCA function indicates that SERCA has a comparable affinityfor DWORF than for PLB, consistent with the hypothesis that DWORF com-petes effectively with PLB for binding to SERCA. This is consistent with thehypothesis that DWORF competes effectively with PLB for binding to SER-CA2a. Site-directed mutagenesis of DWORF identifies specific residues inDWORF that are important for this competitive binding. A central factor in heartfailure is an increase in basal cytosolic [Ca2þ], resulting in decreased cardiaccontractility. Therefore, augmentation of SERCA2a activity by overexpressionof DWORF is a promising therapeutic approach, and these SERCA2a/DWORFand SERCA2a/PLB biosensors could be helpful for screening small moleculesfor potential heart failure therapy. This work was supported by NIH grants(GM27906, HL129814, AR07612, and DA037622).

2473-Pos Board B489Stopped-Flow CalciumAssociation Kinetics of Hypertrophic Cardiomyop-athy Associated Troponin T MutationsMatthew M. Klass, Sarah J. Lehman, Jil C. Tardiff.Physiology, University of Arizona, Tucson, AZ, USA.Hypertrophic cardiomyopathy (HCM) is a complex genetic disorder affecting1/500 individuals. Though generally characterized by thickening of ventricularwalls and impaired relaxation, the phenotypic heterogeneity and variableseverity also characteristic of HCM highlight the complex biophysical patho-genic disease mechanisms. The N-terminus of cardiac troponin T (cTnT),known to play a critical role in regulation of contraction and located withinthe cardiac thin filament (TF), includes a known HCM mutational hotspot atsite Arg92 where three point mutations (R92L, R92Q, and R92W) are eachassociated with differential phenotypes, disease severity, and downstreamcalcium dysregulation. Previous characterization of steady-state calciumsensitivity of force generation revealed comparable increases for these muta-tions and is therefore insufficient to explain the distinct phenotypes. Giventhat calcium binding affinity of the TF can be altered by changes in rates ofcalcium association and dissociation, we investigated these dynamic measure-ments via stopped-flow kinetics. We and others previously characterized thecalcium dissociation rates for these mutations given the proposed physiologicalrelevance to alterations in myocardial relaxation. To more thoroughly charac-terize these mutation-specific alterations in calcium handling, we measured the

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calcium association rates of fully reconstituted TFs over a range of calciumconcentrations ([10mM-100mM]). The observed association rate for WT TFswas calculated to be 1.6x10�6 M�1s�1. R92L filaments showed no change inthe association rate (KON=1.8x10

�6 M�1s�1), suggesting the increased calciumsensitivity is due primarily to a decrease in dissociation rate as previouslyreported. Alternatively, R92Q filaments exhibit an increased association rate(KON=3.8x10

�6 M�1s�1) suggesting that increased calcium sensitivity resultsfrom increased association kinetics when coupled to a previously reportednormal dissociation rate. These data suggest that alterations in myofilamentcalcium kinetics represent a mutation-specific mechanism of disease and arethus a potential target for therapeutic intervention.

2474-Pos Board B490Membrane Domains and cAMP Compartmentation in Cardiac MyocytesShailesh R. Agarwal1, Jackson Gratwohl1, Mia Cozad1, Pei-Chi Yang2,Colleen E. Clancy2, Robert D. Harvey1.1University of Nevada, Reno, NV, USA, 2University of California, Davis,Davis, CA, USA.Confining the production of the diffusible second messenger cAMP to distinctsubcellular locations allows different G protein-coupled receptors to regulatedistinct biological responses in a given cell. In this study, we examined therole that different membrane domains play in producing compartmentalizedcAMP responses in adult rat ventricular myocytes by using genetically encodedFRET-based biosensors targeted to the bulk cytosolic compartment, as well aslipid raft and non-raft domains of the plasmamembrane.We found that b-adren-ergic receptors, which are expressed in both lipid raft and non-raft domains,produced similar cAMP responses in all subcellular locations. However, lipidraft disruption by cholesterol depletion selectively affected FRET responsesassociated with lipid rafts. On the other hand, activation of E-type prostaglandinreceptors, which are excluded from lipid rafts, had a greater effect on cAMPpro-duction associated with non-raft domains. This indicates that separating recep-tors into different membrane domains contributes to the generation of distinctpools of cAMP.We also found that inhibition of basal adenylyl cyclase (AC) ac-tivity produced a decrease in cAMP in all subcellular locations, but the effectwasgreatest in non-raft locations, indicating that there are differences in basal cAMPlevels associatedwith different membrane domains. Differences in sensitivity todirect activation of adenylyl cyclase (AC) suggest that this is due to differencesin basal AC activity. Selective inhibition of different phosphodiesterase (PDE)isoforms indicated that PDE2, 3, and 4 all play a role in regulating cAMP activityassociated with lipid raft and non-raft domains of the plasma membrane, whilePDE3 plays a more prominent role in activity in the bulk cytoplasmic compart-ment. These results support the idea that different membrane domains provide ameans to isolate receptors and relevant downstream signaling proteins tomediate specific cAMP responses.

2475-Pos Board B491Aberrant Cardiac Muscle Mechanics in a Hypertrophic CardiomyopathyTroponin T ILE79ASN Transgenic MouseKarissa M. Dieseldorff Jones1, David Gonzalez-Martinez1,Maicon Landim-Vieira1, Yeojung Koh2, Bjorn C. Knollmann3,P. Bryant Chase4, Hyun S. Hwang2, Jose R. Pinto1.1Biomedical Sciences, Florida State University, Tallahassee, FL, USA,2Nutrition, Food and Exercise Sciences, Florida State University,Tallahassee, FL, USA, 3Division of Clinical Pharmacology, VanderbiltUniversity School of Medicine, Nashville, TN, USA, 4Biological Science,Florida State University, Tallahassee, FL, USA.The missense mutation Ile79Asn in human cardiac troponin T (HcTnT-I79N)has been associated with phenotypic outcomes of hypertrophic cardiomyopathy(HCM), arrhythmias, and sudden cardiac death. Previous characterization of anHcTnT-I79N transgenic mouse, which expresses 52% of the mutant protein,demonstrated increased Ca2þ-sensitivity of isometric force. Little else is knownabout any changes in cardiac muscle mechanics caused by the mutation. Thisstudy investigates cross-bridge kinetics of skinned papillary muscle bundles(isometric force, sinusoidal stiffness and rate of tension redevelopment, kTR),post-translational modifications (PTM) in sarcomeric proteins, and in vivocardiac function. For fiber mechanics and PTM analysis, cardiac muscle wasisolated from the left ventricle of non-transgenic wild-type (WT) mice or trans-genic mice bearing the HcTnT-I79N mutation. Sarcomere length (SL) was setat pCa8 using HeNe laser diffraction after the attachment points of eachskinned fiber bundle were fixed with glutaraldehyde to minimize end compli-ance. Sinusoidal stiffness (0.2% PTP length oscillation) and kTR were obtainedafter recording steady-state isometric tension. As previously reported, theHcTnT-I79N mutation led to increased Ca2þ-sensitivity of isometric force.Length-dependent activation at the longer SL (2.1 vs 1.9 mm) resulted inincreased Ca2þ-sensitivity of isometric force and sinusoidal stiffness and

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slower kTR for both WT and HcTnT-I79N. However, HcTnT-I79N exhibitedslightly faster kTR and greater sinusoidal stiffness at sub-maximum Ca2þ-acti-vation (�40% of maximal isometric force) compared to WT regardless of SL.These data indicate that the HCM HcTnT-I79N mutation, and SL, markedlyinfluence the dynamics of individual regulatory units as well as the kineticsof cross-bridge cycling. Next steps include echocardiographic, sarcomeric pro-tein phosphorylation and further fiber mechanic analyses. NIH-HL128683.

2476-Pos Board B492Kinetic Implications from a Model of Cardiac Length-dependentActivationWilliam C. Hunter, Timothy Alcid.Biomedical Engineering, New Jersey Inst. of Technology, Newark, NJ, USA.Cardiac length-dependent activation (LDA) is commonly reported as a steady-state phenomenon (Dobesh et al, 2002). However, during a cardiac twitch, thecalcium transient significantly precedes the force that sarcomeres develop. Inour model of cardiac LDA, a change in SL acts rapidly, as data suggest (Mateja& deTombe, 2012). SL produces its effect by altering the interaction betweentitin and super-relaxed myosin heads on the thick filament. The rapid shift inequilibrium between super-relaxed and unencumbered detached heads thenrapidly alters the equilibrium between detached myosin heads and those weaklybound to actin – an equilibrium which varies as calcium interacts with regula-tory proteins. Calcium and SL interactions thus occur rapidly without signifi-cant kinetic delay. Changes in SL exert maximal effect as a twitch begins, ascalcium rises rapidly. On the other hand, as the calcium transient fades, theeffect of changes in SL are (surprisingly) predicted to almost disappear. How-ever, strongly attached crossbridges provide an additional mechanism by whichthe thin filament is activated. Due to the maintained force that lags the decay ofcalcium, strongly attached crossbridges thus provide a mechanism to maintainactivation even as calcium concentration fades. During a cardiac contractionwith shortening, a cardiac sarcomere can thus ‘‘remember’’ the longer SL atwhich it was activated and initially developed force. These two kinetic LDAphenomena predicted by the model ( loss of SL effect as a twitch proceeds,and memory of longer SLs earlier in a contraction) are consistent with theobserved behavior of sarcomeres in the wall of an intact left ventricle (Hunter,1983, 1989). (Supported in part by the Center for Engineering MechanoBiologythrough a grant from NSF’s STC program (CMMI): 15-48571).

2477-Pos Board B493Time-Resolved FRET Detection of the Myosin Super-Relaxed off State inCardiac Thick FilamentSami Chu, Lien A. Phung, Joseph M. Muretta, David D. Thomas.BMBB, University of Minnesota - Twin Cities, Minneapolis, MN, USA.We developed time-resolved fluorescence resonance energy transfer (FRET)sensors to detect the structural state of myosin in cardiac thick filaments. Car-diac myosin thick filaments are hypothesized to isomerize between an activestate, termed RX (relaxed) that interacts with actin and generates force in thesarcomere, and an inactive state, termed SRX (super-relaxed) that does notbind actin and exhibits inhibited ATP hydrolysis compared to soluble myosinwithout actin. Regulation of the transition between these states is hypothesizedto underlie the Frank-Starling law of the heart, and dysregulation of the transi-tion to lead to genetically-driven hypertrophic cardiomyopathy; however,exactly how remains unclear, because the structural basis of the SRX is poorlyunderstood. To investigate the structural transition between RX and SRX, wedeveloped a time-resolved FRET assay that measures the intermolecular dis-tance between two adjacent myosin regulatory light chains. Based on proposedstructural models, formation of the SRX increases FRET while disruption de-creases FRET. We tested this model with known modulators of the SRXbiochemical kinetics, including temperature and ionic strength, and with twophysiologically relevant SRX modulators, RLC phosphorylation and myosin-binding protein-C. This work was supported by National Institute of Healthgrants AR032961 and AR057220 (DDT), and American Heart AssociationScientist Development Grant (JMM).

2478-Pos Board B494Turning the Azimuthal Motions of Adjacent Tropomyosins into aCoupled N-body Problem in a Brownian Model of Cardiac Thin FilamentActivationYasser Aboelkassem1, Kimberly J. McCabe1, Gary Huber2,Joakim Sundnes3, Andrew D. McCulloch1.1Bioengineering, University of California San Diego, La Jolla, CA, USA,2Chemistry and Biochemistry, University of California San Diego, La Jolla,CA, USA, 3Scientific Computing, Simula Research Laboratory, 1325 Lysake,Norway.During thin filament activation process, tropomyosin (Tm) oscillates overthe surface of actin filament in the azimuthal direction to regulate the access

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of myosin head to actin binding sites. This motion is characterized by theexistence of three distinct equilibrium positions, namely; blocked (B),closed (C), and open (M) regulatory states. The exact dynamics and energylandscape that governs Tm motions between these conformational states isnot yet fully understood. In this study, we developed a Brownian flashingratchet model to mathematically describe Tm motions over the surface ofactin filament. The model approximates the Tm flexible chain by a systemof discrete and coupled Brownian bodies. In other words, the thin filamentregulatory units (RUs) are prescribed by a set of N-body stochastic ODEsthat govern the azimuthal motion of adjacent tropomyosins. The modelexplicitly accounts for spatial interactions among nearest neighbor RUs,which were thought to appear from structural coupling of adjacent tropomy-osins. This coupling is achieved by assigning each Brownian Tm-body witha local multi-well potential. The energetic barrier, asymmetry, and bias ofeach individual energy landscape are hypothesized to be influenced by theTm status of its neighbors. This coupling approach is anticipated to be suf-ficient to produce observed cooperativity in both force-Ca2þ relationshipand twitch dynamic. The present analysis is expected to perform betterthan many of current phenomenological myofilament models which weremostly derived based on the classical mean-field approximation. Addition-ally, this analysis draws a more detailed molecular connection between Tmdynamics and sarcomeric twitch production. Therefore, the Brownianratchet modeling methodology of Tm motions during thin filament activa-tion may offer an enhanced mechanistic approach to describe cardiac mus-cle contraction.

2479-Pos Board B495Protein Kinase D Modulation of Cardiac Protein PhophatasesMarie R.L. Verberckmoes, Bruno B. Jackobsen, Logan R.J. Bailey,Brent M. Wood, Julie Bossuyt.Pharmacology, UC Davis, Davis, CA, USA.Protein Kinase D1 (PKD1) is an important stress stimuli transducer affectingmyriad cellular functions. Despite a crucial role in pathological cardiac re-modeling, few cardiac PKD1 targets have been identified. Recent studies sug-gest PKD1 could target protein phosphatases (PP) other than the well-knownsubstrate slingshot. Here we test this by measuring phosphatase activity andexpression in ventricular homogenates (n=10) from cardiac-specific PKD1knockout mice (cKO) vs. their wildtype littermates (WT). Using the EnzChekphosphatase assay kit with the 6,8-difluoro-4-methylumbelliferyl phosphatesubstrate (DiFMUP), PP1, PP2a, PP2b and PP2c phosphatase activity wasmeasured as tautomycin-, LB100-, calcineurin- and sanguinarine chloride-sensitive DiFMUP fluorescence respectively. PP1 activity was slightly higherin cKO vs. WT homogenates (1.4 5 0.1 vs. 1.0 5 0.1) at baseline. Inhibitionof the other PKD isoforms with 25 nM CRT0066101, reduced PP1 activity inboth WT and cKO (to 0.8 5 0.4 and 0.7 5 0.1 respectively). PP2a activitywas slightly reduced in cKO homogenates (15 0.2 vs. 0.7 5 0.2) at baseline,and strongly reduced with CRT treatment in both WT and cKO (0.3 5 0.1 vs.0.2 5 0.1 respectively). PP2b activity was unaltered in WT vs. cKO homog-enates (1.050.2 vs. 1.050.1) but CRT strongly reduced PP2b activity(0.250.1 vs. 0.150.2 in WT and cKO respectively). PP2c activity wasalso similar at baseline and decreased following CRT inhibition (0.2 5 0.1vs. 0.1 5 0.1 in WT and cKO). Interestingly PP2A protein expression wasreduced in cKO vs. WT homogenates. Our results are a first step in definingPKD isoforms as regulators of cardiac protein phosphatase activity. Future ex-periments will be critical to uncover the molecular mechanisms that underliethe fine-tuning of phosphatase activity in health and disease by PKDsignaling.

2480-Pos Board B496Methylglyoxal Modifications are Elevated in the Myofilament of DiabeticCardiomyopathy Patients and Reduce Myofilament FunctionMaria Papadaki1, Ronald J. Holewinski2, Nikolai Smolin1,Marisa J. Stachowski1, Cheavar A. Blair3, Kenneth S. Campbell3,Seth L. Robia1, Jonathan A. Kirk1.1Physiology, Loyola University Chicago, Maywood, IL, USA, 2HeartInstitute, Cedars Sinai Medical Center, Los Angeles, CA, USA, 3Physiology,University of Ketucky, Lexington, KY, USA.Methylglyoxal (MG), a byproduct of glycolysis and a reactive carbonyl species(RCS), is significantly elevated in diabetes. MG adds post-translational modi-fications on proteins by reacting with arginine and lysine residues to form irre-versible carbonyl adducts. Previous studies found that MG acts on SERCA andthe ryanodine receptor, but the effect of MG on the cardiac myofilament is un-known. The myofilament was enriched in left ventricular tissue samples fromexplanted diabetic cardiomyopathy patients (dbCM) and non-failing rejected

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donor hearts. Using dot blot and mass spectrometry, dbCM patients exhibiteda�33% increase in total methylglyoxal modifications on myofilament proteins.To investigate the functional effect of methylglyoxal on the myofilament,force-Ca2þ experiments on mouse skinned myocytes were performed. Twentyminutes exposure to 100 mMMG reduced maximal Ca2þ activated force (Fmax)by 12 5 4 % (n = 8, p = 0.048) and Ca2þ sensitivity by 45 5 11 % (n = 8,p = 0.02). With 10 mM MG, Ca2þ sensitivity was decreased by 67 5 17 %(n = 9, p<0.01) but there was no effect on Fmax. MG had no effect on passivetension or protein phosphorylation. Mass spectrometry on MG-treated mouseas well as human dbCM isolated myofilament identified several MG modifica-tions on actin and myosin. Structural analysis found that several MG sites are inthe actin-myosin interface region, on residues that are likely important in cross-bridge formation. MG modifications of these sites are predicted to decreaseactin-myosin interaction and force, as we observed experimentally. OtherMG sites on actin are between the monomers, and may affect actin polymeri-zation. Overall, the data shows that in human diabetic cardiomyopathy, myofil-ament MG modifications are elevated, reducing contractile function likely byinterfering with actin-myosin interaction.

Posters: Microtubules, Structure, Dynamics andAssociated Proteins I

2481-Pos Board B497Computational Modeling of Tubulin-Tubulin Lateral Interaction: Molec-ular Dynamics and Brownian DynamicsMahya Hemmat1, David J. Odde2.1Mechanical Engineering, University of Minnesota, Minneapolis, MN, USA,2Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA.Microtubules’ (MTs) dynamic self-assembly behavior is an important medi-ator of motor-based transport in axons and of cell division and mitosis.Several research studies have been done to understand the underlying energylandscape driving their dynamic behavior, i.e. continually switching fromgrowth to shortening, and back to growth, a process known as ‘‘dynamicinstability’’, both at atomic levels (structural data) and dynamic molecularand cellular level (thermokinetic and mechanical models). However, therehas always been a gap between these models at different scales, preventingthe translation of information from the atomic level up to the cellular level,leaving this field with a need for a multi-scale modeling approach that can beused to understand microtubules’ behavior in physiological contexts. In thisresearch, we use molecular dynamics (MD) to simulate the tubulin-tubulinprotein interactions at atomic s cale, by obtaining the potential of mean force(PMF) of their interaction, a system’s energy change as a function of a reac-tion coordinate and then incorporating the MD results at molecular scale viaBrownian dynamics (BD) modeling. It was found that nucleotide state did nothave any significant effect on the tubulin lateral interaction overall althoughthey had different contributions from ionic and hydrophobic interactions. Inaddition, Brownian dynamics show that lateral bonds resulted from the PMFsare short-term and have a very high dissociation rate. This knowledge willhelp us to build a framework for a multi-scale approach that can bridgethe microscopic data from MD simulation to macroscopic behavior of dy-namic microtubules.Keyboards: Microtubules, Molecular Dynamics, Tubulin, Brownian dy-namics, Multi-scale modeling, dynamic instabilityGrants: R01 AG053951

2482-Pos Board B498Binding Interactions with Tubulin’s C-terminal Tail as Studied by Solu-tion NMRAllison M. Whited, Kathryn P. Wall, Scott Tilden, Loren E. Hough.University of Colorado-Boulder, Boulder, CO, USA.Microtubules, formed by the polymerization of alpha and beta tubulin heter-odimers, play an essential role in cell division and motility. The disorderedC-terminal tails of each tubulin monomer are major sites of tubulin regula-tion. The post-translational modifications of the C-terminal tails enable mi-crotubules to perform a wide range of functions, including the ability tocontrol the dynamics and flexibility of microtubules as well as their abilityto bind to microtubule-associated proteins (MAPs). Despite the importanceof these C-terminal tails and their modifications, little structural informationis available on how they affect binding to MAPs. Moreover, the functionalityof the C-terminal tails may depend on whether the tail is attached to the or-dered tubulin body or it is present as a free peptide. To address these limita-tions, we developed techniques to use solution nuclear magnetic resonance

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(NMR) as a probe of the C-terminal tails (Wall et al, ACS Chem Bio, 2016).Using this approach, we study interactions between the disordered C-terminaltails and different MAPs with a particular emphasis on how post-translationalmodifications may affect these binding interactions. Of particular interest tous are the þTIP families of MAPs, which localize to the growing end ofmicrotubules.

2483-Pos Board B499All Tubulins are Not Alike: Dimer Dissociation and Monomer ExchangeDiffer Depending on the Biological Source of TubulinFelipe Montecinos-Franjola1, Sumit Chaturvedi2, Peter Schuck2,Dan L. Sackett1.1NICHD / NIH, Bethesda, MD, USA, 2NIBIB / NIH, Bethesda, MD, USA.Tubulin, the subunit protein of microtubules, is a heterodimer composed ofone a- and one b-tubulin monomer. Both a- and b-tubulins are coded by mul-tiple genes or isotypes, so a dimer can in principle have many combinationsof one a- and one b-tubulin. Isotypes are expressed in tissue- anddevelopement-specific manner, allowing a different set of tubulins indifferent tissues. This potential diversity is increased by the many posttrans-lational modificiations that are known on both subunits. We have investigatedtubulins form different tissues and different organisms for a basic physicalcharacteristic: dimer stability and monomer exchange between dimers. Wepreviously showed that mammalian brain tubulin dimers reversibly disso-ciate, obeying a mass action Kd. Dissociation reversibly yields monomersthat are stable for hours and which can exchange with added tubulin toform new dimers. In this study, we compared the reversible dissociation oftubulins purified from mammalian brain (rat in our case) and compare withtubulin from human cells (HeLa), chicken brain, chicken erythrocytes, andthe protozoan Leishmania.We used fluorescence-based analytical ultracentrifugation to measure dimerdissociation over a � 1000-fold range in concentrations and found thattubulin dimers from different sources differ in dissociation. Kd valuesmeasured for these tubulins vary through a range of nearly 500-fold underthe same conditions. Furthermore, when nM fluorescent tubulin is titratedwith unlabeled tubulin from a different source, monomer exchange occursmore readily with some tubulins than others, even more readily than whentitrated with autologous tubulin. These results show that the diversity of tu-bulins extends to the level of dimer stability and monomer exchange betweentubulin types.

2484-Pos Board B500Nucleotide-dependent Conformational Dynamics and Energetics ofTubulinMaxim Igaev, Helmut Grubm€uller.Theoretical and Computational Biophysics, Max Planck Institute forBiophysical Chemistry, Gottingen, Germany.Microtubules (MTs) are key components of the cytoskeleton and play a centralrole in cell division and development. They undergo stochastic switching be-tween phases of growth and shrinkage driven by GTP hydrolysis in ab-tubulindimers. MT assembly requires that tubulin adopts a straight conformation in thelattice, while during disassembly it adopts a kinked conformation that is incom-patible with the MT geometry. The mechanism by which GTP binding rendersindividual tubulin dimers polymerization-competent is still poorly understood.In particular, two models of MT assembly are being controversially discussed:in the allosteric model, GTP binding directly promotes tubulin straightening,whereas in the lattice model tubulin is kinked regardless of its nucleotide state,and it is the assembling MT itself that forces tubulin into a straightconformation.In order to distinguish between these two hypotheses, we have characterizedthe conformational dynamics and energetics of free tubulin in solution usingatomistic molecular dynamics simulations and free energy calculations.Unexpectedly, and contradictory to the above models, we find that GTP-tubulin samples a broad range of intrinsic curvatures all of which are almostisoenergetic. In contrast, GDP-tubulin is confined to low-curvature regions ofthe conformational space. Furthermore, an additional energy barrier is seen inall simulated tubulin ensembles that is associated with collective rearrange-ments in the b-subunit of tubulin but not with the nucleotide state. Thisbarrier separates different bending modi and may be related to the inhibitorymechanism of MT-associated proteins such as stathmin which suppressestubulin polymerization in a nucleotide-independent manner. Overall, ourresults suggest a new combined conformational selection / induced fitmodel in which GTP binding renders tubulin more flexible at the intadimerinterface and allows a specific type of tubulin bending that facilitates MT as-sembly.

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2485-Pos Board B501NaCl Affects Microtubule Persistence LengthBrandon J. Harris1, Jennifer L. Ross2, Taviare L. Hawkins3.1Biology, University ofWisconsin-La Crosse, La Crosse, WI, USA, 2Physics,University of Massachusetts Amherst, Amherst, MA, USA, 3Physics,University of Wisconsin-La Crosse, La Crosse, WI, USA.Microtubules are hollow, polymeric filaments localized in the cytoskeleton.They assist in cellular support, intracellular transportation, and cell division.In vitro, GTP (guanosine triphosphate) microtubules produce 12-13 protofi-laments while GMPCPP (guanylyl 50-a,b-methylenediphosphonate), micro-tubules produce 14 protofilaments. GMPCPP is a slowly hydrolyzableform of GTP. Studies have shown high concentrations of sodium chloride(580 mM) during polymerization decrease protofilament count (9-10) andcreate defects in the microtubule (Dias and Milligan, 1999). We are inter-ested in if these defects affect rigidity. Microtubule rigidity is quantifiedby the persistence length (Lp) which is a form of mechanical stiffness pro-portional to flexural rigidity (EI). EI is the elastic modulus of microtubulesand second moment of area. Persistence lengths were measured for individ-ual in vitro GTP microtubules polymerized in the presence of NaCl. Micro-tubules were stabilized using the chemotherapeutic drug Taxol and confinedto oscillate within a thin 2-D chamber (% 3 mm thickness). Fourier modeanalysis was used to determine the persistence length of individual microtu-bules. Bootstrapping was then applied to produce a statistically more accu-rate Lp.

2486-Pos Board B502Microtubule Self-Organization in the Presence of Crowding AgentsCarline A. Fermino do Rosario.UMass Amherst, Amherst, MA, USA.Microtubules are filaments made of tubulin protein that are used to organizeand arrange the interior of cells. Their specific arrangements in space and timeare responsible for cell development, cell motility, and perhaps most impor-tantly, cell division. Microtubules self-organize in the cell via growth andshrinkage, called dynamic instability, or through the activity of ATP-utilizing enzymes, such as motor proteins. We are interested in how microtu-bules can self-organize when propelled by motor proteins, specificallykinesin-1. Previous work from our lab showed that high densities of microtu-bules propelled causes loops. In this study, we are extending the results todescribe how microtubules self-organize in the presence of crowding agents,specifically the large, charged polymer, methylcellulose. We are also inter-ested to determine if the microtubule length plays a role in the alignmentand organizations microtubules can make. Our results have implications forboth cell biology and cutting-edge materials science, where the use ofenergy-using processes to create materials that can sense and move inresponse to stimuli.

2487-Pos Board B503Microtubule Patterns through Growth and CrosslinkingBianca Edozie.UMass Amherst, Amherst, MA, USA.Microtubules are the underlying structures of essential cellular structures suchas the mitotic spindle, primary cilium, and neuronal axon. Self-organized pat-terns caused by motors have been explored, but very few of these are resultedin biologically-relevant organizations. We will report on recent progress increating biologically-relevant microtubule organizations. We find that thephases and patterns depend very sensitively on the filament length and thepercentage of MAP65 crosslinkers present. We find that there is a range ofconcentrations that result in spindle-like ‘‘tactoids’’ that could act as modelmitotic spindles. This work will allow us to explore the effects of proteinsand enzymes on mitotic spindle organization using this novel in vitro recon-stitution system.

2488-Pos Board B504Label Free High Speed Wide Field Imaging of Single Microtubules usingInterference Reflection MicroscopyMohammedMahamdeh1, Steve Simmert2, Anna quchniak1, Erik Sch€affer2,Jonathon Howard1.1Molecular Biophysics and Biochemistry, Yale University, New Haven, CT,USA, 2Center for Plant Molecular Biology, University of T€ubingen,T€ubingen, Germany.When studying microtubules, label free imaging of single microtubules isnecessary when the quantities of purified tubulin is too low for efficientfluorescent labeling. Commonly used techniques for observing unlabeledmicrotubules, such as video enhanced differential interference (VE-DIC),dark-field (DF) and more recently laser-based interferometric scattering

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(iSCAT) microscopy, suffer from a number of drawbacks. For example,contrast dependence on microtubule orientation (DIC), high sensitivity toimpurities and to misalignments (DF), and complexity and limited fieldof view (iSCAT). In addition, all of these techniques require costly opticalcomponents such as DIC prisms, dark-field condensers, and lasers and laserscanners. Here we show wide-field high-speed imaging of single microtu-bules using interference reflection microscopy (IRM) that does not sufferfrom any of the aforementioned drawbacks. The technique only requiresthe incorporation of a 50/50 mirror in a fluorescence microscope. We opti-mized the microscope settings to achieve highest signal-to-noise ratio. Wealso compared IRM to DIC and fluorescence imaging. Finally, we demon-strated the strength of the technique by high speed (100 fps) imaging andtracking of dynamic microtubules. In conclusion, the image quality ofIRM is comparable to aforementioned techniques and, with minimal micro-scope modification, can be used to study the dynamics of unlabeledmicrotubules.

2489-Pos Board B505Catastrophic Depolymerization of Microtubules Driven by Subunit ShapeChangeJonathan A. Bollinger, Mark J. Stevens.Center for Integrated Nanotechnologies, Sandia National Laboratories,Albuquerque, NM, USA.Microtubules are key component of cells and understanding their behavior iskey to cellular biophysics. While microtubules possess high mechanicalstrength (sufficient for driving, e.g., mitosis), they possess a distinctive dy-namic instability between growth and catastrophically-fast depolymerization.GTP-tubulin (a tubulin dimer bound to GTP) self-assembles, but dephosphor-ylation of GTP- to GDP-tubulin within the tubule results in destabilization.The molecular origins of this instability have remained unclear despite theirimportance in cellular processes. One hypothesis is that dephosphorylationcauses tubulin to change shape, frustrating bonds and generating stress. Totest this idea, we perform molecular dynamics simulations of whole microtu-bules built from a new coarse-grained model of tubulin, and we implementconformational changes in tubulin thought to drive the instability. We findthat this shape change induces depolymerization of otherwise stable polymersvia unpeeling ‘‘ram’s horns’’ characteristic of microtubules. Depolymerizationcan be averted by caps with undeformed dimers, i.e., GTP-rich end regions, andmodel microtubules exhibit mechanical responses consistent with experiments.This demonstrates that the shape change is sufficient to yield microtubuledepolymerization.

2490-Pos Board B506Microtubule Depolymerization Inhibited by Macromolecular CrowdingVirginia VanDelinder, Nathan Bouxsein, Randy Ko, George Bachand,Rishi Jain.Sandia National Laboratories, Albuquerque, NM, USA.Microtubule filaments are one the fundamental structural components of thecytoskeleton in eukaryotic cells. They play an important role in manycellular functions, including cellular division, intracellular transport, andmotility. Microtubules have been a prime target of anti-cancer drugs becauseof their essential role in mitosis. From a broader perspective, microtubulesare complex biopolymers that displays dynamic instability, in which the mi-crotubules stochastically switch between states of polymerization (growth)and depolymerization (shrinkage). The process of depolymerization isbelieved to be induced by conformational changes to the tubulin dimersthat create mechanical strain within the microtubule lattice. We investigatehow macromolecular crowding contributes to the regulation of depolymer-ization in microtubules through osmotic pressure. The addition of polyeth-ylene glycol (PEG) in vitro mimics the macromolecular crowding thatoccurs in cells. In addition to increasing the growth rate of microtubulesand promoting the formation of microtubule bundles, we find that PEGalso stabilizes microtubules against depolymerization in the absence of anyother stabilizer; this stabilization effect lasts for more than 30 days. Wefurther show that PEG-related molecular crowding stabilizes microtubuleseven in antagonistic environments of extreme temperatures (i.e. <15�C)and destabilizing chemical agents (calcium). Collectively, these data providecritical insights towards understanding microtubule dynamics, specificallythe role of mechanical strain in the catastrophic depolymerization of micro-tubules. Further, as the osmotic pressure in vivo is higher than in typical invitro experiments, these results are salient for translating between microtu-bule stability experiments in vitro and in vivo and studying proteins anddrugs that interact with microtubules. Sandia National Laboratories is a mul-timission laboratory managed and operated by National Technology and En-gineering Solutions of Sandia LLC, a wholly owned subsidiary of Honeywell

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International Inc. for the U.S. Department of Energy’s National Nuclear Se-curity Administration under contract DE-NA0003525.

2491-Pos Board B507Age-dependent Catastrophes and Macroscopic Switching Transition inDynamic MicrotubulesJ.S. Aparna1, Ranjith Padinhateeri2, Dibyendu Das3.1Centre for Reasearch in Nanotechnology and Science, Indian Institute ofTechnology Bombay, Mumbai, India, 2Dept. of Biosciences andBioengineering, Indian Institute of Technology Bombay, Mumbai, India,3Dept. of Physics, Indian Institute of Technology Bombay, Mumbai, India.Microtubules are biopolymers assembled from tubulin proteins and are knownto perform several vital cellular functions. These filaments exhibit a phenom-enon known as dynamic instability which is characterised by cycles of rapidshortening (catastrophes) followed by slow growth (rescues) of filamentlength, as a result of non-equilibrium conversion of tubulin monomers fromthe GTP- to the GDP-bound state. Experimental evidences suggest a remark-able dependence of frequency of catastrophes on the age of microtubule.Although previous theoretical studies have captured this age-dependenceand the multi-step mechanism behind it, catastrophe frequencies are measuredbased on certain apriori assumptions, and they do not provide length versustime data of suitable scales. In this work, we employ a kinetic model whichaccounts for GTP/GDP-state dependent polymerisation, depolymerisationand hydrolysis to produce catastrophes of micrometer lengthscales and time-scales of a few minutes, as observed in experiments. Further, from our model,the age-dependence of catastrophe frequencies emerges naturally from thelength versus time data, without any ad-hoc assumptions for the existenceof catastrophes. Interestingly, we find that in the parameter regime of age-dependent catastrophes, an ensemble of filaments show an abrupt switch-like transition in the mean velocity, from a growth phase to a shrinkage phase,with a regime of co-existence of the two phases, as envisaged by Mitchisonet al (Cell, 1986). The extent of the discontinuity is probed using character-istic features such as bimodality in velocity distribution, divergence of highercumulants and hysteresis. Overall, our results highlight a strong dependenceof multi-step mechanism of catastrophe and the nature of transition onchanges in kinetic rates. Hence, the model can provide significant insightsinto the mechanism of Microtubule Associated Proteins and motor proteinswhich are known to regulate microtubule dynamics via alteration in kineticparameters.

2492-Pos Board B508AFM Studies of the Structure and Physical Properties of MicrotubulesProduced with Drugs and GTP AnaloguesKaren Richardson1, Hsein-Shu Liao1,2, Maryam Raftari1, Citlally Garcia1,Dan Sackett3, Albert J. Jin1.1LCIMB, National Institute of Biomedical Imaging and Bioengineering/NIH,Bethesda, MD, USA, 2Department of Mechanical Engineering, NationalTaiwan University, Taipei, Taiwan, 3National Institute of Child Health andHuman Development /NIH, Bethesda, MD, USA.Atomic Force Microscopy (AFM) is a sophisticated tool that can evaluate thenanoscale structural properties of biological samples in physiological condi-tions. We have applied AFM to evaluate the structural and mechanical proper-ties of microtubules (MT), cytoskeletal structures with significant roles in celldivision, intracellular communication, locomotion, and other functions. MT areimportant targets for many drugs, including MT stabilizers such as Taxol (Tax)and Peloruside (Pel). We used AFM to study MT polymerized with Tax andPel. We also studied MT polymerized with GMPCPP and other GTP analogueswith or without the drugs. Quantitative Nanomechanical Mapping (QNM)mode was used to map and distinguish between nanomechanical propertieswhile simultaneously imaging sample topography at high resolution. The pointand shoot function was used to determine force curves at specific locations onthe microtubules under fluid, while high speed and high resolution imaging wasused both under fluid and in air. We have compared GMPCPP-MT with Tax-MT and with Pel-MT, as well as GMPCPP-Tax-MT to more fully understandthe mechanical properties of microtubules.

2493-Pos Board B509Skeletal Muscle Contraction Alters Microtubule Properties that ImpactFunctionCamilo Vanegas1, Humberto Joca2, Jack Vandermeulen3, Ramzi Khairallah3,W. Jonathan Lederer2, Joseph Stains1, Christopher W. Ward1.1Orthopedics, University of Maryland, Baltimore, Baltimore, MD, USA,2BioMet, University of Maryland, Baltimore, Baltimore, MD, USA,3Myologica LLC, Baltimore, MD, USA.We discovered the microtubule (MT) dependent mechanotransduction pathwaylinking contraction/stretch to NADPH oxidase 2 (Nox2) derived reactive

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oxygen species (ROS) signals (X-ROS) in skeletal muscle. We further showedthat X-ROS target calcium (Ca2þ) channels to regulate Ca2þ influx. Our workin healthy muscle aligned the properties of the MT (i.e., density and stability) tothe stiffness of the cytoskeleton (CSK) which regulated the magnitude ofmechano-activated X-ROS and Ca2þ influx. Our work in dystrophic muscle re-vealed that disease driven alterations in MT properties impact CSK stiffness todrive the deleterious excess in contraction elicited X-ROS and Ca2þ signalsresponsible for contraction injury. In recent work, we sought to reveal mecha-nisms that drive alterations in MT stability and density and further define howthese MT alterations impact function. Using established in vivo and in vitrocontraction paradigms, we show MT alterations align with the functional def-icits in healthy muscle exposed to non-injurious and injurious contractions; def-icits previously linked to altered Ca2þ and ROS signaling. We further identifiedROS and pro-inflammatory cytokines as signals that promote alterations in MTstability and density. Extending our understanding of X-ROS as a mechano-activated signal, we now show that contraction, pharmacologic or geneticdriven alterations in MT properties act to increase CSK stiffness which is suf-ficient to increase X-ROS signaling and Ca2þ influx in resting muscle. Togetherthese new discoveries add important insight the MT network as a dynamicregulator of skeletal muscle function in health and disease.

2494-Pos Board B510Generation of Electrical Oscillations by Different Microtubule StructuresMaria del Rocio Cantero1,2, Paula L. Perez1,2, Cecilia Villa Etchegoyen3,Noelia Scarinci1,2, Horacio F. Cantiello1,2.1IMSaTeD Conicet, Santiago del Estero, Argentina, 2Facultad de CienciasM�edicas, UNSE, Santiago del Estero, Argentina, 3Segunda Catedra deFarmacologıa, Facultad de Medicina, UBA, Buenos Aires, Argentina.Microtubules (MTs) are unique components of the cytoskeleton formed by hol-low cylindrical structures of ab tubulin dimeric units. MTs form intracellularsuperstructures of variable complexity and defined biological role(s) in cellfunction. MTs are able to amplify and axially transfer electrical signals, thusbehaving as macromolecular transistors. However, the molecular aspects ofthese electrical signals are yet to be disclosed. We applied the patch clamp tech-nique to different MT superstructures, including two-dimensional MT sheetsand macrotubes, to characterize their electrical properties. Brain MTs were iso-lated from either rat or cow brains by polymerization-depolymerization cycles.MT sheets were particularly amenable to voltage-clamping as gigaseal forma-tion was achieved in most cases. Electrically-biased MTs generated highly reg-ular cation-selective oscillatory currents whose magnitude depended on theholding potential, ionic composition and ionic strength. Oscillations progressedthrough various regimes, including single and double periods, and more com-plex behaviors, being prominent a fundamental frequency at 29 Hz (in MTsheets but not macrotubes). In physiological Kþ (140 mM), oscillations repre-sented a 700% change in conductance. Interestingly, the peak conductance at29 Hz obtained from the Fourier spectra, also showed strong nonlinearity,and a clear negative resistance region that was modeled with the Esaki diodecurrent equation displaying a ‘‘tunneling’’ effect. Current injection to MTsalso induced voltage oscillations similar to action potentials. The electrical os-cillations were entirely blocked by taxol, with pseudo Michaelis-Menten ki-netics (KD = 1.29 mM). The findings demonstrate that MTs are capable ofgenerating electrical signals and behave as electrical oscillators and amplifiersthat may be relevant to the various biological features of the neuronalcytoskeleton.

2495-Pos Board B511Microrheology of Microtubule Aqueous SolutionKazutaka Satou1, Daisuke Takeuchi1, Syuzi Fujii1, Hiroshi Orihara1,Kentarou Kayano2, Arif Md Rashedul Kabir3, Ituki Kunita4, Akira Kakugo5.1Division of Applied Physics, Hokkaido University, Sapporo, Japan,2Division of Chemistry, Hokkaido University, Sapporo, Japan, 3Departmentof Chemistry, Hokkaido University, Sapporo, Japan, 4Department ofInformation Engineering, University of Ryukyus, Nakagami, Japan,5Graduate School of Chemical Sciences and Engineering, Department ofChemistry, Hokkaido University, Sapporo, Japan.The dynamics of microtubule is characterized by treadmill-like motion, whichis a result of polymerization and depolymerization processes. Such non-equilibrium motion of the microtubule can be suppressed by adding paclitaxel.Thus, the paclitaxel stabilized microtubule is regards as an equilibrium system.In this study, we investigate an effect of the non-equilibrium treadmill-like mo-tion on the viscoelasticity. In the particle tracking experiments, the meansquared displacement (MSD) of microbeads in non-equilibrium microtubulesystem is larger than that of equilibrium system with paclitaxel. Effect of thepaclitaxel on the diffusivity of microbeads corresponds to the result of the dy-namics viscoelasticity, where the storage modulus of the equilibrium system is

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larger than that of non-equilibrium system. In the presence of the paclitaxel, thevariance of MSD is also larger compared to the non-equilibrium system. Theseresults indicate that equilibrium microtubule system forms inhomogeneousmicrotubule network structure, while non-equilibrium microtubule systemhas relatively homogeneous network. We will also present experimental resultsof confocal rheometry.

2496-Pos Board B512Formation of Shear Band in a Microtubule SolutionKei Hamasaki1, Daisuke Takeuchi1, Shuji Fujii1, Hiroshi Orihara1,Katsuhiko Sato2, Itsuki Kunita3, Kentaro Kayano4, Arif Md Rashedul Kabir5,Akira Kakugo5.1Division of Applied Physics, Hokkaido University, Sapporo, Japan,2Research Institute for Electronic Science, Hokkaido University, Sapporo,Japan, 3Department of Information Engineering, University of the Ryukyus,Okinawa, Japan, 4Graduate School of Chemical Sciences and Engineering,Hokkaido University, Sapporo, Japan, 5Department of Chemistry, HokkaidoUniversity, Sapporo, Japan.Flow instability under shear flow is commonly observed in many soft-biomaterials. This instability causes the formation of macroscopic band referredto as ‘‘shear bands’’, which can be regarded as non-equilibrium phase transi-tion. This shear band formation has been reported in a variety of systems,including DNA, polymer and surfactant systems. Very recently, we foundthat biological materials such as actin filament and microtubule systemsshow the shear band formation. These biological materials are a componentof the cytoskeleton. It is interesting to know their mechanical instability undershear deformation because their structure formations are very important in anumber of cellular processes. In this study, we directly visualize the sheardeformation of a microtubule solution systems, and discuss the physical originof the shear banding. Microtubule movement is characterized by dynamic insta-bility and treadmilling. This non-equilibrium phenomena can be suppressed byadding microtubule stabilizing agent ‘‘Paclitaxel’’. In the absence of paclitaxel,the shear band formation is not observed. In microtubule solution with pacli-taxel, on the other hand, we observed the shear band formation at shear rateof 0.01-0.1 s�1, which roughly corresponds to cell migration speed. As thepaclitaxel concentration is increased, the shear band formation is observed inwide shear rates. Rheological property of the microtubule may play a role inmechanical propagation of the force for the cell migration.

2497-Pos Board B513NuMA Recruits Dynein Activity to Microtubule Minus-Ends at MitosisChristina L. Hueschen1, Samuel J. Kenny2, Ke Xu2, Sophie Dumont1.1Cell and Tissue Biology, UCSF, San Francisco, CA, USA, 2Chemistry, UCBerkeley, Berkeley, CA, USA.To build the spindle at mitosis, motors exert spatially regulated forces on mi-crotubules. We know that dynein pulls on mammalian spindle microtubuleminus-ends, and this localized activity at ends is predicted to allow dynein tocluster microtubules into poles. How dynein becomes enriched at minus-endsis not known. Here, we use quantitative imaging and laser ablation to showthat NuMA targets dynactin to minus-ends, localizing dynein activity there.NuMA is recruited to new minus-ends independently of dynein and morequickly than dynactin, and both NuMA and dynactin display specific, steady-state binding at minus-ends. NuMA localization to minus-ends involves aC-terminal region outside NuMA’s canonical microtubule binding domain,and it is independent of direct minus-end binders g-TuRC, CAMSAP1, andKANSL1/3. Both NuMA’s minus-end-binding and dynein-dynactin-bindingmodules are required to rescue focused, bipolar spindle organization. Thus,NuMA may serve as a mitosis-specific minus-end cargo adaptor, targetingdynein activity to minus-ends to cluster spindle microtubules into poles.

2498-Pos Board B514Geometrical Properties of Antiparallel Arrays Regulate MicrotubuleSliding and Stalling by PRC1 and Kif4ASithara Wijeratne, Radhika Subramanian.Genetics & Molecular Biology, Harvard Medical School & MassachusettsGeneral Hospital, Cambridge, MA, USA.Motor and non-motor crosslinking proteins play critical roles in determiningthe size and stability of microtubule-based architectures. However, we have alimited understanding of how the geometrical properties of microtubule ar-rays, in turn, regulate the output of crosslinking proteins. Here we investigatethe relative sliding of antiparallel microtubules by two interacting proteins:the non-motor crosslinker PRC1 and kinesin Kif4A. The collective activityof PRC1 and Kif4A also results in the accumulation of both proteins atthe microtubule plus-end (‘end-tag’). Sliding stalls when the end-tags onthe antiparallel microtubules collide. Interestingly, we find that structuralproperties of the initial array regulate two aspects of PRC1-Kif4A mediated

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microtubule organization. First, sliding velocity scales with initialmicrotubule-overlap length. Second, the width of the final stable overlapscales with microtubule lengths. Our analyses reveal how nanometer-sizedproteins can decode micron-scale geometrical features of antiparallel bundlesto define the structure and mechanics of microtubule-based architectures.

2499-Pos Board B515Kinesin Binding Expands and Stabilises the GDP-Microtubule LatticeDaniel Peet, Nigel Burroughs, Robert A. Cross.CMCB, Warwick University, Coventry, United Kingdom.Kinesin-1 is a walking machine that steps processively towards the fastgrowing (plus) ends of microtubules, hauling molecular cargo to specific reac-tion sites in cells. Recent work hints that kinesin-1 may also play a role inmodulating the structure and stability of its microtubule track, but results areconflicting and mechanisms as yet unclear. By capping dynamic microtubuleswith GMPCPP tubulin, tethering them in a microfluidic flow and introducingkinesin, we have found that strong-state (ATP- and apo-) kinesin-1 motor do-mains inhibit the shrinkage of GDP-MTs by up to 2 orders of magnitude andexpand their lattice spacing by 1.6%. Our data reveal an unexpected new mech-anism by which the mechanochemical cycles of kinesin and tubulin interlock,allowing motile kinesins to influence the structure, stability and mechanics oftheir microtubule track.

2500-Pos Board B516Mechanism of Microtubule Stabilization by Kinesin-5Geng-Yuan Chen1, Ana B. Asenjo2, Hernando J. Sosa2,William O. Hancock1.1Biomedical Engineering, Pennsylvania State University, State College, PA,USA, 2Physiology and Biophysics, Albert Einstein College of Medicine,Bronx, NY, USA.In addition to their capacity to slide apart antiparallel microtubules duringspindle formation, the mitotic kinesin-5 motor Eg5 has been shown to pauseat microtubule plus-ends and enhance microtubule polymerization (Chen andHancock, Nature Comm. 2015:8160). The goal of the present work is to un-derstand the Eg5 microtubule polymerase mechanism by studying how themotor alters the lateral and longitudinal tubulin-tubulin interactions that stabi-lize the microtubule lattice. Transient kinetics and single-molecule trackingexperiments demonstrate that dimeric Eg5 motors reside predominantly in atwo-head-bound strong-binding state while stepping along the microtubule(Chen et al., JBC 2016:291(39), 20283-94). This suggests that when Eg5pauses at a growing microtubule plus-end, the motor acts as a two-head-bound ‘‘staple’’ to stabilize the longitudinal bonds of incoming tubulin di-mers. The on-rate for Eg5 binding to free tubulin is slow, suggesting thatend-bound Eg5 motors do not bind free tubulin in solution; rather they stabi-lize incoming tubulin dimers that have bound to the plus-end. Because tubulinin the microtubule lattice resides in a ‘‘straight’’ conformation, while freetubulin resides in a ‘‘kinked’’ conformation, a second (non-mutually exclu-sive) model is that Eg5 stabilizes the straight conformation of tubulin. Consis-tent with this, monomeric Eg5 motors, which bind to the microtubule latticewithout crosslinking tubulin dimers, also stabilize microtubules against depo-lymerization. Furthermore, the affinity of Eg5 for free tubulin is reduced inthe presence of ‘‘wedge inhibitor’’ drugs that stabilize the kinked conforma-tion of tubulin; conversely Eg5 binding to tubulin diminishes drug binding.Thus, we propose a microtubule polymerase mechanism in which bindingby one Eg5 motor domain at the microtubule plus-end straightens tubulin di-mers and stabilizes lateral tubulin-tubulin bonds, while the second Eg5 headbinds incoming tubulin and acts as a staple to stabilize longitudinal tubulin-tubulin bonds and enhance microtubule growth.

2501-Pos Board B517Molecular Requirements for the Transition from Lateral to End-onMicrotubule Binding and Dynamic CouplingEkaterina L. Grishchuk1, Manas Chakraborty1, Anatoly V. Zaytsev1,Maxim Godzi2, Ekaterina Tarasovetc1, Ana C. Figueiredo3,Fazly I. Ataullakhanov2.1Dept. of Physiology, Perelman School of Medicine, University ofPennsylvania, Philadelphia, PA, USA, 2Center for Theoretical Problems ofPhysicochemical Pharmacology, Russian Academy of Sciences, Moscow,Russian Federation, 3Chromosome Instability & Dynamics Laboratory,Universidade do Porto, Porto, Portugal.Accurate chromosome segregation relies on the ill-understood ability of kinet-ochores to convert their lateral microtubule attachment into the microtubuleplus-end association, capable of the processive motion with tubulin dynamics.Here we report that this transition can be recapitulated in vitro using only twocomponents: the plus end-directed kinesin CENP-E and the microtubule wall-binding Ndc80 protein. CENP-E’s primary role is to establish the end config-

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urations for Ndc80, while Ndc80 mediates the maintenance of end attachment.To gain insights into the molecular requirements for end-conversion we pairedCENP-E with other microtubule-binding proteins. Ska1, CENP-E Tail, EB1and CLASP2 differed in their ability to retain the microtubule ends, andnone performed as robustly as Ndc80. Likewise, a non-mitotic transporterKinesin-1 failed to support the Ndc80-mediated end-conversion, implyingthat the pair of CENP-E kinesin and Ndc80 is optimally suited for this activity.To investigate the underlying mechanistic differences between these motorsand MAPs, we developed a Brownian dynamics model for the molecularensembles of proteins engaged stochastically in walking and diffusion on themicrotubule wall. Modeling demonstrates that the observed differences in theend-retention by different MAPs can be largely attributed to their different res-idency times and rates of diffusion on the microtubule wall. The model alsorecapitulates the strikingly different behavior of Kinesin-1 and CENP-E,suggesting that it is rooted in their distinct force-detachment characteristics.Following a model prediction, we were able to achieve robust end-conversion with Kinesin-1 by amending its dynamic response via the reducedATP concentration. Together, our results argue strongly that microtubuleend-conversion is an emergent property of the ensemble of transporting motorsand diffusingMAPs, limited by the microtubule end boundary. We propose thatsimilar mechanism ensures microtubule end conversion at mitotickinetochores.

2502-Pos Board B518Microtubule Structural State Recognition by End Binding Protein 1Taylor A. Reid1, Courtney Coombes1, Holly Goodson2, Melissa K. Gardner1.1University of Minnesota, Minneapolis, MN, USA, 2Notre Dame, SouthBend, IN, USA.Microtubules are structural polymers that participate in a wide range ofcellular functions and are the site of localization and activity for a host ofproteins. End binding protein 1 (EB1) localizes primarily to GTP-boundtubulin subunits at the growing ends of microtubules, where it facilitates in-teractions with other key cellular proteins. However, reports of sublocaliza-tion of EB1 within the GTP-rich region at growing microtubule ends suggestthat there may be an additional layer of regulation for EB1 binding to themicrotubule. Using both bulk and sub-microtubule correlation TIRF micro-scopy experiments, we found that, independently of nucleotide state, EB1 ex-hibits preferential binding for non-lattice structures such as those found atmicrotubule ends or defects. To predict the mechanism for this preference,we performed 3D molecular diffusion simulations, and found that the uniquebinding location of EB1 at the pocket-like interface between four adjacenttubulin dimers results in a form of structural state recognition due to ahigh steric hindrance to binding within the lattice, which is reduced at micro-tubule ends or lattice defects. Additionally, our experiments and simulationsusing a tubulin face binding protein resulted in elimination of microtubulestructure recognition, suggesting a general principle for protein associationto cellular polymers based on the location and conformation of the bindinginterface.

2503-Pos Board B519Structural Model for Preferential Microtubule Minus End Binding byCAMSAP CKK DomainsJoseph Atherton1, Kai Jiang2, Marcel Stangier3, Yanzhang Luo4,Shasha Hua2, Klaartje Houben2, Guido Scarabelli5, Agnel Joseph1,Anthony Roberts1, Barry Grant6, Maya Topf1, Michel Steinmetz3,Marc Baldus2, Anna Akhmanova2, Carolyn Moores1.1Birkbeck College, London, London, United Kingdom, 2Utrecht University,Utrecht, Netherlands, 3Paul Scherrer Institut, Villigen, Switzerland, 4UtrechtUniversity, U, Netherlands, 5University of Michigan, Ann Arbor, MI, USA,6University of California, San diego, San diego, CA, USA.Microtubules are polar polymers, with minus and plus ends exhibiting differen-tial dynamics and regulated by different cofactors. The evolutionarilyconserved calmodulin-regulated spectrin-associated protein (CAMSAP) familyare minus end binding proteins that modulate minus end microtubule dynamics.All CAMSAPs share a characteristic conserved CKK microtubule bindingdomain, which defines their minus end specificity. However the mechanismof this specificity is not understood. To shed light on this question, we deter-mined the CKK structure by X-ray crystallography and characterised CAM-SAP1 and 3’s CKK binding site on microtubules by cryo-EM. TheCAMSAP CKK binds at a previously undescribed binding site at the microtu-bule intra-dimer inter-protofilament interface. TIRF microscopy was used toconfirm the contributions of conserved residues at the CKK-microtubule inter-face. The nucleotide state of tubulin was also shown not to influence minus-endspecificity. Interestingly, CKK binding imposes a right-handed ‘supertwist’, onmicrotubule protofilaments, even on the taxol-stabilized microtubules used for

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our reconstructions. Further cryo-EM investigations, including of a CKKmutant, and another CKK domain with reduced minus-end specificity, supportsthe possibility that subtle alterations in CKK positioning relative to tubulinpolymer confer minus-end specificity. Cryo-electron tomography of minus-ends reveals curved lattice to sheet regions that retain lateral protofilament in-teractions and present unique minus-end tubulin conformations to which CAM-SAPs may preferentially bind.

2504-Pos Board B520Structural Changes in Tau Underlie Static and Diffusive Binding to theMicrotubule LatticeAlisa Cario, Jamie Stern, Christopher L. Berger.University of Vermont, Burlington, VT, USA.The microtubule associated protein (MAP) Tau, primarily expressed in neu-rons, is known to have a variety of functions such as regulation of microtubuledynamics, modulation of kinesin motor motility and participation in signalingcascades. Research on the microtubule binding behavior of Tau reveals thatTau binds to the microtubule surface in an equilibrium between static and diffu-sive states. These functional states have been shown to be important in regu-lating kinesin motility during axonal transport. However, the structuralrelationship between the states has not been characterized. Therefore, using To-tal Internal Reflection Fluorescence (TIRF) microscopy, we developed a three-color imaging assay to study the structural changes underlying Tau’s dynamicbehavior while bound to the microtubule surface using single molecule Fluores-cence Resonance Energy Transfer (smFRET). Additionally, Alternating LaserExcitation (ALEX) is used to distinguish between single labeled populationsand low FRET efficiency states. We have generated three 3RS-Tau FRET con-structs to measure distances between distinct locations within Tau, between theN and C termini (N-C), between the microtubule binding repeats and the C ter-minal (3-C), and between the N terminal and the microtubule binding repeats(N-3). Initial studies indicate 3RS-Tau possesses distinct N and C termini inter-actions that allow for static versus diffusive binding. The examination of addi-tional interactions will define overall structural changes in Tau on themicrotubule surface. The smFRET-ALEX approach we have developed has ap-plications for studying differences in other highly dynamic MAPs as well.

2505-Pos Board B521The Effect of Site-Specific Tau Mutations on Microtubule Bundle Struc-turesChristine Tchounwou.UCSB, Goleta, CA, USA.Microtubules (MTs) are biological nanostructures that exhibit GTP-hydrolysiscontrolled polymerization-depolymerization transitions which enable manyimportant cellular functions. In neuronal axons, inherently dynamic MT archi-tectures are functionalized by binding of the MT-associated phosphoproteintau. Changes in tau-MT associations have been noted in the literature as aconsequence of post-translational modifications to tau such as hyperphosphor-ylation leading to the hallmarks of several neurodegenerative disease states.We hypothesize that post-translational alterations to specific residues in closeproximity to the MT-binding region of tau may influence how tau mediatesMT architectures in axons. Furthermore, we demonstrate that purification ofsite-specific mutagenic tau is now readily achievable with expression of mutantrecombinant tau containing a poly-His-tag. Similar to phosphoprotein tau, wehave previously reported that biologically relevant polyamines such as sper-mine also directly modulate electrostatic interactions between MTs leadingto unique bundling architectures. Here we probe dynamical MT bundling archi-tectures in the presence of site-specific mutagenic tau with and without poly-amines using transmission electron microscopy (TEM) and small angle x-rayscattering (SAXS).

2506-Pos Board B522Role of Anti-tau Antibodies on Microtubule Polymerization and StabilityIva Ziu, Matthew Imhof, Saba Anwar, Sanela Martic.Chemistry, Oakland University, Rochester, MI, USA.Tau is an intrinsically disordered protein which in neuronal cells promotes thepolymerization and stability of microtubules (MT). In neurodegenerative dis-eases, tau undergoes post-translational modifications (PTMs), misfolds andforms toxic aggregates. Currently, tauopathies remain without a cure. Immuno-therapies targeting tau protein in animal models induced clearance of tau pa-thology. However, the mechanisms of antibody-based inhibition ofneurodegeneration remain mostly unclear. We have previously reported onthe inhibition of tau aggregation in vitro by antibodies to non-phosphorylatedtau441. We also have shown that phosphorylation of tau441 at Ser199 byGSK-3ß protein kinase may be inhibited by antibodies to phosphorylated tau.Here, we evaluated the polymerization of tubulin in the presence of non-phosphorylated tau and antibodies to non-phosphorylated tau [targeting epi-

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topes in the N-terminus, R4, and C-terminus]. ELISA was used to determineantigen-antibody binding affinities. The microtubules were characterized bytransmission electron microscopy. Tubulin fluorescence polymerization assayindicated that the antibodies reduced tubulin polymerization. In the presenceof tau441, the tubulin polymerization was rescued even in the presence of an-tibodies. In addition to MT formation, the stability of paclitaxel-stabilized MTwas also evaluated. Data indicate that tau protein and its antibodies play regu-latory and/or competitive roles in MT formation/stability.

2507-Pos Board B523N-terminal Inserts Impact the Global Conformation of Tau and the Tau-Tubulin ComplexKristen McKibben1, Elizabeth Rhoades2.1Biochemistry and Molecular Biophysics Graduate Group, University ofPennsylvania, Philadelphia, PA, USA, 2Department of Chemistry, Universityof Pennsylvania, Philadelphia, PA, USA.Tau is an intrinsically disordered, microtubule associated protein best knownfor its role neurodegenerative tauopathies such as Alzheimer’s disease. Tau’sphysiological roles include stabilizing microtubules and regulating microtubuledynamic instability. There are six different isoforms of tau arising from alter-native splicing of two acidic N-terminal insert regions (0N, 1N, and 2N) and thesecond microtubule-binding repeat (3R and 4R). While significant attention hasbeen given to differences in the interactions of 3R and 4R isoforms with tubulinand microtubules, less is known about the role of the N-terminal inserts. In thisstudy, we seek to understand the impact of the N-terminal inserts on the globalconformation of tau in solution and upon binding to tubulin using single mole-cule Forster resonance energy transfer (smFRET) spectroscopy. Our measure-ments show that the overall global architecture between the N-terminalisoforms is similar in solution. However, there are also isoform specific differ-ences; within the N-terminus, there is an inverse correlation between theaverage per residue distance and the number of inserts. Upon binding totubulin, there is a large expansion of the region between the N-terminus andthe third microtubule-binding repeat. While the magnitude of this expansionper residue is similar in 2N4R and 0N4R, 1N4R shows a greater per residueexpansion. Combined with the solution data, this suggests there is a shortsequence or sequences in the N-terminal inserts responsible for maintaininga homologous conformation between isoforms in solution that is disruptedupon binding to tubulin. Additional studies are required to pinpoint this regionand its corresponding interacting sequence.

2508-Pos Board B524Disparate Roles of Alpha and Beta CTTs in Microtubule SeveringRohith Anand Varikoti.University of Cincinnati, University of Cincinnati, Cincinnati, OH, USA.Microtubules (MTs) are responsible for major cellular processes like cellmotility, transport, and mitosis. Modulation of their structure and function inin vivo conditions is achieved by various microtubule associated proteins(MAPs). Among these MAPs are the MT severing enzymes, which belong tothe ATP-dependent homo-hexamerases of ATPases associated with variouscellular activities (AAAþ) family of enzymes. These enzymes are known tobind to MTs in various orientations and for functional reasons they shouldbind to the acidic residue rich carboxy-terminal tails (CTTs) of tubulins.CTTs are known to be crucial for the severing of MTs, but the actual mecha-nism requiring CTTs is unknown. The current view is that severing proteinsbind to CTTs and thread them through the center pore of the hexamer, thusfacilitating severing. Using a combination of docking studies with moleculardynamics simulations and Normal Mode Analysis (NMA), we investigatedthe binding and dynamics of different CTT isoforms on severing proteins.We found that binding of Beta isoforms is preferred over Alpha isoforms,which recapitulates the behavior of katanin binding to CTTs from the experi-mental literature. Using the structural information resulting from docking fordynamic simulations can subsequently give insight into how the action ofsevering enzymes resembles that of AAAþ proteins in general, which involvesthe cooperative action of monomers or groups of monomers in a hexamer toperform the unfolding and translocation of substrates.

2509-Pos Board B525Modulation of Macromolecular Biological Structures by Divalent IonsBretton J. Fletcher1, Chaeyeon Song1, Phillip Kohl1, Peter Chung1,2,Herbert P. Miller1, Youli Li1, Myung Chul Choi3, Leslie Wilson1,S.C. Feinstein1, Cyrus R. Safinya1.1UCSB, Santa Barbara, CA, USA, 2University of Chicago, Chicago, IL, USA,3KAIST, Daejeon, Republic of Korea.Divalent ions can mediate condensation of highly charged biological macromo-lecular assemblies by producing attractions and suppressing repulsions betweenlike-charged structures. Combining small-angle x-ray scattering with x-ray

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fluorescence techniques has allowed us to directly and quantitatively probe thecorrelation between the structural transitions of like-charged biomolecules andthe density of counterions bound to the supramolecular structures. We reporthere on the dependence of this attractive electrostatic regime on counterion con-centration and type (i.e. ion-specific effects) for two different in vitro systems:tau protein-coated microtubules in dissipative, out-of-equilibrium conditionsand DNA molecules confined to two dimensions by cationic liposomes at equi-librium. The functional implications of these ion-mediated transitions in biolog-ical assemblies are poorly understood, creating a need for further research.

2510-Pos Board B526Determining the Important Parameters in Biological Models using Numer-ical Parameter CompressionChieh-Ting Hsu, Gary Brouhard, Paul Francois.McGill University, Montreal, QC, Canada.Building physical models for biological systems is usually difficult due to thenumber of parameters required. In the end, the models themselves usuallydepend on and are sensitive only to a very small number of parameters. Mostexperimental observables are strongly determined by one parameter, meaningthat complete parameter inference from realistic biological data is fundamen-tally challenging. Recent works have explored the method of parametercompression to identify those necessary parameters. In the literature, systemsare often solved analytically, but the complexity of biological models limitsthis approach. Here we will present how we overcome this problem by usinga numerical approach to the parameter compression method. We validate ourapproach against particle diffusion and apply the method to biological modelssuch as protein production-degradation system and a simple dynamic microtu-bule model. The analysis done here shows how the importance of a givenparameter is strongly dependent on the measured experimental observable.

Posters: Kinesins, Dyneins, and OtherMicrotubule-based Motors I

2511-Pos Board B527Biased Binding Mechanism Alone Can Explain the Preferential ForwardStepping of Kinesin-1Kohei Matsuzaki1, Hiroshi Isojima1, Hiroyuki Noji2, Michio Tomishige3.1Department of Applied Physics, The University of Tokyo, Tokyo, Japan,2Department of Applied Chemistry, The University of Tokyo, Tokyo, Japan,3Department of Physics and Mathematics, Aoyama Gakuin University,Sagamihara, Japan.Kinesin-1 moves along microtubule by alternately moving two motor domains(‘‘heads’’); the unbound trailing head is prohibited from rebinding to the rear-binding site and is allowed to binds to the forward-binding site 16-nm ahead.The ‘‘neck linker-docking (or biased-diffusion) model’’ has been proposed toexplain the preferential forward stepping, however this model cannot explainhow the rebinding of the unbound head is prohibited while kinesin waits forATP-binding. Alternate ‘‘biased-binding model’’ proposes that an unfavorableincrease in the tension on the neck linker prohibits the unbound head from re-binding to the rear-binding site. To distinguish between these two models, wepreviously engineered an asymmetrically joined two-headed monomer, inwhich the neck linker of the first head (N-head) is connected to N-terminusof second head (C-head), such that this motor alternately uses biased-binding(when N-head steps forward) and biased-diffusion (when C-head steps) mech-anisms to move in a hand-over-hand manner. In this study we observed the mo-tion of each heads during the processive movement of the two-headedmonomer using the total internal reflection-based dark-field microscopy with50 ms temporal resolution. We found that the stepping of C-head was less effi-cient because the unbound C-head frequently rebound to the rear-binding siteeven at saturating ATP condition, while N-head stepped forward efficientlybecause the unbound N-head rarely showed rebinding under various ATP con-ditions. These results indicate that the diffusional-biased of the unbound head isnot necessary, and that the biased-binding mechanism that prohibits rebindingof the unbound head is sufficient, for the preferential forward stepping ofkinesin-1, at least under low external load.

2512-Pos Board B528Nonequilibrium Energetics of Single Molecule Motor, Kinesin-1Takayuki Ariga1, Michio Tomishige2, Daisuke Mizuno1.1Department of Physics, Kyushu University, Fukuoka, Japan, 2Department ofPhysics and Mathematics, Aoyama Gakuin University, Kanagawa, Japan.Kinesin is a molecular motor that convert chemical energy to mechanicalwork while carrying cargos on the microtubule rail. Although the molecular

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process of the kinesin has been widely investigated, exploring the energeticshas been challenging because it requires to address the nonequilibrium dy-namics of the fluctuating small molecules. In this study, we investigatedthe nonequilibrium dissipation of the walking kinesin [1], by utilizing anovel nonequilibrium equality [2] that relates violation of fluctuationresponse relationship (FRR; the relationship between the fluctuation of thevelocity and the linear response to a small perturbation force) to thenonequilibrium dissipation rate consumed by the system. We observedsingle-molecule kinesin movement by using a feedback-controlled opticaltweezers setup that applied small sinusoidal perturbation in addition to con-stant force to a probe bead attached to the kinesin. We found that the kinesinclearly violates the FRR. We examined the origin of the FRR violation witha theoretical model that couples the Langevin dynamics of the probe to astochastic two-state Markov steps performed by kinesin. The mathematicalanalysis reproduced the experimentally observed FRR. Surprisingly, it turnedout that the sum of the work performed and the heat dissipated via theobserved probe motion were significantly smaller than the total input free en-ergy, indicating that hidden dissipations are dominant. By analyzing thetransmission of the motor action to the probe fluctuations, we concludethat the hidden dissipation is originated from the internal machinery of thekinesin motor performing the biased unidirectional motion. [1] T. Ariga,M. Tomishige and D. Mizuno arXiv:1704.05302 [2] T. Harada and S. -i.Sasa, Phys. Rev. Lett. 95, 13602 (2005).

2513-Pos Board B529A Fluid Membrane Enhances the Velocity of Cargo Transport by SmallTeams of Kinesin-1Qiaochu Li1, Kuo-Fu Tseng2, Stephen J. King3, Weihong Qiu2, Jing Xu1.1Physics, University of California Merced, Merced, CA, USA, 2Physics,Oregon State University, Corvallis, OR, USA, 3Burnett School of BiomedicalSciences, University of Central Florida, Orlando, FL, USA.Kinesin-1 is a major molecular motor driving the fundamental process of trans-port in live cells. While the single-molecule functions of kinesin are well char-acterized, the physiologically relevant transport of membranous cargos bysmall teams of kinesins remains poorly understood. A key experimental chal-lenge remains in the quantitative control of the number of motors driving cargotransport. Here we utilized ‘‘motile fraction’’ to overcome this challenge, andexperimentally accessed transport by single kinesins through the physiologi-cally relevant transport by small teams of kinesins. We use a fluid lipid bilayerto model the cellular membrane in vitro, and employed optical trappingto quantify transport of membrane-enclosed cargos versus traditionalmembrane-free cargos at the same motile fraction (motor number). We foundthat coupling motors via a fluid membrane significantly enhances the velocityof cargo transport by small teams of kinesins. Importantly, enclosing a cargo ina fluid lipid membrane does not impact single-kinesin transport, indicating thatmembrane-dependent velocity enhancement for team-based transport resultedfrom altered interactions between kinesin motors. Our study demonstratesthat membrane-based coupling between motors is a key determinant ofkinesin-based transport. Enhanced velocity may be critical for the timely deliv-ery of cargos in live cells.

2514-Pos Board B530Kinesin Rotates Unidirectionally while Walking on Microtubules Trans-ferring Torque onto CargoAvin Ramaiya1, Basudev Roy2, Michael Bugiel1, Erik Sch€affer1.1Center for Plant Molecular Biology (ZMBP), Universit€at T€ubingen,T€ubingen, Germany, 2Department of Physics, Indian Institute of Technology,Madras, India.Cytoskeletal kinesin motors drive many essential cellular processes. Forexample, kinesin-1 is a dimeric, adenosine-triphosphate-(ATP)-driven molec-ular machine transporting vesicular cargo while stepping in a hand-over-handfashion along microtubules. Because of the identical subunits, the motor hasbeen proposed to rotate during stepping. However, experiments done at lowATP concentrations only revealed occasional, random motor rotations. Directevidence for rotational motion at physiological ATP concentrations is lacking.Here, we used high-resolution optical tweezers combined with a sensitive op-tical micro-protractor and torsion balance employing highly birefringent,liquid crystalline probes to directly and simultaneously measure the transloca-tion, rotation, and generation of force and torque of single kinesin-1 motors.While, at low ATP concentrations, motors did not generate torque, we foundthat motors translocating along microtubules at saturating ATP concentrationsrotated unidirectionally producing significant torque on the probes. Account-ing for the rotational work, makes kinesin a highly efficient machine. Theseresults imply that the motor’s gait follows a rotary hand-over-hand

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mechanism. Our method is generally applicable to study rotational motion ofmolecular machines and our findings have implications for kinesin-drivencellular processes.

2515-Pos Board B531New Structure and Energy Cycles of Kinesin Dimers Walking on Microtu-bules Revealed from Molecular SimulationsAllicia Pan1, Allen Pan1, Bernard R. Brooks2, Xiongwu Wu2.1Madison High School, Vienna, VA, USA, 2NHLBI, National institutes ofHealth, Bethesda, MD, USA.Kinesins are motor proteins that move unidirectionally along microtubules asthey hydrolyze ATP. Although the general features of the kinesin walkingmechanism are becoming increasingly clear, some key questions remain unan-swered, such as how they convert the chemical energy of ATP into mechanicalenergy and walk processively. In this study, through molecular simulations andfree energy calculations, we found that in aqueous solution, kinesin favors anextended form with its microtubule-binding interface (MTBI) motif unfolded,as seen in a recent x-ray structure of kinesin-8. Through the flexible fitting oftwo newly released cryo-electron microscopy (cryo-EM) maps, we derivedatomic structures of the kinesin dimer-microtubule complexes in both two-head-bound and one-head bound states. Free energy calculations showed thatkinesin bound to microtubules has a lower free energy than the extendedform and that the free energy difference is in the range of the free energyreleased by ATP hydrolysis. The transition between the extended and compactforms, the structural differences of the leading and trailing heads, and atomicforce simulations lead us to a completely new mechanism by which kinesin di-mers walk on microtubules.

2516-Pos Board B532Microtubules can Influence Kinesin’s Forestep-Backstep DecisionAlgirdas Toleikis, Nicholas J. Carter, Robert A. Cross.University of Warwick, Coventry, United Kingdom.Kinesin-1 is a processive motor responsible for transporting cargos inside cellsover large distances. At low loads kinesin typically takes about a hundred 8 nmsteps per diffusional encounter with the microtubule, with a very strong direc-tional bias towards the microtubule plus end. Under hindering loads, kinesinstarts to take backsteps, and at stall force, it takes an equal number of backstepsand forward steps. Neither the mechanism of choosing the stepping directionnor the mechanism of backstepping is fully understood. In this work, we are us-ing optical trapping to apply loads to single kinesins in vitro to study the mech-anism of (back)stepping. We are counting forward steps, backward steps anddetachments at particular forces, and investigating how different factors influ-ence the forestep-backstep decision process. We found that when stabilisedyeast (S. pombe) microtubules are used, Drosophila kinesin-1 dwells for longerat any particular load, and stall force is reduced to�6pN. Also, detachments arerelatively less likely at any particular force, indicating an increase in processiv-ity. We further found that for pig brain microtubules, kinesin had a reduced stallforce on microtubules stabilized with GMPCPP compared to those stabilisedwith taxol and was similar to unstabilized GDP-microtubules. These findingsshow that kinesin’s forestep-backstep-detachment decision process is not anintrinsic feature of the motor only, but also is influenced by the microtubuletrack.

2517-Pos Board B533Structural Characterization of the ATP-waiting and Post-hydrolysis Statesof Dimeric Kinesin-1 using Cryo-EMHyo Keun Cha, Xueqi Liu, Garrett Debs, Daifei Liu, Charles Sindelar.Yale University, New Haven, CT, USA.Kinesin functions as a dimeric motor protein that walks along microtubules in aprocessive manner. Over the years, numerous structures of the monomeric mo-tor domain alone and in complex with microtubules have been solved usingconventional structural methods. However, the structural states of the kinesindimer during actual stepping remain poorly understood. In particular, the struc-tural basis by which kinesin detaches its trailing head (ADP-bound following Pirelease) and propels this ‘tethered’ head forward to the next binding site(following hydrolysis in the other ‘bound’ head) remain controversial, andthe structural intermediates corresponding to these events have been elusive.To address these questions, respectively, we generated high-quality samplesof the microtubule-bound kinesin dimer in low ADP conditions (200nMADP) and in the post-hydrolysis ADP-Pi state (200nM ADP þ 10mM Pi),and imaged > 10000 kinesin dimers for each condition by cryo-EM using aTitan Krios microscope. Using the FINDKIN algorithm recently developedby our group1, along with other recent methodological improvements, we areworking towards achieving near-atomic resolution for these structural states.

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These reconstructions will provide insight into how interhead coordination be-tween the two heads promotes processive motility.[1] Liu, D., Liu, X., Shang, Z., Sindelar, C. V. eLife 6:e24490 (2017).

2518-Pos Board B534Enhanced Stability of Kinesin-1 as a Function of TemperatureKatelyn J. Chase1, Florence Doval2, Michael Vershinin2.1Physics, Oregon State University, Corvallis, OR, USA, 2Physics andAstronomy, University of Utah, Salt Lake City, UT, USA.Kinesin-1 is the motor which drives a significant fraction of microtubule-basedtransport in cells. In addition, because of its accessibility and robustness, kine-sin has been seen as a promising motility driver for nano-engineering. Howev-er, degradation of kinesin-1 sets in around 30 oC for typical in vitro assays,much below �40 oC which can be sustained by the motor in vivo. Why arethe in vivo and in vitro results so different? We have examined kinesin motilityassays in the presence of trimethylamine N-oxide (TMAO), which is known tostabilize secondary structure and enzymatic activity of many proteins, and itsmode of action in many ways mimic molecular crowding in cells. We showthat the addition of TMAO results in a dramatic enhancement of kinesin ther-mal range (up to �50 oC). Hence, intracellular crowding emerges as the likelyfactor which allows kinesin-1 to function efficiently in cells at mammalianbody temperatures. This helps address a long standing discrepancy betweenin vivo and in vitro kinesin assays and furthermore positions TMAO as a keynew ingredient for engineered kinesin-based nanodevices.

2519-Pos Board B535Atomic Force Simulations Reveal that the Leading Head of Kinesin DimersGenerates the Cargo Moving ForceAlicia Pan, Allen Pan, Xiongwu Wu.Laboratory of Computational Biology, National Heart, Lung, and BloodInstitute, National Institutes of Health, Bethesda, MD, USA.Kinesin belongs to a family of molecular motors characterized by unidirec-tional movement along microtubules from the center of a cell to its periphery.Numerous experimental and theoretical studies have been dedicated to kinesinssince their discovery in 1985. Kinesin dimers walk along the microtubule trackby binding and unbinding their two heads (the motor domain) to and from step-ping sites on the microtubule surface. It is believed that the cargo moving forceis generated by the trailing head by docking its neck linker. Through flexiblefitting of two newly release cryo-EM maps, we derived atomic structures ofthe kinesin dimer-microtubule complexes in both two-head-bound and one-head bound states. To identify which head generating the cargo moving force,we designed atomic force simulations to examine the responses of the twoheads to dragging forces. Our simulation results show the leading head can pro-vide a necessary force to perform the power stroke while the trailing headcannot stand for even a 5pN dragging force. A structure comparison betweentwo-head-bound and one-head bound states also supports the conclusion thatthe leading head is the source of the cargo moving force.

2520-Pos Board B536Computational and Biochemical Analysis of Disease-Causing Mutations atthe Kinesin-Microtubule InterfaceChelsea Kelland, Lauren Thornton, Hana Alkhafaf,Madhusoodanan Mottamal, Thomas M. Huckaba.Biology, Xavier University of Louisiana, New Orleans, LA, USA.Hereditary Spastic Paraplegia (HSP) is a genetically and clinically heteroge-neous disease that involves the progressive degeneration of axons in the cor-ticospinal tract. To date, 76 distinct genetic loci and 59 separate human geneshave been implicated in the onset of HSP. This work focuses on HSP-causingmutations in Kif5A, a neuronally-enriched transport kinesin. Of the 25 knownmutations in Kif5A that cause HSP, 13 localize to kinesin’s interface withalpha- or beta-tubulin subunits based on crystallographic data. Our workinghypothesis is that these mutations decrease Kif5A’s microtubule affinity dueto altered conformation changes induced by the mutations. To test this hy-pothesis, we performed molecular dynamics simulations by adding theHSP-causing mutations to the published co-crystal structure of kinesin boundto the alpha-beta-tubulin heterodimer. The results of these simulations predicta range of altered kinesin conformations, each losing wild type contacts withthe microtubule but gaining novel compensatory interactions. Usingrecombinantly-expressed purified proteins with the HSP-causing mutations,we performed steady state microtubule binding assays, as well as surface plas-mon resonance analysis to test the affinity of each kinesin construct for micro-tubules. As predicted, each mutation induces a decrease in the protein’saffinity for microtubules, with a range of deficits observed. Finally, wehave performed in vitro assays to study the impact of this altered affinity

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on kinesin motility. In our standard conditions, none of the mutant proteinsexhibit motility; but we can rescue individual mutants by decreasing the ionicstrength of our buffers. This suggests that altered electrostatic interactions arethe root cause of the decreased affinity observed in our assays. These resultslend insight both into the ongoing studies of the kinesin-microtubule interface,as well as providing a mechanistic basis for the cause of HSP resulting fromKif5A mutation.

2521-Pos Board B537KINESIN-2 Motors Adapt their Stepping Behavior for Processive Trans-port on Axonemes and MicrotubulesWilli L. Stepp1, Georg Merck1, Felix Mueller-Planitz2, Zeynep Okten1.1Department f€ur Physik, Technische Universit€at M€unchen, Garching b.M€unchen, Germany, 2Molecular Biology, Biomedical Center, LMUMunich,Martinsried, Germany.The fundamental stepping mechanism of kinesins was investigated using theFluorescence Imaging with One Nanometer Accuracy (FIONA) technique.Kinesin-2 stands out due to it’s employment on both axonemes in cilia and mi-crotubules in the cytoplasm. We used FIONA to track head-labeled kinesin-2motors with high precision on both filaments. Differences in the distributionof step sizes and in the traces show a specialisation of the stepping behaviorto the respective track. The motors take off-axis steps to neighbouring protofila-ments on microtubules but not on axonemes. In combination, both observationsexplain the direction-dependent allocation of cargo that is essential for intrafla-gellar transport. The intrinsic tendency of kinesin-2 to take off-axis steps andstructural barriers specific to the axonemal structure restrict the motors towalk on the B-tubules. These results help to explain, why heterodimerickinesin-2 motors co-evolved with the machinery of cilia to achieve unhinderedtwo-way traffic on axonemes.

2522-Pos Board B538Novel Kinesin-3 Motor Behavior is Regulated by TauDominique V. Lessard, Christopher L. Berger.University of Vermont, Burlington, VT, USA.Axonal transport is a highly regulated process responsible for site-specificneuronal cargo delivery, and many neurodegenerative diseases result fromdysfunction of the axonal transport machinery. This vital process is mediatedin part by the kinesin-3 family of long-distance anterograde molecular motors.A potential regulator of kinesin-3 mediated axonal transport is the microtubuleassociated protein Tau. Our lab has extensively characterized Tau’s effect onkinesin-1 and kinesin-2 family members, however, Tau’s role in regulatingkinesin-3 transport is unexplored. Using single-molecule total internal reflec-tion fluorescence (TIRF) microscopy, we present the first evidence of Tau-mediated inhibition of kinesin-3. Addition of Tau to microtubules resulted ina decrease of kinesin-3 run length and landing rate when compared to runson undecorated microtubules. Further analysis revealed that kinesin-3 is ableto undergo extensive pausing and diffusive events during a run on the microtu-bule. Strikingly, when Tau is added to the microtubules kinesin-3’s pausing anddiffusive behavior is almost abolished. Furthermore, Tau does not decrease thelength of kinesin-3 processive runs between pauses, suggesting that Tau-mediated inhibition of kinesin-3 is due to a reduction in pausing behavior,not inhibition of active, directed movement along the microtubule. It is furtherproposed that Tau’s diffusive behavioral state on the microtubule acts as amechanism to regulate cargo delivery by impeding pausing behavior andlimiting kinesin-3’s ability to string multiple processive events in a singlerun length. Moreover, kinesin-3’s ability to resume processive behavior afterpausing and/or diffusing appears to be essential for successful long-range cargotransport.

2523-Pos Board B539Morelloflavone as a Novel Inhibitor for Kinesin Eg5Tomisin Happy Ogunwa1, Kenichi Taii2, Shuya Yano2, Kei Sadakane3,Yuka Kawata1, Shinsaku Maruta3, Takayuki Miyanishi1.1Graduate School of Fisheries and Environmental Sciences, NagasakiUniversity, Nagasaki, Japan, 2Department of Bioinformatics, Faculty ofScience and Engineering, Soka University, Hachioji, Japan, 3Dapartment ofBioinformatics, Graduate School of Engineering, Soka University, Hachioji,Japan.Morelloflavone, one of the known biflavonoids from Garcinia dulcis leaves,was evaluated in the current study as a novel inhibitor for kinesin Eg5. Bifla-vonoids are reported for their anticancer potency. As part of our search forpromising natural compounds that can suppress cancer cell proliferation aswell as unraveling the possible mechanism for such activity, we employed insilico tools to screen forty (40) plant-derived biflavonoids as possible inhibitorsfor human kinesin Eg5, based on the binding energies, binding modes, molec-

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ular interactions and functional groups which might participate in the interac-tion of biflavonoids with the protein target. Morelloflavone was identified as apotential inhibitor which might bind the putative allosteric pocket of Eg5. Thebiflavonoid was embedded within the cavity formed by amino acid residues Ile-136, Glu-116, Glu-118, Trp-127, Gly-117, Ala-133, Glu-215, Leu-214, Tyr-211 with binding energy value �8.4 kcal/mol while residues Glu116 andTyr211 were found to be essential for hydrogen bond formation betweenmorelloflavone-Eg5 complex. The binding configuration of morelloflavonewas comparable to that of control ligand, STLC. The experimental validationsusing in vitro biochemical analysis confirmed that morelloflavone inhibitedboth basal and microtubule-activated ATPase activity of Eg5 with IC50 valuesof 98 mM and 83 mM respectively. In addition, morelloflavone suppressed Eg5motor gliding along microtubules. These results suggest that the interaction ofmorelloflavone at the L5/a2/a3 allosteric binding site of Eg5 may have eliciteda conformational change which could have led to the inhibition of the enzymeactivity. It would be interesting to find whether proliferation of tumor cells isaffected by this biflavonoid through its inhibitory effect on Eg5.

2524-Pos Board B540Novel Photochromic Potent Inhibitor of Mitotic Kinesin Eg5 Composed ofSpiropyran DerivativesKei Sadakane1, Kenichi Taii2, Shinsaku Maruta1.1Department of Bioinformatics, Soka University Graduate School ofEngineering, Hachioji, Tokyo, Japan, 2Department of Bioinformatics, SokaUniversity Faculty of Science and Engineering, Hachioji, Tokyo, Japan.The mitotic kinesin Eg5, a member of the kinesin-5 family, is a plus-enddirected homotetrameric molecular motor. It is essential for the formation ofbipolar spindles during eukaryotic cell division. It is known that kinesin Eg5overexpress in some of cancer cells. Kinesin Eg5 has been considered as atarget of anticancer therapy. Several small molecules were recognized aspotent inhibitors of Eg5. Interestingly the inhibitors bind to the common pocketin Eg5 motor domain. STLC is one of the well-known potent inhibitor of Eg5.The crystallographic structure of Eg5-STLC complex revealed that STLC bindsto the pocket composed of a2, a3 helix and loop L5. The inhibitory mechanismof STLC has been well studied. Photochromic molecules such as azobenzeneand spiropyran derivatives, which change their structures and properties revers-ibly by light irradiation, are expected to be applicable to photo-switches of bio-nanomachines. Previously we have demonstrated that STLC analoguescomposed of azobenzenen (ACTAB) inhibit Eg5 ATPase activity and motoractivity photo-reversibly upon UV and visible light irradiations. Moreover,HeLa cell division was photo-regulated with ACTAB. In this study, we synthe-sized novel photochromic Eg5 inhibitors (SP)2-APA composed of two spiro-pyran derivatives. (SP)2-APA exhibited spiro and neocyanine isomerizationupon visible and ultraviolet light irradiations, respectively. Spiro isomer of(SP)2-APA inhibited more significantly basal ATPase and microtubule depen-dent ATPase activities than merocyanine isomer. Moreover, microtubulegliding motor activity was inhibited by spiro isomer more significantly thanmerocyanine isomer. We also analyzed which step in ATPase cycle is influ-enced by SP-APA using stopped-flow method.

2525-Pos Board B541Photo-Regulation of Mitotic Kinesin Eg5 using Novel PhotochromicInhibitor that Forms Three Isomerization StatesIslam M.D. Alrazi, Kei Sadakane, Shinsaku Maruta.Bioinformatics, Maruta lab., Soka University, Hachioji, Tokyo, Japan.Kinesin Eg5 is a plus-end-directed microtubule-based motor that is essentialfor bipolar spindle formation during eukaryotic cell division. Loop L5 ofmitotic kinesin Eg5 is a key region determining ATPase activity and motorfunction. During cell division, the bipolar organization of mitotic spindles isessential for proper segregation of chromosomes. Inhibition of mitotic-spindle formation is an interesting target in cancer chemotherapy. Previously,to control the function of mitotic kinesin Eg5 stepwisely, we have synthe-sized thiol group reactive photochromic compound which exhibit three isom-erization states using photo-responsive spiropyran and azobenzenederivatives. ATPase activity of the Eg5 modified with the photochromiccompound at the loop 5 was successfully regulated photo reversibly. It iswell known that there are several specific inhibitors for Eg5. The structureof the inhibitors is not conserved but shows diversity. In this study, wehave synthesized a novel photochromic Eg5 inhibitor composed of spiro-pyran, azobenzene and propionic acid to inhibit Eg5 function photo-reversibly. The photochromic compound SP-AB-propionic acid formedMerocyanine-Cis, Spiro-Trans and Merocyanine-Trans states upon ultravio-let (UV) irradiation, Visible (Vis) Light irradiation, and in the dark, respec-tively. The three isomerization states showed each specific absorption spectra

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upon UV light irradiation, VIS light irradiation and in the dark, respectively.Basal ATPase activity of Eg5 was significantly inhibited in the presence ofthe inhibitor. Photo reversible effects on the ATPase activity and motility ac-tivity of Eg5 were also examined.

2526-Pos Board B542Two Opposing Modes of Cytoplasmic Dynein Regulation by Lis1Zaw M. Htet1,2, Morgan E. DeSantis2, Michael A. Cianfrocco2,3,Phuoc T. Tran2, Andres E. Leschziner2,4, Samara L. Reck-Peterson2,5.1Biophysics Graduate Program, Harvard University, Boston, MA, USA,2Department of Cellular and Molecular Medicine, University of California,San Diego, La Jolla, CA, USA, 3Life Sciences Institute, Department ofBiological Chemistry, University of Michigan, Ann Arbor, MI, USA,4Section of Molecular Biology, Division of Biological Sciences, Universityof California, San Diego, La Jolla, CA, USA, 5Section of Cellular andDevelopmental Biology, Division of Biological Sciences, University ofCalifornia, San Diego, La Jolla, CA, USA.Cytoplasmic dynein-1 (dynein) is a motor protein that is essential for main-taining the proper spatial and temporal organization of the cellular interiorduring both interphase and cell division. Dynein achieves this task by trans-porting various cargoes such as organelles and mRNAs towards microtu-bule minus ends or anchoring cargos at specific subcellular location.Regulation of dynein is important for carrying out its diverse functions.Lis1 is a conserved dynein regulator, which binds directly to dynein’s mo-tor domain and slows down its motility by inducing dynein to tightly bindmicrotubules. Here, using a combination of biochemistry, single-moleculeassays and cryo-electron microscopy, we discovered that Lis1 has twoopposing modes of regulating dynein. We showed that Lis1 can induceeither a weak or tight microtubule-binding state of dynein depending onthe nucleotide state of dynein’s AAA3 domain. Single-particle cryo-electronmicroscopy revealed that these opposing modes of Lis1 regulation are setby the stoichiometry of Lis1 b-propellers bound to dynein. In the Lis1-mediated tight microtubule-binding state, a single Lis1 b-propeller bindsto dynein at the previously described binding site on the motor domain.In addition to this site, the Lis1-mediated weak microtubule-binding staterequires a second Lis1 b-propeller binding at a novel conserved site on dy-nein’s stalk. The novel Lis1 binding site we identified is required fordynein function in vitro and in vivo.

2527-Pos Board B543Cargo Adaptors Regulate the Mechanical Properties of Dynein/DynactinComplexMohamed Elshenawy1, Ahmet Yildiz2.1Molecular and Cell Biology, University of California Berkeley, Berkeley,CA, USA, 2Molecular and Cell Biology, University of California Berkeley,Berkeley, CA, USA.Cytoplasmic dynein is the principle motor responsible for the transport ofintracellular cargoes towards the minus end of microtubules in eukaryoticcells. Recent studies have shown that activation of dynein motility requiresthe assembly of a ternary complex of dynein, dynactin and a cargo adaptor,such as BiCD2, BicDR-1 and HOOK3. The effect of these cargo adaptors onthe dynein/dynactin stoichiometry, motility, and force generation remains un-clear. Here, we use single-molecule approaches to show that the cargo adap-tors can recruit one or two dynein dimers, which alter the velocity and forcegeneration of the complex. Attaching two dynein dimers using an artificiallinker increases the force production, but not the velocity, suggesting amechanism that is independent of the number of dynein dimers responsiblefor controlling the velocity of the complex. We found that various cargoadaptors could regulate dynein’s velocity by tuning its catalytic propertiesand stepping pattern. Additionally, cargo adaptors that recruit two dynein di-mers compete more efficiently against kinesin-1 in a one-to-one mechanicalcompetition. Our results provide a mechanistic basis for how different cargoadaptors modulate dynein’s mechanical properties to perform a wide rangeof cellular functions.

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2528-Pos Board B544Stepping Behavior of Mammalian Dynein-Dynactin ComplexesLiya F. Oster1, John Canty2, Mohamed Elshenawy3,4, Ahmet Yildiz3,4.1Physics and Astronomy, University of California, Los Angeles,Los Angeles, CA, USA, 2Biophysics Graduate Group, University ofCalifornia, Berkeley, Berkeley, CA, USA, 3Physics, University of California,Berkeley, Berkeley, CA, USA, 4Molecular Cell Biology, University ofCalifornia, Berkeley, Berkeley, CA, USA.Cytoplasmic dynein is an AAAþ motor that generates force and motility to-wards the microtubule minus-end. Yeast cytoplasmic dynein is constitutivelyactive and moves through uncoordinated stepping of its two motor domainswith a high frequency of backwards and sideways steps. Unlike yeast dynein,mammalian dynein is autoinhibited and activation of its motility requires dy-nactin and a cargo adapter protein such as BICD2 and BICDR-1. Previousstudies have shown that the complexes formed with BICDR-1 (DDR) recruittwo dynein dimers instead of one and walk nearly twice as fast than complexesformed with BICD2 (DDB) that primarily recruits a single dimer in vitro. It hasbeen proposed that dynactin and cargo adapter binding may restrict sidewaysand backwards movements to allow dynein to take a more direct and fasterroute along the microtubule, and the recruiting of two dyneins side by side inDDR further promotes forwards steps. To determine whether dynactin and acargo adaptor have a net effect in dynein stepping behavior, we tracked themotility of quantum dot-labeled dynein complexes at nanometer precision us-ing fluorescence imaging with one nanometer accuracy (FIONA). We observedthat mammalian dynein, DDB, and DDR have a similar step size distributionwith frequent sideways and backward steps. At limiting ATP concentrations,we did not see a major difference in the stepping characteristics of DDB andDDR. Dynactin and cargo adaptors do not restrict the high variability in dyneinstepping behavior, presumably because the motor domains are still flexible andmove freely in the ternary complexes. We propose that faster motility of DDRthan DDB is due to either mechanical coupling or allosteric interactions be-tween dynein dimers, rather than tighter regulation of the dynein steppingbehavior.

2529-Pos Board B545Single Molecule Study of Long-Range Electrostatic Binding Affinity ofCytoplasmic Dynein’s Microtubule Binding DomainSubash C. Godar1,2, Hailey Lovelace1, Jared Eller1,3, Mattheu Spencer1,4,Lin Li1,5, George Hamilton1, Hugo Sanabria1, Emil Alexov1, Joshua Alper1,2.1Physics and Astronomy, Clemson University, Clemson, SC, USA,2Eukaryotic Pathogen Innovation Center, Clemson University, Clemson, SC,USA, 3Biochemistry, University of Texas at Austin, Austin, TX, USA, 4CivilEngineering, Virginia Tech University, Blacksburg, VA, USA, 5Physics,University of Texas at El Paso, El Paso, TX, USA.Motor proteins are biological macromolecules involved in various dynamiccellular phenomena from cell division, intracellular transport, and cell motilityto muscle contraction and flagellar bending. Dyneins, the largest and least un-derstood family of motor proteins, are responsible for processive transport ofvariety of cargos towards the minus end of microtubules and to drive thebeat of motile cilia and flagella in non-processive manner. As processivity islargely a function of motor protein-microtubule interaction, it is likely thatprocessivity of dynein is, at least in part, due to its microtubule binding domains(MTBDs). Through the application of a multi-scale computational method, wefound that the motor-filament interaction is likely to be mediated by long rangeelectrostatic forces. To investigate this effect further, we expressed dyneinMTBDs with the connecting coiled-coil stalk in the a-registry (high-bindingaffinity) and in the bþ-registry (low-binding affinity). The coiled-coil stalk sep-arates the MTBD from the ATP hydrolysis site, and the changing of its registryis believed to mediate communication between MTBD and ATP hydrolysissite. We investigated the role of electrostatic interactions and applied forceto the MTBD’s binding affinity- using optical tweezers, TIRF microscopy,microscale thermophoresis, and fluorescence correlation spectroscopy. The re-sults have implications on the role of motor-filament interactions in the proces-sivity and regulation of cytoplasmic dynein motility.

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Posters: Cell Mechanics, Mechanosensing, andMotility II

2530-Pos Board B546Desmoplakin Bears Tension under Externally Applied Load but not dur-ing Epithelial Monolayer HomeostasisAndrew J. Price1, Anna-Lena Cost2, Carsten Grashoff2,Alexander R. Dunn3.1Biophysics, Stanford University, Stanford, CA, USA, 2Group of MolecularMechanotransduction, Max Planck Institute of Biochemistry, Martinsried,Germany, 3Chemical Engineering, Stanford University, Stanford, CA, USA.Desmosomes are intercellular adhesion complexes that link the intermediatefilament cytoskeletons of neighboring cells. Mutations in desmosomal proteinslead to severe cardiomyopathies and skin blistering diseases, indicating thatdesmosomes play an essential role in defining tissue mechanical properties.However, how and even whether desmosomes bear tension in living tissues re-mained unclear. In this study we inserted a Forster resonance energy transfer(FRET)-based tension sensing module (TSM) into both major isoforms of des-moplakin (DPI and DPII), a scaffolding protein that links desmosomal cadher-ins to intermediate filaments. Live-cell measurements yielded FRET valuesconsistent with negligible mechanical tension transmitted through DPI inconfluent monolayers, sparse colonies, and the edge of expanding monolayersof Madin-Darby canine kidney (MDCK) epithelial cells. Analogous measure-ments indicated negligible tension transmitted through DPII in confluent mono-layers of wild-type murine epidermal keratinocytes (MEK) and in MEKsdepleted of endogenous DP. External mechanical stretch applied to MDCKand MEKmonolayers, however, yielded significant decreases in FRET, consis-tent with stretch-induced mechanical tension on DP. We suggest that, inepithelia, desmosomes function to protect tissues from mechanical disruptionwhile still allowing the cellular movements and shape changes that are essentialin the contexts of tissue homeostasis and collective cell migration.

2531-Pos Board B547Anisotropic Mechanical Properties of Living Cells Revealed by IntegratedSpinning Disk Confocal and Atomic Force MicroscopyYuri M. Efremov, Mirian Velay-Lizancos, Daniel M. Suter,Pablo D. Zavattieri, Arvind Raman.Purdue University, West Lafayette, IN, USA.Recent developments in fluorescent live-cell imaging and biophysicalmethods have significantly advanced our understanding of the dynamicbiochemical and mechanical processes underlying cellular functions such ascell migration. One of most frequently used techniques for assessment ofcell mechanical properties is the indentation experiments conducted withthe atomic force microscope (AFM), due primarily to AFM’s relative easeof operation, its high precision of force measurement, and high spatial resolu-tion. We used the AFM in conjunction with a spinning disk confocal (SDC)microscope to directly visualize AFM indentation of living cells with highspatial and temporal resolution. With novel live cell imaging probes to fluo-rescently label F-actin, microtubules, and membrane, we were able to directlyobserve structural changes during the indentation process of a living cell (NIH3T3 fibroblasts) with a spherical indenter. The most notable observation was adeformation of single apical stress fibers (ASF) under the action of the probe.The presence of ASF (also known as perinuclear actin cap) correlated withlow cell height and high stiffness. Moreover, the presence of ASF causedan anisotropic indentation geometry as observed from surface displacementprofiles calculated from the SDC images. Isotropic indentation profile wasfound in cells without ASF: cancerous cells MDA-MB-231 which are natu-rally lacking actin cap and NIH 3T3 cells in which ASF were disrupted bylatrunculin A. Anisotropic indentation behavior suggests an anisotropic de-formability and stiffness of the cell. We performed finite element simulationsto extract anisotropic properties from the surface displacement data. Simula-tions suggest a very strong level of anisotropy in cells which role should befurther elucidated.

2532-Pos Board B548Tumor-Associated Macrophages Drive Astrocytoma Spheroid Formationthrough Mechanosignal TransmissionHsiao-Ming Chang1, Yung-Chu Chuang1, Kuo-Hsiang Hung2,Yu-Ming Chen1, Chiao-Lun Chen1, Shun-Chi Wu2, Chi-Shuo Chen1.1Department of Biomedical Engineering and Environmental Sciences,National Tsing Hua University, Hsinchu, Taiwan, 2Department ofEngineering and System Science, National Tsing Hua University, Hsinchu,Taiwan.Tumor spheroids play important roles in cancer progression. Colonization ofcancer cells enhance stem-like properties and elevate the capabilities of pro-

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liferation, anti- apoptosis, therapeutic resistance, metastasis and cancer recur-rence. During the tumor development, tumor-associated macrophages(TAMs), one kind of special macrophages, accumulated around the tumorand secrete certain cytokines including TGF-b and VEGF which improveangiogenesis and tumor invasion. Previous studies of TAMs mainly focuson its chemical signaling. However, the detail of TAMs mechanosignalingon tumor formation is still unclear. Here, we found that the macrophages(BV2) can drive murine astrocytomas (ALTS1C1) to aggregate into spheroidthrough mechanosignaling processes. We demonstrated two major mechano-signal transduction processes between BV2 and ALTS1C1. First, BV2 isresponsible for the regulation of the intercellular traction force and themigration speed of ALTS1C1. Inhibition of ALTS1C1 traction and move-ment by blebbistatin and Y-27632 suggested a role of BV2 adhesion inALST1C1 actin/myosin contractility. Second, substrate stiffness alters theexpression of E-cadherin and focal adherens junction that are associatedwith cell adhesion and motility. Immunostaining results showed theintegrin-dependent adhesion has effects on E-cadherin-based adhesion andsuggested that BV2 adhesion modulates ALTS1C1 aggregation capability.Both macrophage contact and environment rigidity are critical in cell aggre-gation and tumor formation. Traction force field via intracellular tension sup-port that mechanosignaling leads to neighboring cell aggregation. Inconclusion, BV2-ALTS1C1 contact triggers downstream mechanicalsignaling, and the formation of BV2-ALTS1C1 aggregation may facilitatecancer malignancy.

2533-Pos Board B549Nuclear Lamina Stress Measured with FRET Based Stress SensorThomas M. Suchyna1, Fanjie Meng2, Frederick Sachs1, Wilma Hofmann1.1Biophysics, SUNY at Buffalo, Buffalo, NY, USA, 2Laboratory of ChemicalPhysics, NIDDK, National Institutes of Health, Rockville, MD, USA.Cellular stress transmitted to the nuclear lamina can modulate gene expressionand affect DNA repair. While static cell-type dependent nuclear stiffness differ-ences have been measured, the dynamic changes in nuclear stress that occurduring the cell cycle, cell adhesion and differentiation have been more difficultto determine. To measure dynamic changes in nuclear stress we createdchimeric Lamin A and B expression vectors with the fluorescent cpstFRETstress sensing probe inserted into the linker region between helical rod domains1B and 2A. C2C12 myoblast cells expressing approximately a 1:1 ratio of exo-genous:endogenous lamin A progress through the cell cycle and differentiateinto multinucleated myotubes. Lamin stress increased depending on cell stiff-ness as shown when expressed in cells ranging from soft HEK cells to stifferC2C12 myoblasts and differentiated myotubes. Lamin showed higher vari-ability in stress compared to other cytoskeletal proteins, but this variationwas reduced in differentiated myotubes. Monitoring nuclear stress during thecell cycle shows that lamin stress transiently decreases during G1-phase fol-lowed by stress recovery during S-phase suggesting the cell cycle contributesto stress variations. Lamin stress increases rapidly when trypsinized cells aredeposited onto glass coated with different extracellular matrix proteins, butthe kinetics were sensitive to the substrate chemistry. The stress on laminincreased when isolated nuclei were swollen by cation depletion and wereconsistent with probe sensitivity in the pN range. Aspiration of isolated nucleishowed inconsistent stress changes, possibly due to lamina rupture and plasticproperties from lamin interactions with the nuclear membrane and the DNA.The magnitude of these stress changes was significantly muted when thecpstFRET cassette was linked to the C-terminus. This new probe will proveuseful for correlating lamina stress changes associated cell division, differenti-ation and pathology.

2534-Pos Board B550Swarming Migration of Co-attracting Mesenchymal Cells into Fractal-Like Epithelial ClustersSusan E. Leggett, Zachary J. Neronha, Dhananjay Bhaskar,Theodora M. Perdikari, Ian Y. Wong.Brown University, Providence, RI, USA.Cell-secreted factors can function as biochemical ‘‘signal relays’’ to coordi-nate collective migration over long distances. For instance, cells ‘‘co-attract’’by releasing chemokines that they also chemotax towards, driving aggregationand cohesion during neural crest development, mesenchymal condensation,and neutrophil homing. Here, we show that mammary epithelial cells culturedin reduced serum conditions display a mutual attraction that results in self-organizing migration patterns. Ordinarily, the migration of spatially dispersedcells in EGF-supplemented media can be understood as a persistent randomwalk. Instead, cells cultured in EGF-depleted media display directed migra-tion towards each other through co-attraction, resulting in multicellularaggregation. Subsequently, aggregated cells establish cell-cell junctions with

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E-cadherin, reminiscent of a mesenchymal-to-epithelial transition (MET).Furthermore, cells exhibit substantial morphological changes, extending out-ward protrusions towards other cells in the proximity. As a consequence, theseaggregates display a highly branched, fractal-like architecture. This biologicalbehavior has physical analogies with diffusion-limited aggregation ofcolloidal particles, which results in a gelation or jamming transition even atvery subconfluent cell densities. We implement a computational model basedon dissipative particle dynamics that recapitulates clustering and aggregation.These results may reveal new quantitative insights into swarming cell migra-tion during neural crest development, mesenchymal condensation, and tumormetastasis.

2535-Pos Board B551Effects of Ionizing Radiation on the Mechanosensitivity of Single CellsAndrew E. Ekpenyong, Michael Mimlitz, Noah Zetocha, Kaamil Abid,Bong Han Lee.Physics, Creighton University, Omaha, NE, USA.The relationship between radiosensitivity and mechanosensitivity, two sepa-rate properties of cells, tissues and organs that are both important in cancer,is unclear. Radiosensitivity- the relative susceptibility to the harmful effectsof ionizing radiation, is the decisive factor used in radiation therapy. Mecha-nosensitivity- the specific response to mechanical stimuli such as forces, de-pends on the mechanical properties of cells themselves which have beenused, inter alia, in mechanophenotyping for cancer diagnosis. Here, weshow a relationship between radiosensitivity and mechanosensitivity at thesingle cell level.We used a microfluidic microcirculation mimetic (MMM) which mimicsthe capillary constrictions of the pulmonary and peripheral microcirculation,to determine if in-vivo-like mechanical stimuli can evoke differentresponses from cells subjected to low dose and high dose ionizing radiationby means of radiotherapy-grade cell irradiator (CellRad, Faxitron). We alsoused migration assays and CdSe/ZnS core-shell nanoparticles (quantumdots) to quantify functional changes in cells following radiotherapy andMMM advection.The transit times of cells K562 (erythroid) and HL60 (myeloid) cells advectedthrough the MMMwere altered by the X-rays. Our results are first steps in eval-uating the pro-metastatic effects of radiotherapy based on their induced alter-ations in cell mechanosentivity, potentially providing a new rationale for theimprovement of radiotherapy protocols.

2536-Pos Board B552Exopolymer Dynamics Driven by Sessile FlagellatesTyler N. Shendruk1, Andrew K. Balin2, Andreas Zottl2, Julia M. Yeomans2.1Center for Studies in Physics and Biology, Rockefeller University, NewYork, NY, USA, 2Rudolf Peierls Centre for Theoretical Physics, Universityof Oxford, Oxford, United Kingdom.Microbial flagellates typically inhabit complex suspensions of polymeric bio-materials which can impact the swimming speed of motile microbes, filter-feeding of sessile cells, and the generation of biofilms. Extracellular poly-meric substances are continually secreted for a wide variety of purposes,including anchoring to surfaces by long mucous stalks, bioaccumulation ofcontaminants, and as the polymeric matrix within veils and other collectivestructures. While motile swimmers must transverse these complex fluid me-dia, sessile microorganisms that affix themselves in place can individuallyexert net forces on surrounding fluids and collectively produce large-scaleflows that entrain nutrients, detritus and other large high-conformational-entropy biopolymers. There is currently a need to better understand howthe fundamental dynamics of exopolymers near beating flagella impactstransport. Our Stokesian dynamics simulations provide insight on the hydro-dynamic interactions of a model monoflagellate on suspended polymers. As astationary rotating helix pumps fluid along its long axis, polymers migrateradially inwards while being elongated. We observe that the actuation ofthe helix tends to increase the probability of finding polymeric materialwithin its pervaded volume, implying that a dilute suspension of polymerstend to become locally concentrated in and around the flagellum ratherthan depleted. This accumulation of polymers within the vicinity of themodel flagella is more pronounced for higher molecular weight polymers(or equivalently higher Weissenberg number). By measuring the workperformed by the helix, we see that all of the work done to deform thepolymer is ultimately dissipated, with no elastic reclamation by the helixof the polymer’s free energy. By studying this simplified model system,we gain fundamental phenomenological insight into the effects of hydrody-namics activity on the polymeric biomaterials that are ubiquitous inmicrobial environments.

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2537-Pos Board B553Mechanical Analysis of Cells via Electrodeformation-RelaxationYasir Demiryurek1, Miao Yu1, David I. Shreiber2, Jeffrey D. Zahn2,Ramsey Foty3, Jerry W. Shan1, Liping Liu4, Hao Lin1.1Department of Mechanical and Aerospace Engineering, Rutgers University,Piscataway, NJ, USA, 2Department of Biomedical Engineering, RutgersUniversity, Piscataway, NJ, USA, 3Department of Surgery, Rutgers RobertWood Johnson Medical School, New Brunswick, NJ, USA, 4Department ofMechanical and Aerospace Engineering and Department of Mathematics,Rutgers University, Piscataway, NJ, USA.An electrodeformation-relaxation technique is developed to characterize me-chanical properties of cells in suspension. The experimental platform was es-tablished using an indium-tin-oxide (ITO) coated glass slide and parallelelectrodes were lithographically defined by etching ITO. Various types of cellsin suspension, including MCF-10A, MCF-7, MDA-MB-231, and GBM wereexposed to high frequency and amplitude electric pulses. The pulsing protocolensures the occurrence of deformation and the absence of electroporation.Owing to the design of the electrodeformation chip, cells were trapped at theedge of the electrodes under dielectrophoretic forcing. The subsequent defor-mation and relaxation (upon pulse cessation) were captured via synchronizedhigh-speed camera. Images were analyzed and the shape factor (ratio betweenlong and short axes) was extracted as a function of time. In the relaxation pro-cess, two distinctive regimes, dilational and constant-tension, were observed.Data were analyzed with a mathematical theory that we developed previously,which provides a closed-form solution for ellipsoidal deformation-relaxation.The properties of the various cell types were analyzed and extracted usingthis model. The technique promises ease of implementation and high-throughput for large-scale cell mechanical analysis.

2538-Pos Board B554Live Cell Tracking of Human NK Cell Precursors Identifies ComplexModes of Cell Migration Throughout DifferentiationBarclay Lee1,2, Emily Mace2.1Bioengineering, Rice University, Houston, TX, USA, 2Baylor College ofMedicine, Houston, TX, USA.Determining the complex interactions between immune cells and theirmicroenvironment is key to understanding their functions in human healthand disease. In particular, defining the nature and regulation of cell migrationand its role in the development and functional maturation of immune cellswill lead to improved outcomes of immune therapy and transplantation.Natural killer (NK) cells are innate immune effectors that play an importantrole in the control of viral infection and tumorigenesis. The developmentof human NK cells from hematopoietic precursors has not been welldefined, in part because of a lack of satisfactory models of humanhematopoiesis.We have developed a model for in vitro differentiation of human NK cells ondevelopmentally supportive stromal cells that includes continuous phase-contrast imaging of NK cell developmental intermediates (NKDI) with hightemporal and spatial resolution. Here, we combine this model with trackingand analyses to define the modes of migration that mark human NK cell devel-opment from CD34þ hematopoietic stem cells over the period of differentia-tion (28 days). We find that cells progress from a largely constrained modeof migration to increasingly random and directed modes. Overall heterogeneityof cell behavior increased throughout the time of imaging. This was in part dueto an increase in the frequency of cell tracks that included multiple modes ofmigration (random, constrained, directed) within a single track. These complexmodes were reminiscent of Levy-walk tracks previously demonstrated toimprove efficiency of seeking behaviors. The consistency of these behaviorsand those of freshly isolated NK cell subsets demonstrates that acquisition ofcomplex modes of migration is a key component of human NK celldifferentiation.

2539-Pos Board B555Single-Cell Analysis of Complement-Mediated Chemotaxis: Anaphyla-toxic Clouds, and Neutrophil Sensitivity to ChemoattractantEmmet A. Francis, Volkmar Heinrich.Biomedical Engineering, University of California at Davis, Davis, CA, USA.When locating pathogens in tissue, human neutrophils must navigate a varietyof competing chemical gradients. One such gradient is formed when comple-ment proteins from human serum bind to a pathogen and release highly potentchemoattractants known as anaphylatoxins, forming a localized anaphylatoxiccloud. Neutrophils respond to this cloud by making a final course correction to-wards nearby fungal and bacterial pathogens. In this work, we integrate exper-iments and theory to analyze spatial and temporal features of anaphylatoxicclouds, and to quantify the sensitivity of neutrophils to anaphylatoxins. First,

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we formulate a diffusion-reaction problem describing the evolution of a chemo-attractant concentration gradient around a source particle, and derive a closed-form solution. We apply this model to characterize the formation of an anaphy-latoxic cloud under realistic physiological conditions, finding that the cloudforms rapidly (within a few seconds), and that its steady-state concentrationprofile drops off sharply around the particle. Furthermore, the size of the sourceparticle correlates positively with both the chemoattractant concentration andthe spatial reach of the cloud. We leverage these theoretical predictions againstsingle-cell, pure-chemotaxis experiments, using neutrophils to map out the ana-phylatoxic cloud around differently sized aggregates of b-glucan particles. Asthe model predicts, neutrophils generally sense larger aggregates from fartheraway. By comparing the measured response distances for different aggregatesizes with our model, we are able to quantify the neutrophil sensitivity to ana-phylatoxins in terms of the ratio between the concentration and the productionrate of chemoattractant. Together, our model and experiments show thatcomplement-mediated chemotaxis is a short-range homing mechanism. Theyalso imply that simply being small is an effective protective strategy for path-ogens against complement-mediated discovery.

2540-Pos Board B556Three-Dimensional Traction Force Measurement using Planar Epifluores-cence Microscopy for Cell Mechanics StudiesMohak Patel, Susan E. Leggett, Ian Y. Wong, Christian Franck.Engineering, Brown University, Providence, RI, USA.Traction force microscopy (TFM) has become a standard biophysical tool forquantitatively measuring mechanical forces in cell-cell and cell-extracellularmatrix interactions. Currently, 3D TFM techniques require a three-dimensional imaging modality, e.g., a confocal microscope, to capture thethree-dimensional cell-imparted motion fields of a substrate embedded withfiducial marker particles. While full, volumetric three-dimensional imagingtechniques provide the greatest spatial resolution in all three directions, theirhigh upfront cost has kept their proliferation into the mainstream communitieslimited as compared to epifluorescence microscopy. To provide the communitywith full access to three-dimensional measurements without the burden ofrequiring a truly volumetric imaging modality, we present a new 3D TFM tech-nique to measure the three-dimensional displacement of maker particles using aplanar epifluorescence microscope. The known mechanical properties andthree-dimensional deformation measurement of the substrate are used tocompute three-dimensional tractions in a standard manner using a previouslyestablished forward approach. With the use of different color fluorescent beads,deconvolution and position-based single particle tracking (P-SPT), we canaccurately and reliably reconstruct 3D deformation fields from two-dimensional epifluorescence images. As a test case, we measure the three-dimensional tractions produced by breast adenocarcinoma cells and mammaryepithelial cells. Our techniques’ ability to use epifluorescence microscopy tomeasure three-dimensional deformation makes traction force measurementmore accessible to a much broader biological community.

2541-Pos Board B557Neural Signaling Regulates Cancer Cell Physical PhenotypesAmy C. Rowat1, Tae-Hyung Kim1, Erica K. Sloan2.1Integrative Biology & Physiology, University of California, Los Angeles,Los Angeles, CA, USA, 2Monash Institute of Pharmaceutical Sciences,Monash University, Monash, Australia.The physical phenotypes of tumor cells are critical in metastasis. To dissem-inate from the primary tumor, cancer cells generate protrusive and contractileforces to move through the surrounding extracellular matrix. Once in circula-tion, the physical properties of tumor cells allow them to survive fluid shearstresses. Identifying factors that regulate physical properties of tumor cellswill identify important points of therapeutic leverage to stop metastasis.Our recent studies found that neural signaling regulates tumor cell physicalphenotypes. We identified that activation of b-adrenergic (bAR) signaling reg-ulates the physical properties of breast cancer (MDA-MB-231-HM) cellsthrough increased myosin II activity; this makes cells stiffer and more inva-sive. When cells are treated with the bAR agonist, isoproterenol, we findincreased levels of diphosphorylated myosin light chain (ppMLC) at serine19 and threonine 18, indicating increased myosin II activity. We also deter-mine the impact of bAR-induced myosin II activity on tumor cell contrac-tility, invasion, as well as viability after exposure to physiologically-relevant fluid shear stresses. Importantly, activation of myosin II by bARsignaling is blocked by b-blocker treatment; these widely used drugs areshowing exciting potential to be repurposed for cancer treatment. Our findingsdemonstrate the neural regulation of tumor cell physical phenotypes and pro-vide new mechanistic insight into how modulating bAR signaling can be aneffective anti-metastasis strategy.

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2542-Pos Board B558Matrix Rigidity Myosin-II and Lamin-A Regulate Curvature Induced Nu-clear Rupture Causing Repair Factor Mislocalization and DNA DamageYuntao Xia, Jerome Irianto, Kuangzheng Zhu, Cory Alvey, Lucas Smith,Charlotte Pfeifer, Dennis Discher.University of Pennsylvania, Philadelphia, PA, USA.Mechanosensing of matrix stiffness can increase the activity and/or expressionof major structural proteins, including myosin-II and lamin-A, both of whichare sometimes dysregulated in cancer. Genome instability is a hallmark of can-cer, and here we show that nuclear rupture of cancer cells with low lamin-A orhigh myosin-II increases with matrix stiffness, which causes multiple DNArepair factors to mis-localize to the cytoplasm and DNA damage to increase.Soft matrix and inhibition of cytoskeletal stress relax nuclear curvature, sup-press rupture, and minimize the pan-nucleoplasmic pattern of DNA damage.Partial knockdown of multiple repair factors causes similar DNA damagewithout rupture. 3D-migration of wildtype cancer cells through highly curvedrigid pores also causes myosin-dependent nuclear rupture and DNA damagewhich is rescued by co-overexpression of multiple repair factors. A cyto-plasmic nuclease has no effect when overexpressed. Tumors with low lamin-A likewise exhibit elevated nuclear rupture and DNA damage, consistentwith tumor rigidity effects and the effects of nuclear stress in vivo.

2543-Pos Board B559Stress Fiber Subpopulations Have Distinct Viscoelastic Properties andRoles in Maintaining Cytoskeletal TensionStacey Lee1, Sanjay Kumar2,3.1Graduate Program in Bioengineering, UC Berkeley-UCSF, Berkeley, CA,USA, 2Bioengineering, UC Berkeley, Berkeley, CA, USA, 3Chemical andBiomolecular Engineering, UC Berkeley, Berkeley, CA, USA.Actomyosin stress fibers (SFs) are a major component of the cytoskeleton,conferring cell shape and enabling migration by directing intracellular tensionto the extracellular matrix via focal adhesions. Migrating cells exhibit threesubpopulations of SFs—dorsal, transverse arcs, and ventral—differing basedon their location, connections to focal adhesions, and formation pathway.Each subpopulation is predicted to have unique structural roles, but thesehave not been directly tested at the single SF level and it is unclear whetherthe subpopulations have overlapping mechanical roles. Here, we infer the me-chanical properties of single SFs by examining their retraction upon incisionwith a femtosecond laser. We find that dorsal SFs are non-contractile, trans-verse arc retraction is influenced by the proximity of networked SFs, andventral SF retraction is partially dependent on fiber length. Given that SF sub-populations form an interconnected network, we selectively depleted dorsalSFs or transverse arcs via knockdown of palladin or mDia2, respectively, toexamine the effect of removing a subset of SFs on the remaining subpopula-tions. In these SF-depleted cells, we find that ventral SFs, which may formfrom the fusion of dorsal SFs and transverse arcs, have altered retractionbehavior. To some extent, the altered ventral SF retractions can be phenocopiedin cells that are physically constrained so that they cannot form canonicallamella and hence lack dorsal SFs and transverse arcs. This indicates that SFsubpopulations have distinct roles in the generating cytoskeletal tension andthat dorsal SFs and transverse arcs may lend distinct contributions to the visco-elastic properties of ventral SFs. Taken together, these results show that SF sub-populations form a mechanically interdependent network.

2544-Pos Board B560Multiscale Modeling of Tip-Formation and Damage of Red Blood CellsSqueezing through Submicron SlitsHuijie Lu, Zhangli Peng.Department of Aerospace and Mechanical Engineering, University of NotreDame, Notre Dame, IN, USA.We applied multiscale modeling to study the tip-formation and damage of redblood cells (RBCs) squeezing through submicron slits, including healthy cells,cells in hereditary spherocytosis (HS), and cells in sickle cell disease. Wecompared our simulations with recent microfluidic experiments on healthyand diseased RBCs using PDMS submicron slits. This study is motivated bythe mechanical filtration of RBCs by inter-endothelial slits in the human spleen.First, the multiscale model is validated by comparing the predicted healthy cellvelocity and deformed shapes with experiments. Secondly, we predicted thetip-formation and its recovery of healthy cells and sickle cells under variousconditions. Thirdly, the mechanism of bilayer-cytoskeletal detachment underextreme deformation in HS RBCs was also explored by incorporating the HSmolecular mutations in our multiscale model. Finally, the roles of cytoplasmand membrane viscosities in cell shape recovery were examined, especiallyfor RBCs in sickle cell disease. The multiscale simulations result not only pro-vided insight and explanation of the observed phenomenon in experiments, but

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also offered guidance for future experiments to explore the dynamics of RBCsin the splenic filtration, which is critical for understanding physiological func-tions and pathological mechanisms of the RBC-spleen interaction.

2545-Pos Board B561Influence of Extracellular Matrix Stiffness on Modulating the Phenotypeof MacrophageYung-Chu Chuang, Hsaio-Ming Chang, Yu-Ming Chen, Chong-Chun Liao,Hou-Chun Huang, Shan-Rong Wu, Chi-Shuo Chen.National Tsing Hua University, Hsinchu, Taiwan.Macrophages can polarize into pro-inflammatory (M1) or anti-inflammatory(M2) phenotypes, which participate in innate immunity and play importantroles in wound healing. Numerous biochemical triggers for macrophage polar-ization are reported, but the effects of mechanical stimuli are not fully exploredyet. Concerning the rigidity alteration of microenvironment in different tissues,we investigated the M1/M2 differentiations of macrophages using rigidity-tunable engineered polymeric substrates. Macrophages showed differentmorphology and migration patterns, responding to the change of substrate stiff-ness. Quantified gene analysis indicated that cells express higher M1 phenotypemarkers on a softer substrate with increasing inflammatory cytokines secretion.We further induced the macrophage polarization using lipopolysaccharide(LPS) to investigate the inflammasome formation with the apoptosis-associate speck-like protein (ASC) and caspase-1, and our results showed theexpressions of ASC and caspase-1 are mechanosensitive. We speculated mito-chondria ROS may associate with the macrophage M1/M2 transition, and aROS related model was proposed to interoperate the mechanosensitive of in-flammasome formation. Associated with migration alteration, we also noticedthe change of podosome formation and cytoskeleton assembly while cellsgrowing on softer substrates. In conclusion, these results suggest that the mac-rophages are mechanosensitive, and reduced matrix stiffness can modulatemacrophages to polarize to an inflammatory-like phenotype. Our findingscontribute to our understanding about how matrix stiffness can differentiallyregulate macrophages differentiation, and are expected to provide alternativeinterpretations for certain disease pathogenesis.

2546-Pos Board B562Rotational Microscope Visualizes Cell Mechanics under High GravityConditionMasatoshi Morimatsu, Keiji Naruse.Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences,Okayama University, Okayama, Japan.Cells sense gravity during cell migration and proliferation.However, the effectof gravity on cell mechanics is poorly understood due to the lack of observationsystem. Here we describe the system to observe cellular function under highgravity by rotational microscope. We built the rotational microscope to applyhigh gravity (>2G) to cells in a horizon direction of cells and observe cell me-chanics like nucleus deformation and cytoskeleton dynamics in living cells.During the observation, this microscope was installed in the incubator andwas connected to the remote computer. As a result, we observed that smalldeformation of nucleus under high gravity condition in a short time (� 2 s).Furthermore, we found stress fibers localized to the edge of cell under highgravity condition in a long time (�45 min). Our preliminary results showed nu-cleus motion and cytoskeletal organization under high gravity condition, whichleads to cell organization. Ongoing work uses the unique capabilities of gravitycontrol in vitro to clarify the gravity sensing and signaling mechanism.

2547-Pos Board B563Mechanosensing to Protect the Genome from DNA Damage during Devel-opmentSangkyun Cho1, Stephanie Majkut2, Amal Abbas1, Ken Vogel1,Manasvita Vashisth1, Jerome Irianto1, Manorama Tewari1, Andrea Liu2,Ben Prosser3, Dennis E. Discher1.1Molecular & Cell Biophysics Laboratory, University of Pennsylvania,Philadelphia, PA, USA, 2Department of Physics & Astronomy, University ofPennsylvania, Philadelphia, PA, USA, 3Department of Physiology,Pennsylvania Muscle Institute, University of Pennsylvania School ofMedicine, Philadelphia, PA, USA.Mechanosensing by the nucleus involves its main structural proteins, the lam-ins, but mechanisms have been unclear as has the broader biological signifi-cance. In adults, stiffer tissues such as striated muscle that bear highmechanical stress have abundant collagen-I and correspondingly higherlamin-A compared to soft tissues. Here, we demonstrate lamin-A’s mechano-sensitivity in the earliest vertebrate organ, the heart, and relate its regulationto mechano-protection against DNA damage. Proteomic profiling of chickhearts that progressively stiffen from soft embryos show that lamin-A increasesin parallel with collagenous extracellular matrix (ECM) and tissue stiffness, as

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seen with diverse adult tissues. Acute ex vivo perturbation of actomyosincontractility or collagen alters heart stiffness within hours and impacts notonly lamin-A levels but also DNA damage, which impairs rhythmic beating.Low actomyosin stress on the nucleus results in increased interphase phosphor-ylation of lamin-A, which in turn enhances protein mobility and subsequentdegradation by nucleus-localized matrix-metalloproteinase-2 (MMP2).Mechano-sensitive turnover of lamin-A is further confirmed in isolated embry-onic and human iPS-derived cardiomyocytes cultured on soft or stiff collagen-coated gels. When contractile stress is maintained, suppression of lamin-Alevels generally results in increased DNA breaks, and one mechanism is pro-vided by nuclear envelope rupture with loss of DNA repair factors. Nuclearstiffening by lamin-A thus mechano-protects the genome.

2548-Pos Board B564Mapping the Mechanical Cross-Talk between Epidermal Growth FactorReceptor and Focal Adhesion FormationTejeshwar C. Rao1, Tara M. Urner1, Victor Pui-Yan Ma2, Khalid Salaita2,Alexa L. Mattheyses1.1Cell, Development and Integrative Biology, University of Alabama atBirmingham, Birmingham, AL, USA, 2Chemistry, Emory University,Atlanta, GA, USA.Cells can adapt to environmental cues, chemical or mechanical in nature, viafocal adhesions (FAs), which act as command posts connecting the extracel-lular environment to the intracellular mileu. Environmental sensing includesboth integrin-mediated and growth factor receptor-triggered signaling. Thisco-operation between signal transduction pathways is exemplified by the inter-action between integrins and the epidermal growth factor receptor (EGFR). Un-derstanding the nature of this cross-talk is vital to comprehend howenvironmental signals integrate to regulate mechanosensing processes of FAformation and cell spreading. While the forces transmitted via integrin recep-tors have been broadly investigated, there is limited understanding of EGFR it-self acting as a mechanosensor during cell attachment. To map these forcestransmitted by EGFR, we employ novel EGF anchored ‘‘turn on’’ tensiongauge tether (TGT) sensors immobilized on gold nanoparticles. TGTs are me-chanically labile DNA duplexes designed to rupture at forces exceeding its‘‘tension tolerance (Ttol)’’. We show that in Cos-7 cells within minutes of ligandbinding, EGFR can transmit piconewton (pN) forces to rupture the stiffestTGTs (Ttol = 56 pN). RGD mediated integrin binding was required for EGFRmediated force transmission. Using TGTs with Ttol = 12 or 56 pN, we observedthat the 12 pN signal appears closer to cell periphery, possibly co-localizingwith nascent FAs, while the stronger 56 pN signal is less frequently observed,closer to cell center. To simultaneously evaluate the spatio-temporal relation-ship between EGFR and integrin receptor forces, we applied a multiplexTGT approach. We hypothesize that changes in EGFR force transmissioncorrelate with corresponding changes in recruitment of downstream signalingmolecules and constituents of FAs. Characterizing the spatio-temporal inter-play of EGFR-integrin mechanotransduction events has widespread implica-tions to better understand the transformation of cells during tumor cellinvasion and metastasis.

2549-Pos Board B565Single-Cell Mechanical Phenotype is an Intrinsic Marker of Reprogram-ming and Differentiation along the Neural LineageMarta Urbanska1, Maria Winzi1, Katrin Neumann2, Shada Abuhattum1,Philipp Rosendahl1, Paul M€uller1, Anna Taubenberger1,Konstantinos Anastassiadis1, Jochen Guck1.1Cellular Machines, Biotec, TU Dresden, Dresden, Germany, 2Stem CellEngineering, Biotec, TU Dresden, Dresden, Germany.Cellular reprogramming is a dedifferentiation process during which cellscontinuously undergo phenotypical remodeling. While genetic and biochem-ical details of this remodeling are fairly well understood, little is known aboutthe change of cell mechanical properties during the reprogramming process. Inthis study, we investigated changes in the mechanical phenotype of murine fetalneural progenitor cells (fNPCs) during the process of reprogramming toinduced pluripotent stem cells (iPSCs). For this purpose, we employed real-time deformability cytometry (RT DC)—a robust and high-throughput tech-nique allowing for rapid characterization of thousands of cells (Otto et al.,2015)—and confirmed the main findings with atomic force microscopy-enabled nanoindentation. We find that fNPCs become progressively stiffer enroute to pluripotency, and that, with the aid of CD24 and SSEA1 surfacemarkers, mechanical subpopulations corresponding to differently advancedcells can be distinguished at intermediate reprogramming stages. Furthermore,we show that fNPC stiffening is mirrored by iPSCs becoming more compliantduring differentiation towards the neural lineage, and that the mechanicalphenotype of iPSCs is comparable with that of embryonic stem cells. These

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results suggest that mechanical properties of cells are inherent to their develop-mental stage. They also reveal that pluripotent cells can differentiate towards amore compliant phenotype, what challenges the view that pluripotent stem cellsare less stiff than any cells more advanced developmentally. Finally, our studyindicates that the cell mechanical phenotype might be utilized as an inherentbiophysical marker of pluripotent stem cells.

2550-Pos Board B566Is SUN2 Autoinhibited?Zeinab Jahed, Uyen T. Vu, Darya Fadavi, Samuel C.J. Kim,Mohammad R.K. Mofrad.University of California Berkeley, Berkeley, CA, USA.SUN proteins are inner nuclear membrane proteins, which interact with outernuclear membrane KASH proteins to form linkers of the nucleoskeleton andcytoskeleton (LINC) complexes. These complexes are important for severalcellular functions including DNA damage repair, mechanotransduction,meiotic chromosome pairing, and nuclear positioning. A growing list of dis-eases are associated with genetic mutations in LINC complex proteinsincluding ataxia, cancer, and muscular dystrophies. SUN2 is a major mamma-lian SUN protein and consists of two coiled coil domains (CC1-CC2) precedingthe conserved SUN-domain. Recent structural details of short fragments ofSUN2 suggest that CC2-SUN2 unexpectedly adapts an inactive monomericconformation, whereas CC1 is a trimer, and a point mutation in CC2(E471A)can result in the activation of the protein. In that study, the crystal screeningof CC1-CC2-SUN was unsuccessful possibly due to its heterogeneous state,and in order to determine the crystal structures, each protein fragment (CC1and CC2-SUN) was expressed separately; hence any structural changes result-ing from interactions between CC1 and CC2-SUN could not be captured.In thisstudy, we constructed structural models of monomeric and trimeric CC1-CC2-SUN-domain. We observed the dynamics of these structures using�500ns mo-lecular dynamics simulations, and compared it with the dynamics of CC2-SUN_WT and CC2-SUN-E471A. Our results indicate that monomeric CC1-CC2-SUN shows characteristics similar to CC2-SUN-E471A where the resi-dues pairs that supposedly capture the SUN-domain in the inactive conforma-tion are separated, suggesting that the CC1-CC2-SUN fragment does not adaptan inactive conformation, due to associations between CC1 and CC2-SUN.Additionally, we identified an important residue (E438) involved in this asso-ciation . Interestingly this residue has previously been observed in musculardystrophy-associated SUN2 variants. While current experimental attemptshave failed to screen for larger fragments of SUN2 proteins, we believe thatour study provides invaluable insights into the structure of SUN2.

2551-Pos Board B567Correlation and Differentiation Based Algorithms for Cell Mobility Quan-tificationAndreas W. Henkel, Lulwa Al Abdullah, Zoran B. Redzic.Physiology, Kuwait University, Safat, Kuwait.Quantification of cell mobility in confluent cell cultures or on coverslips withhigh cell density is a challenge, because tracking of individual cells is notreadily possible. Two procedures have been developed and tested to solvethis problem, namely a differential (MoveDif) and a correlation (MoveCor) al-gorithm. Both compare successive image frames and calculate a differenceparameter between them.Image preprocessing was required to neutralize brightness changes and inho-mogeneous illumination. Additionally, structures and membranes in the imageshad to be enhanced and standardized by edge-detection filters. We showed thatboth algorithms had advantages in different experimental setups, depending ofthe complexity of the cellular movement. Besides the determination of globalcell mobility, the membrane contractility and movement of single cells, smallcell groups and intracellular structures could be quantified, if individual cellswere separated and magnified. The discriminatory potency and sensitivity ofboth algorithms were tested by drug induced changes of cellular movementspeeds and their robustness against brightness changes were analyzed too.To demonstrate the mobility effects of ketamine on dexamethasone-treated hip-pocampal neurons, we found that the correlation algorithm MoveCor couldquantify the ketamine-dependent increase in cell movement speed after priortreatment with dexamethasone had reduced it distinctively. We also could mea-sure how staurosporine slowed down the mobility of acidic intracellular organ-elles in highly magnified glial cells from chicken telencephalon. Thedifferentiation algorithm MoveDif was employed to demonstrate that thecontractility of pericyte membranes slowed down after induction of oxygenglucose deprivation.The limitations of both methods were given by their inability to produce abso-lute mobility values but these drawbacks can be solved by appropriate imagepreprocessing.

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2552-Pos Board B568Migration and Contraction of Fibroblasts from Normal and Scar VocalFolds with Applications to Wound HealingAnete Branco1, Aashrith Saraswathibhatla2, Jacob Notbohm2,Susan Thibeault1.1Surgery, University of Wisconsin-Madison, Madison, WI, USA,2Engineering Physics, University of Wisconsin-Madison, Madison, WI,USA.Vocal fold scarring has various causes, including inflammation, trauma, radio-therapy and laryngeal surgeries. The treatment for vocal fold scarring is a chal-lenge in Laryngology practice. Scars disrupt the layered structure and inducedisorder in the lamina propria extracellular matrix, which causes significantchange in vocal fold biomechanics, resulting in voice disorders that oftencompromise patient quality of life. Vocal fold fibroblasts are responsible forsynthesis of the extracellular matrix, playing a key role in support of the laminapropria in normal and diseased conditions. During tissue injury, vocal fold fi-broblasts become activated and differentiate into myofibroblasts, initiating con-tractile forces that facilitate wound healing. The correct balance betweencontraction and extracellular matrix deposition is required for optimal healing.Using a scratch assay with live-cell time lapse microscopy and traction forcemicroscopy, we analyze the migration and contraction of fibroblasts fromnormal and scar human vocal folds during wound healing. As expected, mostof the normal and scar vocal fold fibroblasts migrated toward the free areawithin 24 hours, which is a critical time-point after laryngeal surgeries. Scarvocal fold fibroblasts moved less persistently but in a collective mannerwhereas normal cells had more persistent and individual patterns of migration.Additionally, scar cells contracted approximately two times more than normalvocal fold fibroblasts. We expect these results to clarify mechanisms of migra-tion and contraction of normal and scar vocal fold fibroblasts. This data may beuseful to design experiments that increase or decrease contraction and migra-tion to cause faster healing or reduced scar formation.

2553-Pos Board B569Controlling Candidate Physical Inputs to the Spindle Assembly Check-pointJonathan A. Kuhn1,2, Eline G. Ter Steege1,3, Sophie Dumont1,4.1Cell and Tissue Biology, University of California, San Francisco, Oakland,CA, USA, 2Tetrad Graduate Program, University of California, SanFrancisco, San Francisco, CA, USA, 3Cancer, Stem Cells, andDevelopmental Biology, University of Utrecht, Utrecht, Netherlands, 4Celland Molecular Pharmacology, University of California, San Francisco, SanFrancisco, CA, USA.To ensure accurate chromosome segregation, the spindle assembly checkpoint(SAC) prevents anaphase until chromosomes correctly attach to the spindle.The SAC detects some aspect of plus-end microtubule attachment to the kinet-ochore, and generation of tension is not sufficient to satisfy the SAC without it.However, we do not know whether tension is necessary to satisfy the SAC, andthis is in part due to the difficulty of controlling tension in otherwise native end-on attachment scenarios. Here, we use laser ablation to change tension in realtime, and we currently monitor SAC satisfaction. By detaching a kinetochore-fiber from the spindle for timescales relevant to SAC responses, we show thatthe SAC is insensitive to loss of tension when attachments are otherwise notdetectably changed. What about end-on attachment the SAC detects and howit integrates this information remain unclear, and to address these questionswe are developing assays to control end-on attachment features and test signalintegration mechanisms. Together, we hope that the ability to control physicalparameters of attachments will help push our understanding of what cues thekinetochore integrates to control cell cycle progression.

2554-Pos Board B570Mechanical Relaxation of Alpha-Actinin in the Cellular Cytoplasm Probedwith Magnetic TweezersChristopher C. Sitaras, Allen J. Ehrlicher.Bioengineering, McGill University, Montreal, QC, Canada.The actin cytoskeleton is a key determinant of cell force production, mechan-ical integrity and structure, and mechanosensation. An essential yet often over-looked element of the actin cytoskeleton is the crosslinker, alpha-actinin 4(ACTN4), which in vitro has been shown to exhibit catch-bond behavior anddetermine the mechanical relaxation of purified actin networks. ACTN4’srole in determining the mechanical properties of the cytoplasm, however, is un-clear. Here we use Magnetic Tweezers (MTs) to probe the moduli and creepbehavior of the cytoplasm in living cells. We use biolistic injection of 200nm fluorescent super-paramagnetic particles as probes, and then apply a step-stress magnetic field using the MTs, and quantify the time-dependent displace-ment of particles in the viscoelastic cytoplasm. To evaluate the role of ACTN4,

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we employ two cell lines: wild type ACTN4, and a mutated variant (K255E),which has previously been shown to exhibit stronger actin binding, and hasbeen implicated in kidney pathology. These results present some of the firstcytoplasmic creep measurements related to crosslinker binding kinetics, andprovide insight into the detailed cytoskeleton relaxation responsible for cellmechanics.

2555-Pos Board B571Probing How the Mammalian Kinetochore Holds on to Growing versusShrinking MicrotubulesAlexandra F. Long, Dylan B. Udy, Pooja Suresh, Sophie Dumont.University of California, San Francisco, San Francisco, CA, USA.The kinetochore links chromosomes to dynamic spindle microtubules anddrives chromosome segregation at cell division. Although we know many ofthe mammalian kinetochore’s key molecules, our understanding of its me-chanics is limited. We do not know what interfaces it uses to hold on to growingversus shrinking microtubules and how these interfaces are regulated to supportdifferent mitotic functions. To address these questions, here we use laser abla-tion and other physical approaches to isolate kinetochores inside mammaliancells in states where they bind to polymerizing versus depolymerizing microtu-bules. We find that phosphorylation of key load-bearing protein Hec1 (Ndc80)tunes passive friction along polymerizing microtubules, and yet does notcompromise the kinetochore’s grip on depolymerizing microtubules. Thesedata suggest that different interfaces engage with growing and shrinking micro-tubules. To probe the nature of these molecular interfaces, and how they areregulated by force, we are developing assays to apply controlled forces to ki-netochores in live mammalian cells. More broadly, we hope that this workwill help us understand the feedback between molecular composition, microtu-bule dynamics and force at the mammalian kinetochore.

2556-Pos Board B572Inertial Microcavitation as a Neural Cell Damage Mechanism in a 3D InVitro Model of Blast Traumatic Brain InjuryHarry C. Cramer III,1,2, Jonathan B. Estrada2, Mark T. Scimone1,2,Christian Franck1,2.1Center for Biomedical Engineering, Brown University, Providence, RI,USA, 2School of Engineering, Brown University, Providence, RI, USA.Blast traumatic brain injury (bTBI) is a leading cause of injury in the armedforces. Diffuse axonal injury, the hallmark feature of blunt TBI, has been inves-tigated in direct mechanical loading conditions. However, recent evidence sug-gests inertial cavitation as a possible bTBI mechanism. Cavitation occurs via alow-pressure region caused by pressure waves, and is strongly dependent onlocal geometric and mechanical properties. The structural damage features asthe result of cavitation - particularly at the cellular level - are incompletely un-derstood. Microcavitation is induced via single, high-energy laser pulses,wherein single bubbles are generated within the focal plane of the sample, re-corded at high-speed and analyzed with custom image processing algorithms.Initial work characterized distinct strain regimes where different modalitiesof cellular injury dominated, including complete cellular fragmentation, severesomatic and projection fragmentation, limited somatic damage with severe pro-jection fragmentation, and limited projection damage. Preliminary actin frag-mentation results indicated the presence of severe cytoskeletal disruptions upto the fragmentation radius dictated by cavitation bubble dynamics. The focusof this study is the examination of cytoskeletal disruption, including microtu-bule disruption and actin fragmentation. In addition, cellular viability andcellular apoptotic pathway studies are examined within the context of the largerpathology of bTBI.

2557-Pos Board B573Cellular Contraction can Drive Rapid Epithelial FlowsAlex Hamby.University of Arizona, Tucson, AZ, USA.Single, isolated epithelial cells move randomly; however, during wound heal-ing, organism development, cancer metastasis, and many other multicellularphenomena, motile cells group into a collective and migrate persistently in adirected manner. Recent work has examined the physics and biochemistrythat coordinates the motions of these groups of cells. Of late, two mechanismshave been touted as being crucial to the physics of these systems: leader cellsand jamming. However, the actual importance of these to collective migrationremains circumstantial. Fundamentally, collective behavior must arise from theactions of individual cells. Here, we show how biophysical activity of an iso-lated cell impacts collective dynamics in epithelial layers. Although many re-ports suggest that wound closure rates depend on isolated cell speed and/orleader cells, we find that these correlations are not universally true, nor do col-lective dynamics follow the trends suggested by models for jamming. Instead,our experimental data, when coupled with a mathematical model for collective

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migration, shows that intracellular contractile stress, isolated cell speed, andadhesion all play a substantial role in influencing epithelial dynamics, andthat alterations in contraction and/or substrate adhesion can cause confluentepithelial monolayers to exhibit an increase in motility, a feature reminiscentof cancer metastasis. These results directly question the validity of wound-healing assays as a general means for measuring cell migration, and providefurther insight into the salient physics of collective migration.

2558-Pos Board B574Distinct Relaxation Timescales of Neurites Revealed byMicrorheology andRelaxation TestsChao Fang.The University of Hong Kong, Hong Kong, Hong Kong.It is commonly believed that the mechanical response of neurites, finger-likestructures extending from the main body of neural cells, plays a crucial rolein processes such as neurite outgrowth and the formation of the neural network.In this study, by using dynamic AFM (atomic force microscopy) indentation,we systematically examined the relaxation and rheological behavior of well-developed neurites on primary neurons. Interestingly, the response was foundto be strongly rate-dependent, with an apparent initial and long term elasticmoduli of neurite around 780 and 156 Pa, respectively. To better analyze themeasurement data and extract information of key interest, finite element simu-lations (FEM) were also conducted where the neurite was treated as a visco-elastic solid consisting of multiple characteristic relaxation times. It wasshown a minimum of three relaxation timescales, i.e.� 0.01, 0.1 and 1 seconds,are needed to fit the simulation results to the relaxation and rheological data un-der different indentation rates and depths. We further demonstrated that thesethree characteristic relaxation times are likely originated from thermal fluctua-tions of the microtubule, membrane relaxation and cytosol viscosity, respec-tively. By identifying key parameters describing the viscoelastic behavior ofneurites, as well as revealing possible physical mechanisms behind, this studycould greatly help us understand how neural cells perform their biologicalduties over a wide spectrum of time-scales.

2559-Pos Board B575Influence of Mechanical Environmental Factors on Cell Migration Phe-nomenonZbigniew Baster, Tomasz Witko, Zenon Rajfur.Faculty of Physics, Astronomy and Applied Computer Science, JagiellonianUniversity, Cracow, Poland.Current research in cell biology, including cell motility, proliferation or cancerresearch, are based on different cell types cultured on glass or plastic substrate.However, despite of its many advantages, such as ease of use or widespreadapplication, this model system does not account for different tissues stiffness.Several recent studies showed that cells, cultured on substrates with a differentelastic modulus, behave differently in many processes, among which is cellmigration, division, metastasis or response to chemical or mechanical stimuli.This phenomenon is connected to different regulatory pathways, and thereforeto different protein expression, distribution and activation. In our research, weare mimicking stiffness of body tissues using polyacrylamide hydrogels.Our results show that cells behave differently depending on substrate elasticmodulus. Both, cells morphology and physiology are affected by the changeof substrate stiffness. These changes are correlated with variance in cytoskel-etal proteins distribution and migration-related protein (such as Rho family pro-teins) activation patterns in cells. Our work is concentrated on these aspects andtheir connections to cells behavior. We employ advanced optical microscopymethods such as confocal and bright field and fluorescence microscopy todissect the cellular regulatory mechanisms which are responsible for observedchanges in cell morphology and migration.This work is supported by a grant from the Polish Ministry of Science andHigher Education 7150/E-338/M/2017.

2560-Pos Board B576Electrotactic Migration of Chondrocytes in a 3D Collagen MatrixJoshua Bush, Xavier Palmer, Anthony Asmar, Michael Stacey.Old Dominion University, Norfolk, VA, USA.Endogenous direct current (DC) electric fields (EFs) have been measured dur-ing the development and regeneration of tissues. Given that there are endoge-nous EFs and the disruption of these EFs interrupts wound healing anddevelopment, there has been considerable interest in studying cellular re-sponses to EFs. It has long been recognized that EFs enhance the repair of con-nective tissue through elevated production of collagen and proteoglycans. Briefenzymatic digestion at the site of damaged cartilage also increases chondrocytemigration to the damaged area with deposition of a rich extra cellular matrix(ECM), emphasizing the importance of migration in the regeneration of carti-lage. The mechanism whereby chondrocytes sense damage and polarize to

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initiate directional movement is understudied. There are a variety of assays forapplying an EF to cells. The common method uses silver electrodes and glasstubes filled with agar, as a salt bridge between a salt solution and cell media.Cells are plated within a thin channel made of cover glass. Variations of thissetup have been made into small chips formed of laser cut acrylic and/orPDMS. Most of these setups require significant assembly time and are aimedto study cells in a 2D environment. Because cells lose their differentiated prop-erties when grown in 2D tissue culture compared to 3D, it is important to factorthis into experimental set-ups. We propose a 3D printed device that will allowfor the electrotactic study of cells in a 3D collagen matrix. Chondrocytes are ofparticular interest to identify the mechanism by which electrotactic migration isinduced. With knowledge of this mechanism treatments can be optimized foraccelerated cartilage repair.

2561-Pos Board B577Using FLIM-FRET to Measure Force in Zebrafish Embryos using anEpCAM-Embedded Molecular Tension SensorMelanie R. Malinas.Stanford University, Stanford, CA, USA.Mechanical forces direct developmental processes, yet the mechanical stressesdefining embryonic development remain poorly understood at the cellular andmolecular levels. We explored whether an in-vivo tension probe—a geneticallyexpressible Fluorescence Resonance Energy Transfer (FRET)-based piconew-ton Tension Sensor Module (TSMod, Grashoff et al. 2010)—could measuremechanical forces during zebrafish development. We demonstrated the design,in-vivo expression, and localization of the probe by inserting TSMod into anepithelial cell adhesion molecule (EpCAM).We describe protocols for Fluores-cence Lifetime Imaging Microscopy (FLIM)-FRET imaging and quantificationduring zebrafish gastrulation. Our methods achieved a FLIM resolution forEpCAM-TSMod of 90 ps over multiple embryos over multiple days. Due toresolution limits, we only detected trends but not statistically significant signalchanges in tension between cells with our EpCAM-TSMod. This is the first useof FRET tension sensors in zebrafish embryos. Softer TSMod probes or othercarrier molecules in combination with our methods will enable biophysical ten-sion measurements in multicellular systems.

2562-Pos Board B578Modelling the Dynamics and Distributions of Focal AdhesionsLaurent MacKay.Physiology, McGill University, Montreal, QC, Canada.Focal adhesions are dynamic aggregates of the transmembrane receptor integ-rin that serve as a point of force transmission from the cytoskeleton to the extra-cellular environment. Adhesions can be classified according to their geometricand stability properties that vary as they progress along a stepwise pathway ofadhesion maturation. These properties are partially attributable to the presenceand post-translational modification of a variety of adaptor proteins mechani-cally linking adhesions to the cytoskeleton. Immediately after their birth,nascent adhesions exist as small and highly dynamic structures which typicallyturnover within a minute, while those that mature to larger sizes are stable onthe timescale of an hour. There exists a large number of proteins which regulatethe assembly, disassembly, and maturation of adhesions. However, the tran-sient nature of adhesions combined with their extended physical dimensionsmake the effects of regulatory proteins nontrivial to understand. This is partlydue to a lack of models relating the geometric properties of two-dimensionalproteins aggregates to single molecule dynamics. We present a mathematicalmodel of adhesions as geometric objects which assemble, grow, shrink, andeventually disassemble due to spatio-temporal changes in their interactionswith a dynamic cytoskeleton. Stochastic realizations of adhesion dynamicsspecified by these models allow us to compute the time evolution of size andposition distributions of adhesions as well as their lifetime distributions. Thesedistributions are critical for understanding regulatory effects on ensembles ofadhesions that exist only transiently. Moreover, because the cytoskeleton is me-chanically coupled to the extracellular environment through adhesions, thesemodels predict the traction stress distributions produced by cells. This allowsus to test the emergent outcomes of different hypotheses in the literaturerelating to the relationship between adhesion size and traction stress.

2563-Pos Board B579Spatiotemporal Change in Cell Stiffness during Early EmbryogenesisInvestigated by Atomic Force MicroscopyYuki Fujii1, Taichi Imai2, Wataru Koizumi2, Kohji Hotta2, Kotaro Oka2,Takaharu Okajima1.1Hokkaido University, Sapporo, Japan, 2Keio University, Yokohama, Japan.During developmental processes of embryo, cells undergo a large deformationand cell division. The dynamic change in cell shape and size in the embryogen-esis is directly associated with the mechanical properties of cells such as the

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contractile and traction forces [1] and the elastic modulus of cells. However,little is known about the spatiotemporal change in the elastic modulus of cellsin the developmental stages. Here we measured the temporal evolution ofelastic modulus of embryo of C. intestinalis, which is well known as a modelorganism, in an early developmental stage by atomic force microscopy(AFM). The fertilized eggs were cultured on dish with a weak adhesion forceso that they followed a normal development at least until the gastrula stage.The AFM force mapping was examined in living embryo from fertilized eggstage to early gastrula stage, and the cell stiffness was estimated with a Hertziancontact model. We observed that cell stiffness of the early developing embryoincreased around the cell division where the cortical actin was accumulated.The result is similar to that observed in single cells in vitro [2,3]. Such anincreased cell stiffness was also observed in an endodermal cell lineage beforegastrula stage. This indicates that the time evolution of the elastic modulus isquite different between the ectodermal and endodermal regions. Moreover,we find that the change in cell stiffness is suppressed as the actin filamentsare depolymerized, indicating that the cell stiffness in embryo is associatedwith the remodeling of actin filaments. [1] P. Campinho et al., Nat. CellBiol, 15, 1405 (2013) [2] M P. Stewart et al., Nature 469, 226 (2011). [3] SP. Ramanathan et al. Nat. Cell Biol 17, 148 (2013).

Posters: Energy Transducing Membrane ProteinComplexes

2564-Pos Board B580Evolutionary Tradeoffs in Efficiency and Turnover Rate for F0F1-ATPaseJason A. Wagoner, Ken Dill.Laufer Center for Physical and Quantitative Biology, Stony BrookUniversity, Stony Brook, NY, USA.We present an evolutionary model of F0F1-ATPase based on nonequilibriumstatistical mechanics and experimental observations. We aim to describe thetransduction of energy throughout the motor and how this has impacted its evo-lution across different species. We describe how the mechanical and electro-static properties of the motor are optimized for fast and efficient energytransduction. We particularly focus on the c-ring stoichiometry—which variesin number of subunits from 8 to 15 across different species. Each c-ring subunitpumps a single proton, so this variable stoichiometry leads to differences in thenumber of protons pumped per three ATP synthesized by the motor. We findthat this variable stoichiometry reflects an evolutionary optimization againsta necessary tradeoff between thermodynamic efficiency and turnover rate(number of ATPs synthesized per second) in different environments. Wedescribe this tradeoff between efficiency and turnover rate and how it arisesfrom the specific mechanism of F0F1-ATPase.

2565-Pos Board B581A Theory for Rate Constants in Rotation Trajectories of F1-ATPaseSandor Volkan-Kacso, Rudolph Marcus.Chemistry and Chemical Engineering, California Institute of Technology,Pasadena, CA, USA.I present an elastic chemo-mechanical theory to treat single molecule imagingand ‘‘stalling’’ experiments in the F1-ATPase enzyme. Using a molecular grouptransfer approach the theory couples chemical reactions in the stator and thephysics of torsional elasticity in the rotor. In the theory we predicted andcompared with experiment the rate and equilibrium constant dependence ofsteps such as ATP binding as a function of the rotor angle.[PNAS, 112,14230 (2015)] Using independent experimental data from biochemicalensemble and single-molecule imaging experiments, the model correctly pre-dicts the controlled rotation data on fluorescent ATP without any adjustable pa-rameters. We took into account the biasing effect of finite experimental timeresolution in the single fluorescence trajectories and treated these data by devel-oping computational statistical methods.[PNAS, 113 (48), 12029 (2016)] Atheory-based method for the extraction of rate constants for hydrolysis and syn-thesis from controlled rotation data was also provided for angular range whereno such data is currently available [PNAS, 114, 7272 (2016)] The framework isgeneric and we plan to apply it to other biomolecular motors.

2566-Pos Board B582Isolation and Characterization of a Novel ATPase-Photosystem I ReactionCenter Complex in the Chloroplast Thylakoid MembraneSatarupa Bhaduri.Purdue University, West Lafayette, IN, USA.A novel 1 MDa intra-membrane ‘super-complex’ dominated by the chloroplastATP synthase and the photosystem I (PSI) reaction-center complex has beenisolated from spinach thylakoid membranes. This super-complex has beencharacterized by assay of ATPase activity, negative stain electron microscopy,

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tandem mass spectrometry, and Western assay of subunits. High-resolution Or-bitrap mass spectrometry was used to quantitatively analyze the presence andrelative amount of the multiple major and minor polypeptide components inthe super-complex. This is the first isolation of a higher order complexinvolving intact ATP synthase and Photosystem I to be identified and docu-mented by negative stain electron microscopy. The importance of theATPase-PSI super-complex in the thylakoid domain may imply efficient chan-neling of substrates and cofactors utilized for localized CO2 fixation. Studiessupported by NIHGMS-038323 and the Henry Koffler Distinguished Professor-ship (WAC), and NIHDK-063491 (JPW).

2567-Pos Board B583Structural Determination of Beef Heart Mitochondrial Cytochrome cOxidase in Small Unilamellar Liposomes using Small-Angle NeutronScattering (SANS)Lawrence J. Prochaska1, Kenneth A. Rubinson2, Christine N. Pokalsky1.1Biochemistry and Molecular Biology, Wright State University, Dayton, OH,USA, 2National Center for Neutron Research, National Institute of Standards,Bethesda, MD, USA.Our previous work using low angle neutron scattering of mitochondrial cyto-chrome c oxidase (COX) in asolectin unilamellar liposomes resulted in alow-resolution three-dimensional structure defined as a simple parallelepipedwith side lengths of (59 x 70 x 120) A with uncertainties of (11, 12, 20) A),respectively. The reconstituted enzyme was fully functional in mixed-lipid li-posomes which are unique because they allow scattering from the liposomesto be eliminated by using contrast-matched conditions in 15% D2O. The mo-lecular mass calculated for a protein with this volume was estimated to be(410 5 124) kDa, which suggested a COX dimer. One limitation in the datawas that the protein concentration in the liposome was 1-2 mM, which leadto uncertainty in long dimension due to intermolecular scattering. The calcu-lated SANS scattering curve from the three dimensional X-ray structureof the COX dimer (PDB 3AG1) fitted to a parallelepiped measuring(59 x 101 x 129) A with fitting uncertainties of (2, 3, 3) A, respectively, differedsignificantly from the SANS data from COX in the liposomes. These resultstaken together suggested that the structure of the enzyme in the membrane ismore compact than the three dimensional crystal perhaps implying importantfunctional significance. Most recently, we have increased the COX concentra-tion in the liposomes to 5 mM towards the goals of increasing signal to noiseratio for enhanced structural resolution and also providing less uncertainly inour long dimension measurements. The 5 mMCOX liposomes retain maximumfunctional activity and the enzyme is more than 95% incorporated into themembrane bilayer, similar to results obtain at the lower enzyme concentration.Recent SANS results using this preparation will be discussed with functionalimplications indicated.

2568-Pos Board B584Fluorescence Lifetime Imagings Shows that Respiratory SupercomplexesChange with Different Metabolic ConditionsKarin B. Busch.Institute for Molecular Cell Biology, Westfaelische Wilhelms University,Muenster, Muenster, Germany.Respiratory supercomplexes are macromolecular assemblies of diverse com-binations of OXPHOS complexes. Single particle electron microscopy of pu-rified supercomplexes revealed their structure and isolation from differenttissues point to a certain plasticity in their amount and composition. Super-complex assembly factors were identified that chaperone the formation ofsupercomplexes. In addition to these biochemical methods proving super-complex assembly, we recently could show that fluorescence proteins placedspecifically at the contact between a supercomplex CIn/CIII2/CIVm sense thedense environment what can be read out by fluorescence lifetime measure-ments. Fluorescence lifetime imaging microscopy thus allows to monitorchanges in supercomplex formation in life human cells. We found thatincreased supercomplex formation was correlated with increased respirationbut under different metabolic conditions, an observation that implies inter-esting interpretations.

2569-Pos Board B585EPR Detection of Radical(s) in Cytochrome C OxidaseDaniel Jancura1, Marian Fabian2.1Department of Biophysics, Safarik University, Kosice, Slovakia, 2Center forInterdisciplianry Biosciences, Safarik University, Kosice, Slovakia.Catalytic mechanism of cytochrome c oxidase (CcO) involves formation offerryl intermediates P and F. The production of these intermediates is accom-panied by a formation of protein-based radical(s). The reaction of oxidized CcOwith hydrogen peroxide also leads to the formation of P and F and correspond-ing radical species. However, the application of electron paramagnetic spec-

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troscopy (EPR) to detect such radical has resulted only in the observation oflow amounts relative to the concentration of the P intermediate. A possiblereason for this fact is a coupling of the unpaired electron of radical with theparamagnetic metal center(s) within the catalytic site of CcO. We have devel-oped a new approach, a moderate destabilization of the enzyme structure byprotein denaturant, guanidinium chloride (Gnd.Cl), to detect stoichiometricamount of the radical in CcO. In this situation, a coupling between protein-based radical and a metal center(s) is broken. As our results show, the yieldof the EPR observed radical in P is significantly increased in the presence ofGnd.Cl relative to that in the absence of denaturant. In a sample with 2 MGnd.Cl, the yield of the detected radical reached �50% of the P population.The origin of EPR detected radical(s) and their possible roles in the catalyticcycle of CcO is discussed.

2570-Pos Board B586O-O Bond Formation in Photosystem II Oxygen Evolving ComplexYulia Pushkar1, Scott Jensen1, Katherine Davis2.1Physics, Purdue University, Lafayette, IN, USA, 2Department of Chemistry,Princeton University, Princeton, NJ, USA.Photosynthetic water oxidation is a fundamental process that sustains thebiosphere. Light driven water oxidation is facilitated in Nature by the Oxy-gen Evolving Complex (OEC) - Mn4Ca cluster embedded in the photosystemII protein. During its function, the OEC cycles through five redox statestermed S0-S4 after which, oxygen is evolved. Here, time-resolved x-ray emis-sion spectroscopy (XES) was used to observe the process of oxygen forma-tion in real time. These experiments reveal that the oxygen evolution step,initiated by three sequential laser flashes, is accompanied by rapid (within50 ms) changes to the Mn Kb XES spectrum. However, no oxidation ofthe Mn4Ca core above the all MnIV state was detected to precede O-Obond formation, and the observed changes were therefore assigned to O-Obond formation dynamics. Our mechanism proposes O-O bond formationoccurs prior to the transfer of the final (4th) electron from the Mn4Ca clusterto the oxidized TyrZ. This model resolves the kinetic limitations associatedwith O-O bond formation, and suggests an evolutionary adaptation to avoidreleasing of harmful peroxide species. Atomistic model for the S3 state re-ported by us [1, 2] is indistinguishable (within the resolution of XRD)from one of the recently reported by Shen and co-workers [3]. This S3-statemodel incorporates a MnIV=O fragment of radicaloid character and exhibitsantiferromagnetic alignment with opposite spin orientations between two Mncenters and associated oxygens creating an opportunity for a low barrier toO-O bond formation.1. Davis et al. 2015, arXiv:1506.088622. Jensen et al. J Phys. Chem. Letters, 2017, 8, 2584�2589.3. Suga et al. Nature, 2017, 543(7643), 131-135.

2571-Pos Board B587Gravitational Strain as a Driving Mechanism for Cell MetabolismSteve Thorne.Biophysics, Celll.org, Berkeley, CA, USA.Relative to the inertial frame of the sun, all earthbound laboratories possesssmall gravitationally induced patterns of acceleration. Rather than dismisstheir influence outright, this paper considers their possible roll in cell func-tion. What is identified here is how two fundamental values associatedwith cell metabolism can be obtained by applying a simple work functionassociated with mass displacement within the gravitational field of the sun.It is shown first, that at the cellular scale, the unit of energy released by aden-osine triphosphate (ATP) when it undergoes hydrolysis is roughly equal tothe change in gravitational potential energy of an ATP molecule over thecourse of one day. Second, pertinent to energy change at the scale of thewhole organism, it is shown that the minimum energy needed for the organ-ism to maintain metabolic order, referred to as the Basal Metabolic Rate(BMR), is also produced by the computing the change in potential energyfor the mass of the organism over the course of one day. The period of theseenergy changes is fundamentally circadian, and it is hypothesized that thecell energy pathways might be regulated by the induced cycles of gravita-tional stress and strain that correspond to the rotation and orbital periodsof the earth.

2572-Pos Board B588Elucidating the 30-year-Longstanding Bioenergetic Mystery in Alkalo-philic BacteriaJames W. Lee.Chemistry and Biochemistry, Old Dominion University, Norfolk, VA, USA.The decades-longstanding energetic mystery of alkalophilic bacteria as to howthey are able to synthesize ATP has now, for the first time, been clearly solvedusing the Lee proton-electrostatics localization hypothesis [Lee (2015)

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Proton-electrostatic localization: explaining the bioenergetic conundrum in al-kalophilic bacteria, Bioenergetics 4: 121. doi:10.4172/2167-7662.1000121]and proof-of-principle experiments [Saeed and Lee (2015) Experimentaldemonstration of localized excess protons at a water-membrane interface, Bio-energetics 4: 127. doi:10.4172/2167-7662.1000127]. This is a major break-through in understanding proton-coupling bioenergetics over Peter Mitchell’schemiosmotic theory. The widespread textbook Mitchellian proton motiveforce (pmf) equation could significantly underestimate the true pmf valueand thus must be revised. Use of Lee’s newly formulated pmf equation resultsin an overall pmf value that is more than 4 times larger than that calculated fromthe Mitchellian equation for the alkalophilic bacteria. This newly calculatedpmf value is sufficient to overcome the observed phosphorylation potentialDGp of �478 mV to synthesize ATP in the bacteria, which can now explainthe 30-year-longstanding bioenergetics conundrum. This finding may havefundamental implications not only in the science of bioenergetics but also inunderstanding the importance of water to life not only as a solvent and substratebut also as a proton conductor for proton coupling energy transduction. In thispresentation, the results of our latest bioenergetics calculation and analysis willbe presented and discussed in greater details.

2573-Pos Board B589Atomic-Level Characterization of the Structural Dynamics of Azurin Var-iants with Tuned Reduction PotentialsAnthony T. Meger, Steven M. Berry, Alessandro Cembran.Chemistry and Biochemistry, University of Minnesota Duluth, Duluth, MN,USA.Understanding how non-covalent interactions modulate the reduction potentialof redox proteins is of fundamental importance to address design challengesranging from artificial photosynthetic centers to enzymatic biofuel cells.Here, we employed the copper-containing protein azurin as a model to under-stand the mechanism by which mutations affect the copper reductionpotential. In particular, we tested the hypothesis that introducing phenylala-nine mutations in the secondary coordination sphere increases the hydropho-bicity around the copper center, ultimately leading to an increase in thereduction potential. We tested this hypothesis by means of x-ray crystallog-raphy, redox potential measurements, and all-atom molecular dynamics(MD) simulations of several variants of azurin. The variants were engineeredto mimic elements in rusticyanin, and the secondary coordination sphere ofazurin was altered by incorporating up to four mutations to phenylalanine.Although the mutations led to an increase of the reduction potential as largeas 80 mV, the x-ray structures showed negligible overall structural changeupon mutation. Yet, both the x-ray structures and the MD simulations high-lighted a correlation between the reduction potential and the distance betweenthe copper and the carbonyl oxygen of the coordinating Gly-45. The x-raybeta-factor and the simulations also indicated an increase in the dynamicsin the region surrounding the copper in the variants. MD simulations revealedthat, contrary to the hypothesis, the mutations in general led to an increase ofthe water content in the region surrounding the copper. The increased wateraccessibility was attributed to conformational changes caused by steric clashesintroduced by the bulkier phenylalanine side chains. Overall, we have pro-vided an interpretative mechanism of the experimental data that could guidethe design of biomolecular nanoelectronic devices exploiting blue copperproteins.

2574-Pos Board B590Dissipation in a Sequence of Relaxations: The Ladder TheoremPeter Salamon1, Ty N.F. Roach2, Forest L. Rohwer2.1Mathematics and Statistics, San Diego State University, San Diego, CA,USA, 2Biology, San Diego State University, San Diego, CA, USA.We consider one relaxation: the complete equilibration of a system to a bath.We show that replacing one relaxation with two relaxations starting and endingat the same states but reaching an intermediate equilibrium along the way al-ways produces less entropy than the single relaxation. We present a completelygeneral proof of this Ladder Theorem, which asserts that the entropy produc-tion in a relaxation process is decreased when the relaxation proceeds via inter-mediate steps.This is indeed what is observed in the central carbon metabolism of the mi-crobes in coral ecosystems. The microbes in the coral-dominated, energy-limited environments have an enrichment in genes encoding for the EmbdenMeyerhoff Parnas (EMP) pathway, a central carbon catabolic pathway thathas 12 steps involved in the breakdown of sugars to pyruvate. On the otherhand, microbes in algal dominated, energy surplus environments have anenrichment for alternative central carbon metabolism pathways such as thePentose Phosphate (PP) pathway and the Entner Doudoroff (ED) pathway,which contain fewer steps than the EMP pathway.

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2575-Pos Board B591Direct Observation of Polarization in Short Hydrogen Bonds due to ProtonDelocalizationChi-Yun Lin, Steven G. Boxer.Department of Chemistry, Stanford University, Stanford, CA, USA.Based on their relatively shallow potential energy surfaces (PESs), shorthydrogen bonds with heavy atom distances about or less than 2.5 A are believedto exhibit proton delocalization, which has been argued to be beneficial to bio-logical catalysis1. While the degree of delocalization is usually characterizedby neutron diffraction or inferred from broad bands in the IR spectra, these ap-proaches can result in ambiguities2 and information on energetics cannot bedirectly obtained. Here we apply an external electric field to perturb the protonequilibrium position of a short hydrogen bond in previously characterized GFPS65T/H148D mutants with various degrees of proton affinity mismatch (DpKa)between the chromophore and aspartate3. The chromophore serves as a spectralprobe of the proton position, allowing us to perform electronic Stark spectros-copy. The local curvature of the PES is reflected by the ease of proton polari-zation, and the resulting proton displacement effectively amplifies the electricfield experienced by the chromophore, leading to anomalously large Stark tun-ing rates as readouts. These Stark tuning rates are sensitive to the presence ofthe short hydrogen bond, proton position, DpKa and deuteration, in accordancewith large hydrogen bond polarizations predicted by Zundel4. The results rein-force our previous arguments on the absence of low barrier hydrogen bonds inthese variants3: the general shape of PESs is double-welled with a rather highbarrier in between and the proton is delocalized within each well.[1] Perrin, C. L.; Nielson, J. B. Annu. Rev. Phys. Chem. 1997, 48, 511-544.[2] Graen, T. et al. J. Am. Chem. Soc. 2016, 138, 16620-16631.[3] Oltrogge, L. M.; Boxer, S. G. ACS Cent. Sci. 2015, 1, 148-156.[4] Janoshek, R. et al. J. Am. Chem. Soc. 1972, 94, 2387-2396.

2576-Pos Board B592Mimicking Natural Photosynthesis: Charge Transfer in PpcA-Ru(bpy)3ComplexesDaniel R.Marzolf, Matthew O’Malley, Coleman Swaim, Oleksandr Kokhan.Department of Chemistry and Biochemistry, James Madison University,Harrisonburg, VA, USA.We are developing biomimetic molecular architectures for efficient solar en-ergy conversion using artificial photosensitizers combined with natural andgenetically engineered host systems capable to support long-lived charge-sepa-rated states and conduct charges away from the photosensitizers. Convertinglight energy into its electrochemical equivalent requires precise control andfine tuning of relevant kinetic and thermodynamic parameters, including pri-mary charge separation. To this end, we developed a series of 22 cysteine mu-tants of PpcA, a 3-heme cytochrome from Geobacter sulfurreducens. Theseproteins were successfully expressed in E.coli and isolated for covalent labelingwith Ru(bpy)2(bpy-Br). Protein purity and successful posttranslational modifi-cations were confirmed with HPLC-MS. With time-resolved nanosecond andultrafast transient absorbance spectroscopy we have identified 6 constructswith apparent photo-induced charge transfer time constants of 20 ps or faster,including 2 constructs with 1-2 ps time constants. The is a significant result asup to this point only natural photosynthetic systems demonstrated such a fastinitial charge separation, while all artificial covalent biohybrid constructs ex-hibited charge transfer rates 3 or more orders of magnitude slower. To under-stand molecular principles responsible for such a dramatic acceleration ofelectron transfer rates, we used small- and wide angle X-ray scattering andcurrently attempting to obtain X-ray crystallographic and NMR structures ofultrafast constructs. Finally, we performed triplicate 250-300 ns all-atom mo-lecular dynamics simulations of all 6 ultrafast constructs. Based on the obtainedresults we conclude that that photo-induced ultrafast charge transfer requiresvan der Waals contact between heme vinyl groups and photosensitizers whilecontacts with propionates or a small number of covalent bonds between the do-nors and acceptors play much less significant role.

2577-Pos Board B593Regulating Photonic Properties of Lamellar Chloroplast and the Environ-mental AdaptionMing-Chih Shih1, Ping-Yun Tsai2, Ming-Huang Wu1, Jiannyeu Chen3,Chiou-Rong Sheue4.1Physics, National Chung-Hsing University, Taichung City, Taiwan, 2LifeSciences, National Chung-Hsing University, Taichung City, Taiwan, 3Centerof Nanoscience & Nanotechnology, National Chung-Hsing University,Taichung City, Taiwan, 4Life Sciences & Center of Global Change Biology,National Chung-Hsing University, Taichung City, Taiwan.Iridescent structures in photosynthetic organisms has recently been identified topossess enhanced light harvesting capability. It was proposed that interference

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nature of periodic photonic nanostructure results in this enhancement, nonethe-less, it will inevitably accompany with light capture efficiency reduction in thephotonic band gap spectrum range. Enhancement or reduction of light captureefficiency depends on the coupling condition between the electric field spatialvariation and the lamellar structures. This work will provide insight about howphotonic band gap wavelength of the lamellae can be regulated and the possi-bility in correlation between this regulation and different strategies from naturefor environmental adaptation. Most of the plants with iridescent chloroplastsdwell in understorey environment, low light coherence condition might affectthe predicted efficiency enhancement, and some results will be presented.These results will offer new perception about the formation of lamellar struc-ture become a better tactic than accumulate more chlorophyll (or other lightabsorbing materials) molecules together for obtaining better light harvestingefficiency.

2578-Pos Board B594Identification of Red Pigments in the Photosystem I Complex of OxygenicPhotosynthesisYuval Mazor, Hila Toporik, Su Lin.Arizona State University, Tempe, AZ, USA.Oxygenic photosynthesis powers our biosphere using two large reaction cen-ters, photosystem I and photosystem II (PSI and PSII). Both photosystemsare composed of hundreds of light harvesting pigments, mainly chlorophylls,coordinated by several transmembrane protein subunits. All chlorophylls arenot equivalent due to their interactions with their environment, either aminoacids side chains, lipids, or other chlorophylls. The absorption maxima ofsome chlorophylls in the core antenna of PSI is tuned to very low levels, lowerthan that of the final photochemical trap in the complex. Because of this lowexcitation maximum, these red pigments play a crucial role in the path of exci-tation energy through the core PSI antenna. The location of these red pigmentswithin the PSI antenna is unknown. Using chimeric PSI complexes in cyano-bacteria we identified the first red pigment in the PSI antenna. We show thata single added red pigment and can greatly affect energy migration in thecore of PSI, which contain more than 90 other chlorophylls. We also deter-mined the structure of chimeric PSI and observe the configuration of the addedred site.

2579-Pos Board B595A Multiscale Model of PhotosynthesisDoran I.G. Bennett1, Graham R. Fleming2,3, Kapil Amarnath4.1Department of Chemistry and Chemical Biology, Harvard University,Cambridge, MA, USA, 2Department of Chemistry, UC Berkeley, Berkeley,CA, USA, 3Molecular Biophysics and Integrated Bioimaging Division,Lawrence Berkeley National Labs, Berkeley, CA, USA, 4Department ofPhysics, UC San Diego, San Diego, CA, USA.Photosynthetic light harvesting, the conversion of photons into chemicalenergy, is responsible for all food and atmospheric oxygen on earth.Understanding the design principles underpinning light harvesting from theatomic to cellular length scales offers the potential of rationally engineeringincreased photosynthetic yield. The challenge, however, is bridging the largerange of length and timescales involved. We have developed a model ofexcitation energy transfer and light harvesting that accurately spans from iso-lated pigment-protein complexes to the 100 nm length scale of the photo-system II (PSII) enriched portion of the thylakoid membrane. We explorethe emergent features of PSII light harvesting in the presence of quenchersand demonstrate that the phenomenological models used historically fail tocapture essential features of regulation by non-photochemical quenching, aprocess important for plant fitness. This work connects the structure ofpigment-protein complexes to the resulting properties of photosystem II lightharvesting in vivo, providing a first step towards a predictive model ofphotosynthesis.

2580-Pos Board B596Molecular Dynamcis of Light-Harvesting Complex II Embedded in theThylakoid MembraneSebastian Thallmair1, Petteri A. Vainikka1,2, Siewert-Jan Marrink1.1University of Groningen, Groningen, Netherlands, 2University of Turku,Turku, Finland.Plant chloroplasts contain considerable amounts of the antenna protein light-harvesting complex II (LHCII) which is a key player in natural photosynthesis.It is associated to the photosystem II (PSII) and occurs as trimer. Each mono-mer contains a variety of different cofactors: 8 chlorophyll a, 6 chlorophyll b,and 4 carotenoid molecules. They are responsible for capturing photons andtransmitting the excitation energy towards the PSII reaction center. This chal-lenging task requires a highly precise arrangement of the involved chromo-

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phores resulting in a specifically fine-tuned ordering of the energy levels andchromophore couplings. Moreover, LHCII can switch between two states: alow light state which allows for efficient excitation transport and a non-photochemically quenched state which protects the thylakoids from too manyexcitations.We built a coarse-grained model of LHCII using the Martini force field. Basedthereon, we investigated the dynamics of LHCII monomers and trimers in anordinary dipalmitoylphospatidylcholine (DPPC) bilayer as well as in the thyla-koid membrane. The latter constitutes the natural environment of LHCII andcontains a large amount of glycolipids. We discuss the differences betweenthe monomeric and the trimeric form as well as the impact of the membranecomposition on the properties of LHCII. In addition, we simulated LHCII tri-mers under low and high light conditions by adapting the protonation state ofseveral residues exposed to the thylakoid lumen. Our simulations capture smallconfigurational changes and provide insight into the exchange of violaxanthinby zeaxanthin by switching from low to high light conditions. This is in agree-ment with experiments and provides a molecular view on this importantprocess.

2581-Pos Board B597Increase in Dynamical Collectivity and Directionality of Orange Carot-enoid Protein in the Photo-Protective StateYanting Deng1, Catherine H. Luck1, Tod D. Romo2, Alan M. Grossfield2,Sepalika Bandara3, Zhong Ren3, Xiaojing Yang3, Andrea G. Markelz1.1Department of Physics, State University of New York-Buffalo, Buffalo, NY,USA, 2Department of Biochemistry and Biophysics, University of RochesterMedical Center, Rochester, NY, USA, 3Department of Chemistry, Universityof Illinois at Chicago, Chicago, IL, USA.Photo-protection is crucial for photosynthesis efficiency. Cyanobacteria haveevolved a unique photo-protection mechanism mediated by Orange Carot-enoid Protein (OCP). OCP binds a single ketocarotenoid as the chromophore,essential to its photo-protective function. Under strong green-blue (or white)illumination or high chaotrope concentration, OCP converts from the orangestate OCPO to the activated or photo-protective red state OCPR. The OCPR

facilitates dissipation of excess energy via direct interaction with allophyco-cyanin (APC) cores of the light-harvesting antenna Phycobilisome (PB). Pico-second intramolecular dynamics are critical to the photo-protectiveconformational switching, energy transfer between the APC and OCP, and en-ergy dissipation. In particular intramolecular vibrations at THz frequenciescan both provide efficient access to intermediate state conformations andcouple to embedded chromophore vibrations for energy dissipation. Herewe characterize global picosecond flexibility using temperature dependent ter-ahertz spectroscopy on OCP solutions. The THz absorbance decreases andstructural resilience increases in the photoactive state. The dynamical turnon temperature for picosecond dynamics shifts from 200K in OCPO to250K in OCPR, signifying a substantial increase in vibrational collectivityand structural stability. To characterize the nature of the intramolecular vibra-tions in more detail, we employ our recently developed techniquePolarization-Varying Anisotropic Terahertz Microscopy (PV-ATM). Thetechnique isolates specific vibrational bands associated with long range collec-tive motions of the protein structure. For the first time we demonstrate intra-molecular vibrational changes with photoexcitation. In particular we find anincrease in vibrational directionality in the photo-activated OCP in the 60-72 cm�1 and 85-100 cm�1 bands. In addition, the orientation of the vibra-tional motions switches for the 38-48 cm�1 band. We suggest that theincreased dynamical collectivity and directionality changes with photo-statecontribute to OCP efficiently binding and interacting with the APC complexto optimize photo-protective function.

2582-Pos Board B598Single-Molecule Measurements of Quenching and Photophysical Hetero-geneity in PhycobiliproteinsAllison H. Squires1, Peter D. Dahlberg1, Haijun Liu2,Robert E. Blankenship2, W.E. Moerner1.1Department of Chemistry, Stanford University, Palo Alto, CA, USA,2Departments of Biology and Chemistry, Washington University in St. Louis,St. Louis, MO, USA.Phycobilisomes, the membrane-associated light-harvesting antenna system ofcyanobacteria, dynamically adjust to changing irradiation by modulating en-ergy capture and transfer over a few seconds or longer, for example by geneticregulation, structural reorganization, or non-photochemical quenching by Or-ange Carotenoid Protein (OCP). Recent observation of excitation-dependentphotodynamics in single intact surface-immobilized phycobilisomes, including‘‘blinking’’ to dark or dim states, suggests that the phycobilisome itself

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may also possess intrinsic molecular mechanisms to regulate energytransfer. Here, we confine single phycobiliproteins in free solution to a smallmeasurement region for several seconds at a time using an Anti-BrownianELectrokinetic (ABEL) trap, and simultaneously monitor changes in multiplespectroscopic properties (brightness, fluorescence anisotropy, fluorescencelifetime, and emission spectrum). We are able to trap single intact phycobili-somes and isolated substructures thereof, including phycobilisomes quenchedby OCP. We find that even the smallest constituent phycobilisome subunitsexhibit surprisingly complex photodynamics. For example, in monomers ofC-phycocyanin (C-PC), a phycobiliprotein that contains three covalently boundand chemically identical chromophores, we observe reversible switchingamong at least seven distinct photophysical states (Squires and Moerner,PNAS 2017). The spectroscopic properties of these states and the transitionsamong them allow us to identify each state as a unique combination ofchromophores participating in energy transfer. A simple computationalFRET model closely predicts the observed states, and additionally revealsthat the primary acceptor chromophore in C-PC may sometimes act as aquencher. The complex photodynamics and quenching that we observe for in-dividual subunits of the phycobilisome are likely one source of the dynamicsobserved for the full phycobilisome complex, and may provide an intrinsicmechanism for short-timescale modulation of energy transfer to the reactioncenter.

2583-Pos Board B599Removal OF B800 Bacteriochlorophyll A from Two Structure-DeterminedLight-Harvesting Proteins 2 in Purple Photosynthetic BacteriaYoshitaka Saga1,2, Keiya Hirota1.1Department of Chemistry, Kindai University, Osaka, Japan, 2Presto, JapanScience and Technology Agency, Kawaguchi, Japan.The light-harvesting complex 2 (LH2) is a peripheral antenna protein in pur-ple photosynthetic bacteria. This protein possesses two types of bacteriochlo-rophyll (BChl) a termed B800 and B850. These BChl a pigments in the LH2protein play important roles not only in the photosynthetic antenna functionsbut also in folding and maintaining its protein structure. We report hereinremoval kinetics of B800 BChl a from two structure-determined LH2 proteinsderived from purple photosynthetic bacteria Rhodoblastus (Rbl.) acidophilusand Phaeospirillum (Phs.) molischianum (denoted acidophilus-LH2 and moli-schianum-LH2, respectively) as well as physicochemical properties of LH2proteins without B800 BChl a. B800 BChl a was released from molischia-num-LH2 much slower than acidophilus-LH2 under acidic conditions. Thedifference in the removal kinetics of B800 BChl a can be qualitatively ex-plained by the difference in interactions of B800 BChl a with surroundingamino acids in the B800 binding sites between two types of LH2. Based onthe results of removal kinetics of B800 BChl a, we successfully preparedLH2 proteins that selectively lacked B800 BChl a from native LH2 proteinsderived from Rbl. acidophilus and Phs. molischianum. CD spectroscopyindicated that the local structures around B850 BChl a and the contents ofa-helices were barely changed even if B800 BChl a was absent. ExogenousBChl a was reconstituted into both the LH2 proteins that selectively lackedB800 BChl a, and transfer the excitation energy to B850 BChl a in theseproteins.

2584-Pos Board B600Quantum Dot-based Fluorescence Resonance Energy Transfer throughExciton Dynamics in DNA-Templated J-AggregatesSarthak Mandal1, Xu Zhou2, Nour Eddine Fahmi2, Su Lin1,3, Hao Yan2,3,Neal Woodbury1,3.1Center for Innovations in Medicine at the Biodesign Institute, Arizona StateUniversity, Tempe, AZ, USA, 2Center for Molecular Design andBiomimetics at the Biodesign Institute, Arizona State University, Tempe,AZ, USA, 3School of Molecular Sciences, Arizona State University, Tempe,AZ, USA.Highly efficient and rapid exciton dynamics is crucial for building nanoscaleartificial photonic devices with tailored properties for energy related applica-tions. DNA-templated self-assembly of dyes has been shown as a powerfulapproach to gain controls and optimization of positions, interactions and dy-namics of the dyes. The investigation of exciton dynamics in closely packedmolecular J-aggregates has gained significant interests because their opticaland electronic properties often resemble with those of self-assembled biochem-ical molecules in nature. Here, J-aggregates of pseudoisocyanine (PIC) dyeformed spontaneously on the double stranded DNA template are used todevelop a FRET-based donor-J-aggregate-acceptor assembly. In this systemhighly emissive quantum dot is acting as FRET donor and Alexa Fluor 647as terminal acceptor. The donor and acceptor are placed in such a geometric

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arrangement that direct energy transfer from donor to acceptor is prohibited.The photophysical and dynamical properties of the DNA templated J-aggre-gates, as well as the donor-to-acceptor energy transfer through J-aggregatehave been investigated using steady-state fluorescence, time-resolved fluores-cence, and pump-probe absorption spectroscopic techniques. The efficiencyof this quantum dot-based donor-J-aggregates-acceptor ensemble system hasbeen further investigated by varying the length and structure of the J-aggregate,as well as compared with the system where a molecular dye, ATTO 390 hasbeen used as donor. The detailed fundamental understanding of this systemcan help develop more complex nanoscale devices using complex DNAorigami nanostructures.

Posters: Diffraction and Scattering Techniques

2585-Pos Board B601Development of Advanced Diffracted X-Ray Tracking for Single MoleculeIntra- Dynamics with Low Dose and Wide Angular Dynamic RangeHiroshi Sekiguchi1, Koki Aoyama1, Yuji C. Sasaki2.1Japan Synchrotron Radiation Research Institute, Sayo, Japan, 2TheUniversity of Tokyo, Kashiwa, Japan.Diffracted X-ray Tracking (DXT) is one of single molecule techniques forinvestigating internal motion of protein at single molecule level. In DXT, agold nanocrystal is used as motion probe and its motion is investigated by tra-jectory of diffraction spot in time resolved diffraction images. It can detectatomic-scale dynamic motion of the protein with several tens of microsecondstime resolution. Although DXT is expected as a powerful tool, the narrow dy-namic angular range (about one degree in BL40XU/SPring-8), and the X-rayradiation damage for the sample are big issues. The wider energy width ofincident X-ray enhances the dynamic angular range of the method; however,such incident X-ray has serious radiation damage for the sample. In this pre-sentation we report the following developing methods which we have beenworking on to solve problems described above. [A] Sample Angular Scanningtype DXT: Recording time resolved diffraction images with high-speed sam-ple angular scanning, and motions of diffraction spots are reconstructed byangular position information of the sample and its diffraction images (angulardynamic range broadened) [B] Diffraction Intensity Correlation SpectroscopicDXT: Analyzes self and cross correlation of diffraction intensity and derivesmotion of diffraction spots (incident X-ray with broad energy width is notnecessarily required) [C] Time of Arrival (ToA) imaging DXT: Using ToAfunction of TimePix detector, acquires diffracted photon information andderives diffraction spot motion. These methods were applied for motion anal-ysis of AChBP (acetylcholine binding protein) upon agonist biding, andangular motion enhancement were detected. We expect those methods to beable to trace a wide range of angle changes while minimizing damage tothe sample.

2586-Pos Board B602Novel In Vivo Observations of Single Protein Motions Using LaboratoryX-Ray SourceYuji C. Sasaki1,2, M. Kuramochi1,2, H. Sekiguchi3, K. Mio2.1The University of Tokyo, Kashiwa-city, Japan, 2AIST-UTokyoOperando-Oil, Kashiwa-city, Japan, 3SPring-8/JASRI, Hyogo, Japan.In 1998, we proposed a method to observe intramolecular motions by labelinggold nanocrystals with individual single protein molecule and observing themotions of diffracted X-ray spots from labeled individual nanocrystals. ThisDXT (=Diffracted X-ray Tracking) can trace all rotational motion within singleprotein molecule using white X-rays1-5. When using monochromatic X-rays, itis impossible to track all motions of diffraction spots. However, we detected aclear blinking in diffracted X-ray intensity. Now, we call Diffracted X-ray Blin-king(DXB). The observed X-ray blinking intensity from the labeled and mov-ing gold nanocrystals correlated with the velocity of the diffraction spots byautocorrelation function(ACF). Additionally, the standard deviation of the X-ray blinking intensity distribution shifts to the larger side than when thosestopped. Recently, we developed this technique to observe the molecular dy-namics using laboratory X-ray source; Rigaku FR-D (Cu anode, 50kV,60mA) and a high sensitive detector; PILATUS-100K. We try to distinguishmolecular dynamics of AChBP between with or without toxin. In the meeting,we report the details of our analytical laboratory technique. In order to analyzethese motions, the autocorrelation ACF of the diffracted x-ray intensity fromadsorbed gold nanocrystals is calculated. We have recently succeeded in beingable to measure in vivo single molecular observations with DXB even with alaboratory x-ray source for the first time in the world. X-ray blinking is avery efficient and effective measurement means for acquiring dynamic

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information in physical or chemical phenomena on a wide area time scale. 1.Phys. Rev. Lett., 87, 248102(2001). 2. Cell, 132, 67-78, (2008). 3. ScientificReports 4, 6384 (2014). 4. Biophy. J., 108, 350(2015). 5. Scientific Reportsin press (2017).

2587-Pos Board B603Dynamics of Multicellular Assemblies Measured by Coherent LightScatteringBenjamin Brunel.Laboratoire Interdisciplinaire de Physique, Saint-Martin-d’Heres, France.Multicellular aggregates, or spheroids, represent an interesting model to studytumor response towards external stresses. The question of cell organization andflows inside spheroids revealed to be a challenging topic as usual imaging tech-niques are limited in terms of penetration length and/or time resolution. Toanswer this question, we developed a new method based on coherent light scat-tering. First, from static light scattering, we deduce the average cells size andspatial organization (spatial auto-correlation). Secondly, from dynamic lightscattering, we deduce cells displacements histogram as a function of time.This technique gives statistical results over a large field of view, inside spher-oids as thick as 400 mm. In comparison to standard microscopy, no dyes areneeded and analysis does not require the usual processing (segmentation,tracking,/) which can sometimes lead to biases. Application to the study ofosmotic pressure effect on spheroids showed that cells movements are signifi-cantly reduced under pressure. This 3D emergent property seems related to therole of extra-cellular matrix.

2588-Pos Board B604Ultra-Efficient Micromirror Total Internal ReflectionMicroscope with nmSpatial Precision and Microsecond Temporal ResolutionXuanhui Meng, Daniel Cole, Gavin Young, Anne Schumacher,Philipp Kukura.Department of Chemistry, University of Oxford, Oxford, United Kingdom.Total internal reflection fluorescence (TIRF) microscopy has been widelyused in single molecule tracking and imaging for more than 20 years dueto its remarkable background suppression and high axial resolution. Someintrinsic photophysical properties of fluorescent labels, however, such as pho-tobleaching and photoblinking, as well as the presence of biological auto-fluorescence, impede the application of TIRF to particle tracking problems,especially those requiring high spatiotemporal precision. Here, we present amicro-mirror total internal reflection dark field scattering microscopy withunprecedented levels of background suppression and spatiotemporal resolu-tion. Our approach uses micromirror-based coupling of incident andreflected illumination light with optimised mirror diameters and objectivemirror separation. Using this approach, we demonstrate tracking of goldnanoparticles down to 20 nm diameter with simultaneous nm precisionand temporal resolution on the order of 5 microseconds at incident power den-sities comparable to single molecule fluorescence imaging, opening up newavenues to studying structural dynamics and motion of biological macromol-ecules. We demonstrate the capabilities of our instrument by single particletethered motion of nanoscale DNA and high-speed tracking of molecularmotors.

2589-Pos Board B605The High Resolution Diffraction Beamline P08 at Petra III ExpandedTowards a Platform for Structure Characterization of Organic LiquidSurfaces - Results from Lipid MonolayersFlorian Bertram1, Gerald Brezesinski2, Olof Gutowski1, Beate Klosgen3,Milena Lippmann1, Uta Ruett1, Chen Shen1.1Deutsches Elektronen-Synchrotron Desy, Hamburg, Germany, 2Max PlanckInstitute of Colloids and Interfaces, Potsdam, Germany, 3University ofSouthern Denmark, Odense, Denmark.Lipid monolayers are widely used as model systems for biomembranes. Theirstudy yields precious knowledge about the materials used, and may serve to un-derstand some functions of biomembranes. Grazing Incidence X-ray Diffrac-tion (GIXD) and Total Reflection X-ray Fluorescence (TRXF) experimentsreport on structural details, meaning the lateral organisation of the materialon atomic scale, and even atomic composition. The analysis of GIXD data pro-vides the crystallographic in-plane order of the film, and, depending on the en-ergy used, TRXF gives information about the presence of specific species, e.g.ions, at the interface.The High Resolution Diffraction beamline P08 at PETRA III was expanded tooffer combined GIXD-TRXF measurements on organic monolayers at liquidsurfaces. This is achieved by a mirror that bends the X-ray beam (at �8-25keV) to an incidence angle below the critical angle of reflection. Here, wepresent two example studies on lipid systems illustrating potential applications

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for the new setup. The studies involve pure and mixed phospholipid andglycosphingolipid monolayers, studied at different surface pressures, subphasepH, and in the presence of ions. The acquisition of the TRXF signal allows ac-cessing the concentration of ions near the surface, and hence the ionization de-gree of lipids. Combined GIXD-TRXF experiments were done to correlatelateral structures with the electrostatic properties of partially charged lipidmonolayers.

2590-Pos Board B606NE-CAT: Crystallography Beamlines for Challenging Structural BiologyResearchSurajit Banerjee, Malcolm Capel, Igor Kourinov, Anthony Lynch,Frank Murphy, David Neau, Kay Perry, Kanagalaghatta Rajashankar,Cynthia Salbego, Jonathan Schuermann, Narayanasami Sukumar,James Withrow, Steve Ealick.Chemistry and Chemical Biology, Cornell University / NECAT, Lemont, IL,USA.The NorthEastern Collaborative Access Team (NE-CAT) focuses on the designand operation of synchrotron X-ray beamlines for the solution of technicallychallenging structural biology problems and provides an important resourcefor the national and international research community. Currently, NE-CAT op-erates two undulator beamlines: a 6 - 22 keV tunable energy beamline (24-ID-C) and a 12.662 keV single energy beamline (24-ID-E). Both beamlines areequipped with state-of-the-art instrumentation. MD2 microdiffractometersinstalled at both beamlines provide very clean beams down to 5 microns indiameter and are capable of visualizing micron-sized crystals. Large area pixelarray detectors (Pilatus6M and Eiger16M) provide fast noiseless data collectionand make possible it to resolve large unit cells. Both beamlines are equippedwith custom-built ALS-style robotic sample automounters with dewars capableof holding 14 pucks. Our locally developed software suite RAPD provides datacollection strategies, quasi-real time data integration, scaling and simple auto-mated MR/SAD pipeline through a 384-core compute cluster. Towardsimproving the diffraction from low resolution crystals, NE-CAT has installedhumidity controlled device, HC1 in the 24ID-E hutch. Users of the beamlinesare supported 24/7 by experienced resident crystallographers. Funding for NE-CAT is provided through a P41 grant from the NIGMS and from the NE-CATmember institutions.

Posters: Molecular Dynamics II

2591-Pos Board B607A Dynamical Model for Insulin Degrading Enzyme Conformational Tran-sition between Closed and Open StatesMichael F. Cronin1, Wookyung Yu2, Wei-Jen Tang3, Esmael J. Haddadian4.1Department of Mathematics, University of Chicago, Chicago, IL, USA,2Physics, Pusan National University, Pusan, Republic of Korea, 3Ben-MayDepartment for Cancer Research, University of Chicago, Chicago, IL, USA,4Biological Sciences Division, University of Chicago, Chicago, IL, USA.Insulin Degrading Enzyme (IDE) is involved in the deconstruction of insulin,amylin, and glucagon peptides involved in controlling blood glucose leveland has also been found to break down amyloid-b, a peptide involved in Alz-heimer’s disease. IDE has two homologous N- and C-terminal domains, IDE-Nand IDE-C. For IDE to degrade its substrates, both domains must come togetherfor the catalytic cleft to assume closed conformation. New Cryo-Electron Mi-croscopy (Cryo-EM) data suggests IDE passes through five conformations:completely closed with substrate bound, partially closed with substrate bound,partially open with substrate unbound, and open without substrate. Dynamicaldetails of how these conformations connect are unknown. We investigated thisprocess using long all atom molecular dynamics simulations (MD) of IDEmonomer and dimer structures with and without substrate. Our simulationsshowed that IDE without insulin (pdb-code 4IOF) has larger relative motion be-tween four subdomains (D1, D2, D3, D4) in the N- and C-terminal domainscompared to an IDE with insulin (pdb-code 2WBY). Analysis of the insulin in-side the catalytic chamber suggested that insulin was stabilized by interactionsof its residues Gly1, Tyr14, Glu17 form its chain A and Phe1 and Ph25 from itschain B with IDE residues via a combination of hydrogen bonding and p - pinteractions. We used, Essential Dynamics Analysis (EDA) to calculate prin-cipal normal modes of IDE motion from our trajectories. These normal modeswere then applied as coordinate transformations combined with short MD sim-ulations to gradually open the IDE-closed conformation. We used cross-comparison with Cryo-EM and hydrogen-deuterium exchange data of IDE asa guide to obtain a realistic model of how IDE changes between closed andopen conformations.

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2592-Pos Board B608Characterization of the Clustering of PI(4,5)P2-Clusters: All-AtomMolec-ular Dynamics Simulations and Graph-Theoretic AnalysisKyungreem Han1, Richard M. Venable1, Katrice McLoughlin2,Arne Gericke3, Richard W. Pastor1.1NHLBI, NIH, Bethesda, MD, USA, 2Department of Biology, WashingtonUniversity in St. Louis, St. Louis, MO, USA, 3Department of Chemistry andBiochemistry, Worcester Polytechnic Institute, Worcester, MA, USA.Phosphatidylinositol (4,5)-bisphosphate (PIP2) is a lipid in cell membranesthat is critical to a broad range of cellular processes. Its versatile functionsare rooted in both the rich chemical functionality of the phosphoinositideheadgroup and the cooperative action of lipid clustering. However, thedesign principles of PIP2-clusters and their behavior in biological membranesremain unexplored. This study focuses on the identification of the factorswhich induce or stabilize PIP2-clusters using all-atom molecular dynamicssimulations and graph-theoretic analysis. Special attention is paid to theelectrostatic interactions between phosphate groups at PIP2 headgroup andcations and the intra- or intermolecular hydrogen bond formations. Further-more, hypotheses which address the roles of cholesterol in the hydrogenbond network formation are tested by both experiments and simulations ofpure PIP2 and PIP2-rich mixed monolayers. Characterizing such structuraland dynamical properties of PIP2 clustering using simple model membranescould provide fundamental knowledge for a better understanding of the mul-tiplicity and context-dependency of the function of PIP2-clusters in biologicalmembranes.

2593-Pos Board B609The Role of Hydrophobic Interactions and Water Dynamics AroundDystrophin Spectrin RepeatsSarah Moe, Alessandro Cembran.Department of Chemistry and Biochemistry, University of Minnesota Duluth,Duluth, MN, USA.Dystrophin is a cytoplasmic protein that provides stability to the membrane ofmuscle cells by dissipating mechanical stress during muscle contraction andrelaxation. Mutations in the dystrophin gene lead to various forms of musculardystrophy, for which there is currently no cure and in the most severe formslead to premature death. The design of therapeutics is hindered by the lack ofstructural information of full dystrophin, and by the fact that dystrophin’smechanism of function is largely unknown. We hypothesize that dystrophindissipates mechanical energy by undergoing an order-to-disorder transition,during which buried hydrophobic regions are partially exposed to solvent,and transduction of energy leads to an increase in entropy of surroundingwater molecules. To test this hypothesis, we have generated a structuralmodel of a large portion of dystrophin using homology modeling. Next, wehave focused on understanding the role played by methionine-aromatic hydro-phobic interactions, which we identified being present in some but not allspectrin repeats. Our all-atom molecular dynamic simulations show thatintra-spectrin methionine-aromatic interactions provide an additional stabili-zation of 1-2 kcal/mol when compared to similar interactions that do notinvolve sulfur. Additionally, we have investigated the mechanism of unfold-ing in spectrin monomers using computational pulling with steered moleculardynamics. The evaluation of order parameters throughout pulling trajectoriesindicates enhanced orientational ordering of water molecules as hydrophobicresidues become accessible to surrounding solvent. Together, these resultsprovide insight into a possible mechanism by which dystrophin transducesmechanical stress, and present a compelling motif for further mechanisticanalysis.

2594-Pos Board B610Solvent Ions Adversely Affect Binding of pHLIP to Bilayer SurfacesChitrak Gupta, Blake Mertz.Chemistry, West Virginia University, Morgantown, WV, USA.The pH (Low) Insertion Peptide (pHLIP) is an acid-sensitive cell-penetratingpeptide with potential for applications in targeted delivery of cargoes to acidictissues (e.g., cancer cells). pHLIP binds to cell membranes in a coiled confor-mation under neutral/alkaline conditions; upon acidification, the peptide foldsinto an a-helix and unidirectionally inserts into the membrane. Although thegeneral mechanism of pHLIP is known, the molecular interactions that leadto binding of pHLIP to the cell membrane surface are poorly understood. Inthis study, our aim is to determine the role that salt concentration has on theability of pHLIP to bind to a bilayer surface, as most experimental studies onpHLIP are carried out at low buffer concentration. We employed Gaussianaccelerated molecular dynamics (GaMD) simulations [1] to model the binding

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of pHLIP to a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)bilayer. At low salt concentration, our results agree well with a recent solid-state NMR study where it was determined that the N-terminal half of pHLIPbinds more effectively to the bilayer surface [2]. At physiological salt concen-tration (150 mM), we observe a significant decrease in effectiveness of binding.This decreased binding affinity is a combination of electrostatic (i.e., increasedcompetition from ions for complementary binding sites within the PC head-groups) and steric (i.e., decrease in area per lipid) effects. Thus, our study sup-ports the hypothesis that ionic strength plays a role in the function of pHLIP.These results are relevant to our understanding of environmental influenceson the ability of pHLIP to bind to the cell membrane, and are useful in under-standing the binding behavior of other anionic cell-penetrating peptides. [1]Miao, Journ. Chem. Theor. Comput., 11:3584 (2015); [2] Shu, Nat. Comm.,6:7787 (2015)

2595-Pos Board B611Solution Properties of Complex Shape PolymersBeatriz Pazmino Betancourt.NIH, Gaithersburg, MD, USA.Glycosaminoglycans (GAG) of aggrecan are important in the lubricationprocess in joints and constitute the main component in bearing loads.Cartilage is composed mainly by proteoglycans (PGs) which are the mostcommon GAG molecules. In particular, the negative-charged aggrecangoverns the osmotic pressure and the load-bearing property of cartilage.PGs are known to form complexes with a bottle brush topology when theyinteract with hyaluronic acid (HA). Here, we study systematically thesolution properties of a coarse-grained model for bottle brushes in differentsolvents by a combination of molecular dynamics simulations and pathintegration calculations. In particular, we compute the radius of gyration,hydrodynamic radius, and intrinsic viscosity of the polymer brushes,which will be compared with experimental scattering measurements of theGAG.

2596-Pos Board B612Rapid Folding of Trp-Cage in Ionic Liquid: Implications in Protein Rena-turationsMohammad H. Rahman, Kalpanna Manne, Sanjib Senapati.Biotechnology, Indian Institute of Technology, Chennai, India.Ionic Liquids (ILs) have emerged as ideal solvent media for enzymatic reac-tions and enhanced protein stability. Studies have also shown the ability ofILs in renaturation of unfolded proteins. Here, we explore the mechanism ofprotein refolding in ILs by simulating the miniprotein, Trp-cage in varying con-centrations of 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim][BF4])using all-atom Molecular Dynamics (MD) simulations. Our results show thatTrp-cage folded to its native conformation very rapidly in low IL concentra-tions, while it fails to fold in neat water or high IL concentrations. This rapidfolding is attributed to the strong IL ions-water interactions that help in strip-ping significant amount of water from the unfolded protein surface. Further,the strong electrostatic interactions between IL cations and anions and the spe-cific interaction of [Emim] cations with protein N-terminal residues and [BF4]anions with C-terminal residues facilitate the protein hydrophobic collapse.Interestingly, at higher IL concentrations protein was unable to fold due to sig-nificant reduction in the static dielectric constant of the medium that enhancedion-ion associations.

2597-Pos Board B613Electric Fields and Fast Protein Dynamics in EnzymesIoanna Zoi, Steven Schwartz.University of Arizona, Tucson, AZ, USA.In recent years there have been debates about the nature of enzymatic catalysisand whether including protein dynamics is necessary for understanding cata-lytic enhancement or whether electrostatic preorganization of the active siteis its sole origin. An important contribution in this debate was made with theapplication of the vibrational Stark effect for measuring electric fields in theactive site, which provided an atomistic description of the particular interac-tions that lead to this preorganization. We performed computational studieson two enzymes where we have shown that fast dynamics is part of the reactionmechanism and calculated the electric field near the bond breaking event. Wefound that the fast motions we had identified lead to an increase of the electricfield, thus preparing an enzymatic configuration that is electrostatically favor-able for the catalytic chemical step. This shows that there is not necessarily adisagreement between the electrostatic and dynamical views on the enzymaticmechanism.

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2598-Pos Board B614A Multi-Scale Model for Insulin Self-Association Rates and Oligomeriza-tion KineticsRit P. Mishra, Richa Singh, Tirumalarao Kotni, Gaurav Goel.Chemical, Indian Institute of Technology Delhi, New Delhi, India.Insulin, a therapeutic protein, can aggregate during manufacture and is consid-ered detrimental to its quality. Early identification of aggregation inducing fac-tors would be an essential aspect concerning the design of mitigation strategiesduring manufacturing processes. The time-scales and length-scales relevant foraggregation makes the use of all-atom (AA) explicit solvent simulations forprediction of aggregation thermodynamics and/or kinetics very difficult. Sinceaggregation is mainly driven by non-native interactions, we have used the phys-ics based MARTINI coarse grained model to model protein-protein interac-tions. In an earlier work we had identified aggregation-prone, partially foldedintermediates (PFIs) of insulin. Here we have employed a transient-complexmodel to determine self-association rate of native insulin (N) and the PFIs ofinsulin. To this end, a set of most probable conformations of the N-PFI complexwere first obtained using docking and scoring on basis of MMPBSA energies.These docked complexes were then refined by separating the N and PFI mono-mers translationally followed by long molecular dynamics simulations. In twosets of test cases we show that the binding interface of the final N-PFI complexis similar in both the AA simulations and CG-MARTINI simulations withelastic network constraints. We hypothesize that this N-PFI complex can betaken as the diffusion limited transient state for the insulin homodimer on ag-gregation pathway. We use the TransComp server to calculate association ratesfor formation of this diffusion limited complex. The dimer association rates andmonomer conformational transition rates are used to determine all the rates forformation of various small oligomeric species on insulin aggregation pathway.These rates are then input to a kinetic monte carlo (KMC) scheme to determineoligomerization kinetics of insulin.

2599-Pos Board B615Accurate Refolding of Experimentally Determined Protein MechanicalUnfolding Intermediates via All-Atom Molecular Dynamics SimulationsDavid Wang1, Piotr Marszalek2.1Physics, Duke University, Durham, NC, USA, 2Mechanical Engineering andMaterials Science, Duke University, Durham, NC, USA.One of the major goals of all-atom molecular dynamics simulations is torecreate the trajectories of proteins as they fold into their native state. Currently,one of the main methods to validate the accuracy of these folding trajectories isto observe whether the simulated unfolded protein eventually obtains a struc-ture similar to its native conformation. However, it is unknown whether thefolding trajectories produced by simulations accurately represent the foldingpathways actually undertaken by the protein. Thus, incorporating experimen-tally known protein intermediates into these simulations can provide a newmeans to verify the accuracy of these refolding trajectories. We simulatedthe refolding trajectories of titin I91 (I27) domain and consensus ankyrin repeatstructures, NI3C, from their experimentally perturbed (partially denatured) me-chanical unfolding intermediate states generated using atomic force spectros-copy and witnessed the refolding of these intermediates into their finalprotein structures. In doing so we show that we can accurately reproduce theexperimentally-determined partial folding trajectories of proteins using molec-ular dynamics simulations. The creation of these trajectories then provides anew way to test the accuracy of the molecular dynamics force fields and mayprovide valuable information regarding the folding landscape of many proteinsunder investigation.

2600-Pos Board B616Ring Opening Mechanism of Epoxide Inhibitors in Aspartate Proteases: AQM/MM StudyMohd Ahsan, Sanjib Senapati.Biotechnology, Indian Institute of Technology, Chennai, India.Aspartate proteases are reported to be involved in several human diseasesand are potential drug targets. All exisiting drugs/inhibitors against HIVand other proteases bind reversibly and exhibit strong drug-resistance. Irre-versible inhibition through the formation of covalent linkage with proteaseactive site residues by a series of epoxide based compounds has been testedover the decades to combat drug resistance. However, the detailed mecha-nism of epoxide ring opening in protease active site is not yet understood.Here we explore this mechanism in two common aspartate proteases,HIV-1 and pepsin by means of hybrid quantum mechanical and molecularmechanical (QM/MM) methods. Results show that epoxide ring opening fol-lows a unique pathway involving two steps similar to a typical SN1 reaction,but produces transition state (TS) and intermediates that advocate for SN2 ki-netics. More interestingly, the water molecules were found to act as hydrogen

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bond catalyst in the reaction. The detailed understanding and the obtainedstructures of TS and reaction intermediates could pave way for new genera-tion irreversible protease drugs.

2601-Pos Board B617Free-Energy Landscape of Amyloid-Beta PeptidesApichart Linhananta.Physics, Lakehead University, Thunder Bay, ON, Canada.It is well known that Alzheimer’s disease (AD) is correlated with the aggrega-tion of amyloid-beta (Ab) peptides into plaques. Due to the large size of Abplaques (several mm), and long incubation period (hours and days), MolecularDynamics (MD) simulations require coarse-grained models. Our model isbased on the coarse-grained model of Fawzi et al [Biophysical J. 94 (2008)2007-2016] that represents one amino acid as a single unit. However, thenew model will include an improved representation of backbone-hydrogenbonds that stabilize the b-sheet structures of plaques. The van der Waals forceinteractions used in the model will be based on published NMR crystal struc-tures of Ab segments resolved by the Eisenberg group [Nature 447 (2007)453-457]. MD simulations observed a rugged energy landscape, withcooperative monomer state to oligomers state, as the temperature is lowered.This is followed at lower temperature, by trapped states that lead slowly to crys-tal structures. Transition states and trapped states are identified using a metricthat includes the radius of gyration of number of backbone hydrogen bonds.

2602-Pos Board B618The Change in Reaction Coordinate Induced by Directed Evolution ofSynthetic EnzymesXi Chen.Department of Chemistry and Biochemistry, University of Arizona, Tucson,AZ, USA.Rate promoting vibrations (RPVs) are fast protein motions coupled with the re-action coordinate of enzymes, which promote the rate of the chemical step ofenzymatic reactions. Evidence has shown that RPVs exist in a variety of en-zymes. This study investigates the behavior of RPVs in three variants alongthe directed evolution(DE) process of artificially designed enzyme Kemp Elim-inase 59 (KE59). Through this study, we wish to gain an understanding of howthe directed evolution process affects RPVs. Also, we would wish to learnabout protein motions in artificially designed enzymes, and utilize this knowl-edge for further enzyme design. In this study we employ, R1-7/10H, the startingpoint of DE, R5-11/5F, an intermediate stage of DE and R13-3/11H, the endingpoint of DE. Molecular dynamic (MD) trajectories using QM/MM potentials ofthe chemical step of the reaction are acquired using the Transition Path Sam-pling (TPS) method. Transition states of trajectories are determined by theCommittor Analysis method. A Committor Distribution Analysis method isapplied to search for the reaction coordinates of each enzyme. We have suc-ceeded in acquiring an ensemble of reactive trajectories for each enzyme,and have found transition state structures for each enzyme. Trajectories anal-ysis and Transition states structures have revealed a change of the reactionmechanism during the directed evolution process. We have not yet found thecomplete reaction coordinate for each enzyme. Current progress in Committordistribution analysis indicate that the reaction coordinate R1-7/10H follows aconstrained reaction tube in phase space, whereas the reaction coordinates ofR5-11/5F and R13-3/11H contain many more degrees of freedom. This resultis an indication of the presence of RPVs in R5-11/5F and R13-3/11H, andwill thus be the focus of future work.

2603-Pos Board B619Computational Analysis of Small Biological Molecules as a PhysicsProblemYuly E. Sanchez, Jose M. Jimenez.Physics, National University of Colombia, Bogota, Colombia.More than 50 years ago the protein-folding problem was posed trying to under-stand different questions about how can proteins fold so fast? and How to find acomputer algorithm to predict computer structures from their sequences?,among other relevant issues in this topic. In a limited number of cases, com-puter simulations of the physical forces in some chemically detailed modelshave now succeeded the accurate folding of small proteins. There has beenextensive and ongoing work done on biological molecules computationalmodeling prepared by physics undergraduate students, with the aim to getthem interested into biological systems analyzed at multiple levels as theapproach of complex self organizing systems. Using this premise, we per-formed a computational analysis of a peptide that has a biological importanceas possible drug target, and we was able to find a range of conformers that wecan report as potential solution for the biochemical experimental data fromphysics point of view.

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2604-Pos Board B620Colloidal Nanoparticle Translocation through Nanopores: Effect ofExternal Electric FieldNazar Ileri-Ercan.Chemical Engineering, Bo�gazici University, _Istanbul, Turkey.Driving nanoparticles through nanopores in the presence of an external electicfield is critical to understand for efficient biomedical and biodefense applica-tions. It is known that the rate at which particles transit the nanopore is highlydependent on the electric field, which is in turn a function of both the appliedvoltage and the pore geometry. Models developed in this context accuratelydescribe transport through high aspect ratio pores, but are not sufficient toexplain behavior in technologically-important lower-aspect ratio pores whereless pore clogging is experimentally observed. In particular, experimentally re-ported lower capture rates and larger current drops are unexpected. In thisstudy, a colloidal nanoparticle translocation through mid-to-low aspect rationanopores under an external field is investigated by atomistic molecular dy-namics simulations. Our preliminary results show that not only the nanoporesize, but also the nanoparticle size play a critical role on ion dynamics.

2605-Pos Board B621Investigating the Dynamics of Designed Ligand-Binding ProteinsEmilia Pecora de Barros, Rommie E. Amaro.Chemistry and Biochemistry, University of California San Diego, La Jolla,CA, USA.Protein design constitutes a challenging but promising area in biochemistry.Custom-designed ligand-binding proteins, in particular, present promising ap-plications in small-molecule sensing, diagnostics, and in therapeutic scav-enging of toxic compounds. Some of the challenges in designing ligand-binding proteins lie in the creation of a binding site that results in high affinityinteraction but is also pre-organized and does not collapse in the absence ofligand. Here, we use molecular dynamics simulations to assess the cavity sta-bility and binding affinity of designed proteins that have been tested experimen-tally. Our apo simulations provide information on the cavity pre-organizationwhile holo simulations indicate protein-ligand affinity and correlate wellwith experimental observations. The results obtained can be used to guidefurther steps in protein design and to select appropriate metrics for the predic-tion of binding affinity of future un-tested proteins, aiding the protein designmethodology.

2606-Pos Board B622The Localization of Biological Compounds on the Soft Interface of Micro-droplet May Answer the Accelerated Reaction Rates Inside MicrodropletSangMoon Lhee1, Sunhee Kim1, Hong Gil Nam1,2.1Center for Plant Aging Research, Institute for Basic Science, Dalseong-gun,Daegu, Republic of Korea, 2Department of New Biology, Daegu GyeongbukInstitute of Science and Technology, Daegu, Republic of Korea.Many researchers have reported that microdroplet environment is a good reac-tion bath providing the remarkably accelerated chemical reaction rates and isdirecting the reaction path differently from the corresponding bulk-phase reac-tions. Since the related thermodynamic parameters gave the clues to the unevendistribution of molecules inside the small-compartmentalized space, we havevisualized the localization of several biological compounds inside the aqueousmicrodroplet including proteins, nucleic acids, and carbohydrates with the helpof some fluorescence microscopy techniques. Now, we present the localizationof molecules on the water-oil interface is common under physiological condi-tion and the charge states of molecules and interface contributes to the variationof the distribution. The observation of the molecules on the interface by meansof fluorescence anisotropy gives us some suggestions that the molecular move-ment on the interface is not only spatially restricted, but is also guided withorientation. We suggest that such the guided movements with some degreeof order is another contribution factor to accelerating the unfavorable reactionincluding enzymatic reactions.

2607-Pos Board B623Diffusion of Proteins and Lipids in Membranes Corrected for Finite-SizeEffectsMartin Vogele, J€urgen Kofinger, Gerhard Hummer.Department of Theoretical Biophysics, Max Planck Institute of Biophysics,Frankfurt, Germany.Translational diffusion coefficients are primary reporters on the dynamics inlipid membranes. However, diffusion coefficients of lipids and membrane pro-teins calculated in molecular dynamics (MD) simulations suffer from severefinite-size effects. We show that these finite-size effects can be quantified interms of hydrodynamic theory and develop accurate correction formulas. Usinglarge-scale coarse-grained MD simulations of up to 132 million particles, weassess the effects of box size and shape on translational diffusion in lipid mem-

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brane simulations. For the flat periodic boxes commonly used for membranesimulations, we find that the diffusion coefficients of lipids and membrane pro-teins increase logarithmically (and therefore without bound) with the boxwidth. Only if the box size is increased also normal to the membrane surface,the diffusion coefficient converges. This dependence on both the size and theshape of the simulation box is captured quantitatively by a hydrodynamic the-ory. The corresponding correction formulas make it possible to perform mean-ingful comparisons of diffusion coefficients from simulations with differentbox sizes, and facilitate the comparison to experiment.

2608-Pos Board B624Thermodynamics of Membrane Partitioning and Folding of an AnionicCell-Penetrating PeptideAustin R. Clark, Zachary Bonham, Blake Mertz.C. Eugene Bennett Department of Chemistry, West Virginia University,Morgantown, WV, USA.Application of cell-penetrating peptides for selective drug delivery has the po-tential to increase drug distribution and decrease toxicity by lowering dosagesrequired to achieve a therapeutic effect. pH (Low) Insertion Peptide (pHLIP), a36-amino acid anionic peptide, has the ability to selectively penetrate cellmembranes under acidic conditions. Spontaneous, unidirectional insertion ofpHLIP proceeds through three pH-dependent states: 1) coiled pHLIP binds tothe plasma membrane surface; 2) upon acidification, pHLIP folds into an alphahelix on the membrane surface; and 3) pHLIP then inserts across the mem-brane. The binding process is accompanied by a �7.2 kcal/mol release of en-ergy that is followed by an additional release of �2.0 kcal/mol upon foldingand insertion [1]. Theory predicts that partitioning of pHLIP into a membraneis favorable only when either 1) a predominantly nonpolar segment of the pep-tide binds or 2) the acidic residues are titrated, lowering the change in free en-ergy of binding from þ1.52 to �7.74 þ/- 2.24 kcal/mol [2]. A detailed pictureof the molecular interactions between pHLIP and the cell membrane is criticalto bridging the existing gap between theoretical prediction and experimental re-sults. Using a series of enhanced sampling molecular dynamics techniques, wehave characterized the thermodynamic cycle of binding and folding of pHLIP.Folding of pHLIP in solution is energetically unfavorable, and binding ofpHLIP to a 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC) bilayer ismost favorable when the peptide is helical and acidic residues are titrated.Our study builds the initial framework for understanding the thermodynamicsof pHLIP-membrane interactions, providing a unified model between theory,simulation, and experiment that can be broadly applied to cell-penetrating pep-tides. [1] Reshetnyak et al. (2008) PNAS 105:15340. [2] Wimley and White(1996) Nat. Struct. Mol. Biol. 3:842.

2609-Pos Board B625Molecular Simulations of Lipid Electropore Formation and Pore-Mediated Calcium Transport with an Improved Ca2D ModelFederica Castellani1,2, P. Thomas Vernier1.1Frank Reidy Research Center for Bioelectrics, Old Dominion University,Norfolk, VA, USA, 2Biomedical Engineering Institute, Frank Batten Collegeof Engineering and Technology, Old Dominion University, Norfolk, VA,USA.Molecular dynamics simulations of lipid membranes are widely used to facil-itate the understanding of phenomena at the atomic and molecular level thatcannot be observed with conventional experimental methods. Molecular dy-namics tools are particularly useful for the analysis of electric-field-inducedpore formation in lipid bilayers.Atomic and molecular interactions in molecular dynamics simulations are gov-erned by sets of properties and functions called force fields. For our studies ofmembranes in electric fields, to better understand how the physical and me-chanical properties of the membrane constituents and the interactions amongthem are influenced by the force field, we have compared properties such asarea per lipid, lipid order parameter, ion coordination number, and ion binding,specifically for the older GROMOS-OPLS and the newer CHARMM36 forcefields. During these comparisons we noticed significant deficiencies in theCHARMM36 Ca2þ model.Here we describe the unacceptable behavior of the standard CHARMM36 Ca2þ

model in aqueous systems, and we propose modifications to the model thatresult in more realistic interactions between Ca2þ, water, and phospholipids.We also present initial results from simulations of pore-mediated ion transportin these systems. We track the electric field- and diffusion-driven passage ofions through field-stabilized pores over time, calculate the resulting currentsand conductances, and relate these transport properties to the pore geometry.We note, among other things, that equilibration of Ca2þ with a phospholipidbilayer takes at least 10 ms, a much longer time than would be expected frompublished simulation studies of ion binding to phospholipids.

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2610-Pos Board B626Comparing Structure Stability between Earth and Subsurface Ocean onTitan using Molecular Dynamics SimulationKyle Martin1,2, Shannon MacKenzie1, Jason Barnes1, F. Marty Ytreberg1,2.1Department of Physics, University of Idaho, Moscow, ID, USA, 2Center forModeling Complex Interactions, University of Idaho, Moscow, ID, USA.Models of Titan predict that there is a subsurface ocean of water and ammoniaunder a layer of ice. This model could prove important in the search for extra-terrestrial life due to its prediction of a potentially habitable environment. Inthis study, we used molecular dynamics simulations to study proteins in Earthand Titan-like conditions, focusing on the most common secondary structuretypes: alpha helix and beta sheet. The Earth environment was simulated usinga temperature of 300 K, a pressure of 1 bar, and water. The Titan environmentwas simulated using a temperature of 300 K, a pressure of 1000 bar, and aeutectic mixture of water and ammonia. We analyzed protein compactness,flexibility, and backbone dihedral distributions to identify differences betweenthe two environments. Proteins in the Titan environment were more compactand less flexible, and have small differences in backbone dihedral preferences(e.g., in one instance a stable p-helix formed). These differences could impactaffinities between these proteins and other biomolecules.

2611-Pos Board B627A Computational and Experimental Study of Crystallization-DrivenSelf-Assembly and Micelle Formation in Poly(Ethylene Glycol)-B-Oligo(Ethylene Sulfide)Emre S. Sevgen, Juan J. de Pablo, Jeffrey A. Hubbell.Institute for Molecular Engineering, University of Chicago, Chicago, IL, USA.Using a combination of experimental, theoretical and computational methods,we study the structure and properties of semi-crystalline oligo(ethylenesulfide)-b-poly(ethylene glycol) micelles. As few as three ethylene sulfidemonomers are sufficient to form a highly crystalline core, surrounded bywater-soluble ethylene glycol corona of arbitrary size. Sulfur-sulfur interac-tions induce formation of rhombohedral lattice crystalline regions in thecore, having well-defined intramolecular and intermolecular ordering. Anatomistic model is used to determine the free energy of the micelles; forOES3-b-PEG10, we report evidence of critical micelle concentration (CMC)-like aggregation behavior, with a CMC of 1.4E� 8 mol/L. The size distributionof these micelles is near-monodisperse and invariant to large changes in con-centration. The unprecedented stability and small size (6.8nm diameter) thatcrystallinity confers to these micelles, coupled to the existence of amorphousregions that can host drugs, could enable novel applications in drug deliveryand immunology.

2612-Pos Board B628Simulated Strain Response of Two-Dimensional Beta-Solenoid ProteinLatticeRachel A. Baarda, Daniel L. Cox.Physics, University of California Davis, Davis, CA, USA.Self-assembling biomaterials hold much potential for applications such as bio-logical sensors, actuators, and passive molecular sieves. Beta solenoid proteins(BSPs) are a class of rod-shaped proteins that we propose for use in rationallydesigned nano-structured scaffold-like biomaterials. The BSP structure consistsof a solenoid-like coil of parallel beta-sheets, and the solenoid interior is denselypackedwith hydrophobic residues. Since this structure is reinforced by hydrogenbonding between beta-sheet layers and hydrophobic packing, BSPs are quiterobust to a variety of perturbations including high temperatures, introductionto uric acid solution, and additivemodifications to the original sequence. In addi-tion, the BSP sequence-to-structure map is well understood, allowing for a morestraightforward design and functionalization process than is allowed for withmore disordered proteins. We have designed a simulation model of a two-dimensional hexagonal lattice consisting ofBSPs covalently bonded to viral pro-teins (pdb code 4NCV) with C3 symmetry.We use the molecular dynamics toolGromacs to strain the lattice by deforming the periodic simulation cell. Thestress response to the applied strain is monitored to yield the lattice’s elasticbulk and shear moduli as a measure of its material strength.

2613-Pos Board B629Developed Potential across the Bilayers under External Electric FieldCauses ElectroporationAmit Kumar Majhi.Soft condensed Matter, Raman Research Institute, Bangalore, India.In molecular dynamics (MD) simulations, the sequences of the events leading toelectroporation are pore initiation, pore growth and bilayer rupture. This electro-poration have been studied under the external electric field, 0.43V/nm, forDPPCand POPC lipid bilayers. The external electric fieldmodifies the dipole potential,which has been evaluated in every 1 ns; and is turn out to bemuch higher than the

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applied one. The dipole potential reaches 2.24 Vwithin first ns of the field appli-cation and then achieves a value of 4 V within 6 ns in POPC bilayers. It is alsoobserved that the pore initiation take placeswithin 1 ns. InDPPCand POPC lipidbilayers, the pore initiation times are 0.58 and 0.71 ns respectively. Both the poreand the dipole potential continue to increase rapidly and finally they lead todisruption of the bilayers within 10 ns if the external field is maintained.

2614-Pos Board B630Molecular Dynamic and Free Energy Analysis of Doxorubicin and DNAComplexBahaa Jawad, Lokendra Poudel, Wai-Yim Ching.Physics, UMKC, Mission, KS, USA.Doxorubicin is a widely used therapeutic drug for chemotherapy. It used to treata wide range of tumors. In the present work, we performed molecular dynamicsfromNano-scale molecular dynamic (NAMD) package for Doxorubicin with orwithout DNA in solvated environment. We have calculated the non-bondedinteraction energies such as electrostatic energy and van der Waals/Londondispersion energy of DOX, DNA and DOX-DNA complex. TheMD calculationshows that the amino sugar group domain in DOXhasmore flexibility than otherdomain. The binding of DOX in DOX-DNA complex has highly preferable.Furthermore, we have analyzed the free energies of DOX-DNA complexthrough the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method. In MM-PBSA method, the molecular mechanics calculationscombined with continuum solvation models to determine binding free energy.In our study, NAMDused to calculate total energy of gas phasewhich representsMMcalculation. The polar contribution (DGPB)was estimated by aidingDelPhiV. 7.0 by solving the Poisson-Boltzmann (PB) equation, while the nonpolar sol-vation energy (DGnp) was calculated from the solvent-accessible surface area(SASA) using VMD. We found the free energy of DOX_DNA complex is�13.89 (kcal/mol) which in reasonable agreement with the experimental esti-mate �9.4 kcal/mol if the entropic contribution takes into account.

2615-Pos Board B631Anomalous Diffusion as Seen through the Lens of Inverted VariableLength Scale FCSMichael Stolle, Cecile Fradin.Physics and Astronomy, McMaster University, Hamilton, ON, Canada.Variable Length Scale Fluorescence Correlation Spectroscopy (VLS-FCS) pro-vides a novel approach to examine diffusion models and compare these modelsto experimental data at several different length and time scales. Length scalevariation over several orders of magnitude including sub-diffraction limitedlength scales can be achieved using, for example, scanning FCS or STED(STimulated Emission Depletion) FCS. Inverting the autocorrelation functionobtained by FCS further allows extracting the particles mean-squared displace-ment as long as they are undergoing a diffusion process with a Gaussian prop-agator. We have performed simulation of particle diffusion over a large length-scale range in order to examine the signature in VLS-FCS experiments ofseveral complex or anomalous diffusion processes relevant to protein diffusionin cells. We show that VLS-FCS allows discriminating diffusion models basedon the presence of a characteristic length-scale, their self-similarity, andwhether they have a Gaussian propagator. This work thus gives leads on theinterpretation of experimental FCS data in complex biological systems.

2616-Pos Board B632Determining the Interaction Enthalpy of Side Chain and Backbone Amidesin Polyglutamine Monomers and FibrilsRiley J. Workman, Jeffrey D. Evanseck.Chemistry and Biochemistry, Duquesne, Pittsburgh, PA, USA.Polyglutamine fibrils are implicated in the pathology of various neurodegener-ative diseases, the most notorious of which is Huntington’s disease. Under-standing the formation of polyglutamine fibrils is key in developing acomplete molecular picture of the pathology of these diseases. Here we presentmolecular dynamics results in which we calculate the interaction enthalpies ofvarious amide carbonyl groups present in D2Q10K2 peptides. We use severallow energy monomeric D2Q10K2 conformations as well as an amyloid-likefibril conformation. Results from our MD simulations are compared withexperimental results from UV resonance Raman spectroscopy that relate theAmide I vibrational frequency to the interaction enthalpy of the carbonyl group.We find that inter-amide side chain-side chain and side chain-peptide backboneinteractions in fibrils and b-strand monomers are stronger than side chain-waterinteractions in monomers with polyproline II-like (PPII-like) solution confor-mations. In fibrils, we also find that interactions between side chain amidesare enthalpically more favorable than interactions between peptide backboneamides. This work provides direct experimental validation that inter-amideside chain interactions play an important role in driving the formation and sta-bility of polyQ fibrils.

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Posters: Optical Microscopy and SuperresolutionImaging: Novel Approaches and Analysis II

2617-Pos Board B633Identifying the Axial Location of Proteins at the Nuclear Envelope withNanometer ResolutionSiddarth Reddy Karuka1, Jared Hennen1, G.W. Gant Luxton2,Joachim D. Mueller1.1School of Physics and Astronomy, University of Minnesota, Minneapolis,MN, USA, 2Genetics, Cell Biology, and Development, University ofMinnesota, Minneapolis, MN, USA.The nuclear envelope (NE) separates the cytoplasm and nucleus by a double-layered membrane consisting of the inner nuclear membrane (INM) and outernuclear membrane (ONM). The INM and ONM are separated by tens of nano-meters. Specific INM and ONM proteins form LINC (linker of nucleoskeletonand cytoskeleton) complexes, which span the nuclear envelope and mechan-ically couple the nucleoskeleton and cytoskeleton. It has been suggested thatLINC complexes define the spacing of the INM and ONM, which might bemodulated by a wide range of diseases associated with LINC complexes.To test this hypothesis in future work we are seeking to directly measurethe distance between the INM and ONM in live cells. We axially scan thetwo-photon point spread function through a cell with a fluorescently-labeledprotein residing on a nuclear membrane. The scan generates an intensity pro-file that reveals the spatial location of the proteins along the scan path. Tomeasure the axial distance separating two protein systems we label eachwith a distinct color and perform a dual color z-scan, which simultaneouslydetermines the location of each protein species. We experimentally verifythat dual-color z-scan is capable of identifying the axial separation of mem-brane proteins with a few nanometer uncertainty using model systems. Wealso perform direct measurements of the distance between the INM andONM on individual cells. In addition, we extend our studies to the transloca-tion of transmembrane proteins from the ONM to the INM. This work hasbeen supported by a grant from the National Institutes of Health (R01GM064589).

2618-Pos Board B634StudyingBiomolecularSystemsBeyond theDiffractionLimitwithMolecularResolution by STED-MFIS MicroscopyJan H. Budde, Ralf K€uhnemuth, Claus A.M. Seidel.Molecular Physical Chemistry, Heinrich-Heine University D€usseldorf,D€usseldorf, Germany.We combine Stimulated Emission Depletion (STED) microscopy and Multipa-rameter Fluorescence Image Spectroscopy (MFIS) to selectively measure andcharacterize biomolecular systems on surfaces and in living cells with molec-ular resolution.Scanning confocal STED microscopy overcomes the diffraction limit and lo-calizes molecules with a resolution down to 25 nm, while MFIS monitorssimultaneously a variety of fluorescence parameter such as fluorescence inten-sities, fluorescence lifetimes and anisotropy pixelwise in three spatial dimen-sions (3D). MFIS allows for detailed spectroscopic analysis and providesAngstrom resolution via Forster Resonance Energy Transfer (FRET). Thus,macromolecules can be localized in living cells with nanometer accuracy(STED), while monitoring their structure with Angstrom resolution (FRET).Hetero-FRET studies on model systems with small synthetic fluorophore andfluorescent proteins, respectively, demonstrate the benefits of combiningSTED and MFIS techniques to map the localization of macromolecules withhigh precision and to resolve their inter- and intramolecular structural and dy-namic features simultaneously. In a test case, we systematically vary distancewithin a FRET pair up to the practical resolution limits of STED. This demon-strates that a combination of STED and FRET bridges the length scales ofFRET-spectroscopy and microscopy to study cellular architectures in livingcells with Angstrom resolution.

2619-Pos Board B635Super Resolution Method for Fluorescent Image DeconvolutionSandra R. Martınez1, Micaela Toscani2, Oscar E. Martınez2.1Departamento de Matematicas, Facultad de Ciencias Exactas y Naturales ,Universidad de Buenos Aires, Instituto de Investigaciones Matematicas LuisA. Santalo (IMAS), Conicet, Buenos Aires, Argentina, 2Departamento deFısica, Facultad de Ingenierıa, Photonics Lab Universidad de Buenos Aires,Conicet, Buenos Aires, Argentina.A new algorithm for microscope fluorescent images deconvolution is pre-sented that retrieves the information with super resolution. The method isparticularly fit for sparse data, such as encountered in typical intracellular

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experiments, where only a minor fraction of the explored volume has fluo-rescent markers. The technique retrieves by construction only positive valuesfor the spatial density, avoiding the need for nonlinear constrains found inprior deconvolution techniques such as Tikhonov-Miller and Richardson-Lucy. A genetic algorithm is performed to optimize the solution. Themethod automatically subtracts the background from the image. Based onthe measured point spread function with the information of the quality ofthe fit and the information on the noise figure of the camera as a functionof the read signal, the method provides a predictor of the uncertainty inthe reconstruction both in lateral resolution and amplitude. Artificially syn-thesized images including noise were used to test the method, showing thereconstruction below the 50nm spatial resolution when analyzing arrays ofmolecules or fluorescent lines. Images of 100nm and 200nm fluorescentbeads and bead clusters are also efficiently and precisely deconvolved. Forthe images obtained with an Andor Zyla5.5 camera with a measured noisegiven by Noise= 23þsqrt(N) (where N is the count of the pixel) the theoret-ical predictor mentioned before gave a lateral resolution of 44nm consistentwith the experimental and simulated results. Examples of intracellular fluo-rescent image reconstruction are presented using bovine pulmonary arteryendothelial cells with fluorescent labels for the F-actin, microtubules andmitochondria. The extension of the technique to 3D deconvolution willalso be discussed.

2620-Pos Board B636A Protein Tag-Specific Aptamer for Use in Dstorm and Paint Based Super-Resolution ImagingJuan Wang1, Avtar Singh2, Warren Zipfel3.1Graduate Field of Biophysics, Cornell University, Ithaca, NY, USA, 2Schoolof Applied and Engineering Physics, Cornell University, Ithaca, NY, USA,3Department of Biomedical Engineering, Cornell University, Ithaca, NY,USA.Developing fluorescence labeling methods that can densely and continuouslytag specific cellular structures is important for super-resolution imaging.Here we demonstrate the use of fluorophore labeled RNA aptamers asSTORM and PAINT probes by imaging different fusion protein targetsboth inside and outside the cell. Aptamers can have distinct advantagesover antibodies in that they can have equally high binding affinities, butare smaller in size and inexpensive to produce in the laboratory, makingthem useful for targeting the specific proteins they were selected against.For this purpose, it is particularly advantageous to use aptamers selectedagainst commonly used fusion protein tags. With this in mind, we developeda labeling method in which we use an aptamer that binds any GFP fusionprotein with high affinity and specificity. Super-resolution imaging isachieved by either conjugating the GFP-binding aptamers with dSTORMcompatible fluorophores such as Alexa-647, or by using fluorescently labeledDNA oligo probes to the GFP-binding aptamer for PAINT based super res-olution microscopy. Using cell lines expressing EGFP labeled transferrin re-ceptor, we show aptamer-based dSTORM and PAINT imaging as anexample of super-resolution imaging of plasma membrane proteins in bothfixed and live cells. Intracellular aptamer dSTORM and PAINT super-resolution imaging is demonstrated using lamin-A-EGFP expressing cells,in which the lamin structural network of the nuclear membrane can be visu-alized at high resolution.

2621-Pos Board B637A Red Fluorescent Protein for Cryogenic Single-Molecule SuperresolutionImagingAnnina M. Sartor1, Peter D. Dahlberg1, Jiarui Wang1,2, Lucy Shapiro2,W.E. Moerner1.1Department of Chemistry, Stanford University, Stanford, CA, USA,2Department of Developmental Biology, Stanford University School ofMedicine, Stanford, CA, USA.Superresolution imaging techniques routinely resolve fluorescently labelledstructures in cells with resolution more than an order of magnitude beyondthe diffraction limit. The molecular specificity and spatial resolution of thesetechniques make them a powerful tool for the life sciences. In single-molecule superresolution imaging, the precision with which a single moleculecan be localized depends on the number of photons collected before photo-bleaching. It has long been known that photobleaching is significantly reducedat cryogenic temperatures; thus, cryogenic fluorescence imaging can provide aroute to improved superresolution imaging. However, performing superlocali-zation measurements based on stochastic activation of single fluorophores (e.g.PALM, STORM) requires the fluorophores to exhibit the necessary photo-switching properties at cryogenic temperatures. Restricted conformational

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mobility in a frozen solvent prevents many photoactivatable small moleculefluorophores and fluorescent proteins from undergoing the necessary isomeri-zations or other rearrangements required for photoactivation or blinking. Inthis work, we have identified a red fluorescent protein which exhibits photoac-tivation at cryogenic temperatures. Using a custom fluorescence microscopecapable of maintaining a sample at cryogenic temperatures during imaging,we have characterized the photoswitching behaviour and obtained superresolu-tion images of previously studied proteins in the model organism Caulobactercrescentus.

2622-Pos Board B638FluorescenceMicrospectroscopy with Nanometer Peak Position Resolution:Novel Applications of Environment-Sensitive ProbesZoran Arsov, Iztok Urbancic.Jozef Stefan Institute, Ljubljana, Slovenia.Fluorescence microspectroscopy (FMS) with environment-sensitive probesprovides information about local molecular surroundings at microscopic spatialresolution. Until recently, only probes exhibiting large spectral shifts due tolocal changes have been used. Herein, we show that appropriate measuring pro-cedure and data analysis enable nanometer spectral peak position resolution,even for photosensitive fluorophores [1].The reach of our approach is demonstrated in several examples. The first appli-cation shows howwe can distinguish lipid vesicles in different lipid phases withtwo commonly used polarity-sensitive probes. A synthesized NBD-based fattyacid red-shifted its emission maximum by 1.5-2 nm going from gel to liquid-disordered phase in DPPC. Between these two phases Laurdan exhibits a large50 nm red-shift. We therefore chose a more challenging combination - gel andliquid ordered phase, realized by DPPC and DPPC/Chol (40 mol%), respec-tively, where we were able to detect a 3 nm blue-shift with Laurdan [1]. Thesecond example shows application of a synthesized rhodamine-based pH-acti-vatable probe that is sensitive to aggregation. We studied a receptor-mediatedinternalization in dendritic cells and measured a 3 nm aggregation-inducedemission spectral shift due to probe accumulation in endosomes and lysosomes[2,3].The results show that peak position resolution, characteristic for spectrofluori-metric measurements on bulk samples, could readily be achieved at micrometerspatial scale.[1] I. Urban�ci�c, Z. Arsov, A. Ljubeti�c, D. Biglino, J. �Strancar, Opt. Express21:25291–25306 (2013).[2] Z. Arsov, U. �Svajger, J. Mravljak, S. Pajk, A. Kotar, I. Urban�ci�c, J. �Strancar,M. Anderluh, ChemBioChem 16:2660–2667 (2015).[3] Z. Arsov, I. Urban�ci�c, J. �Strancar, Spectrochim. Acta A Mol. Biomol. Spec-trosc., https://doi.org/10.1016/j.saa.2017.09.067 (2017).

2623-Pos Board B639Boosting the Localization Precision in Super-Resolution Microscopy:booSTORMHannah S. Heil1, Benjamin Schreiber1, Marie-Christine Dabauvalle2,Georg Krohne2, Sven Hofling3,4, Martin Kamp3, Markus Sauer5,Katrin G. Heinze1.1Rudolf-Virchow-Zentrum, University of W€urzburg, W€urzburg, Germany,2Department of Electron Microscopy, Biozentrum, University of W€urzburg,W€urzburg, Germany, 3Technische Physik, University of W€urzburg,W€urzburg, Germany, 4SUPA, School of Physics and Astronomy,University of St Andrews, St Andrews, United Kingdom, 5Department ofBiotechnology and Biophysics, Biozentrum, University of W€urzburg,W€urzburg, Germany.Over the last decade, single molecule switching and localization havebecome the key for super-resolved visualization of molecular architectureand dynamics. Here, we show that biocompatible plasmonic coatings onstandard microscope cover glasses significantly improve the resolution ofa direct stochastic optical reconstruction microscopy (dSTORM) experiment.The enhanced signal-to-noise ratio sharpens the location precision by a fac-tor of 1.8 as demonstrated in super-resolution images of the nuclear porecomplex (Heil et al., bioRxiv doi: 10.1101/136739). Together with recentadvances in image processing and analysis (Franke et al., 2016, Nat.Methods, 14, 41-6) we demonstrate the reliability of our optoplasmonicapproach (booSTORM) and proof that the effect leads to an increased totalphoton yield per fluorophore. The strength of booSTORM is that theenhancement is solely induced by the metal-dielectric coatings on the coverglass and does not require any additional modification of the microscopesetup. This makes booSTORM very versatile, and applicable as a boosterfor many other fluorescence approaches including for example live cell Fluo-rescence Correlation Spectroscopy and Fluorescence Resonance EnergyTransfer studies.

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2624-Pos Board B640Force Spectroscopy of Phagocytosis with High Frame Rate 3D Light SheetImagingEvan Nelsen, Chad Hobson, Joe Hsiao, Michael Falvo,Edward T. O’Brien III, Takashi Watanabe, Klaus Hahn, Richard Superfine.Physics and Astronomy, UNC-Chapel Hill, Chapel Hill, NC, USA.A critical part to the immune system is the search and elimination of foreignpathogens by macrophages, neutrophils and other cells. These cells eliminatelarge particles (>0.5mm) by consuming them through a process called phago-cytosis. Understanding the mechanics of how a macrophage engulfs a target re-quires careful monitoring of forces and high quality florescence imaging of themembrane and cytoskeleton. Using an atomic force microscope (AFM) with aversatile optics system, we can monitor piconewton scale forces while imagingthe phagocytosis engulfment from the side using Pathway Rotated Imaging forSideways Microscopy with vertical light sheet (PRISM-LS). In addition, theversatile optics system can be used to record high frame rate three dimensionalimages of macrophage engulfment. In this study, we look at the effects ofdifferent applied forces on phagocytotic uptake and mechanical response ofmacrophage cells. Preliminary results show the macrophage produces a dy-namic response to the application of a several hundred piconewton forcewith an IgG covered AFM tip during its engulfment. Light sheet imagingwith controlled force data will inform mechanical models of phagocytosiswhich will improve understanding of this important immunological processand inform mammalian disease progression.

2625-Pos Board B641Bacterial Proteins Associated with Cell Shape Homeostasis Localize toSpecific 3D GeometriesBenjamin P. Bratton1,2, Zemer Gitai1, Joshua W. Shaevitz2,3.1Molecular Biology, Princeton University, Princeton, NJ, USA, 2Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton,NJ, USA, 3Physics, Princeton University, Princeton, NJ, USA.The bacterial kingdom exhibits a wide variety of cell shapes and sizes. Theshapes of these cells are crucial for their lifestyle. Over the past few yearswe have developed an image-processing framework allowing us to extract pre-cise 3D shapes of bacterial cells from fluorescence microscopy data. Fromthese xyz coordinates, we calculate geometric parameters such as local curva-tures, surface areas, and the relative enrichment of fluorescent signals. We usethis to measure the geometric localization of bacterial proteins responsible forestablishing and maintaining the characteristic shape of various Gram-negativebacteria such as the straight rod Escherichia coli, the curved rod Vibriocholerae and the helical rod Helicobacter pylori. In Escherichia coli, wefocused on the bacterial actin MreB which localizes away from positiveGaussian curvature and toward zero and low curvature. MreB’s many cellshape roles are modulated by interactions with the transmembrane proteinRodZ. In the absence of rodZ, MreB loses its curvature localization and cellsare not able to maintain their uniform rod-like morphology. Additionaly, wehave used antibiotic treatment and genetic perturbations to confirm that ourmethod and normalization strategies perform well in a variety of geometries.In contrast to MreB’s enrichment at low Gaussian curvature, the cholera proteinCrvA localizes to regions of negative Gaussian curvature. We have also devel-oped a cell wall pulse-chase labeling strategy (Quantitative Analysis of Saccul-lus Architectural Remodeling, QuASAR), to determine that CrvA reduces theeffective exponential growth constant for the inner curve relative to the outercurve. This results in cells which curve up over time. Recently, we have begunto examine the localization of the multiple cell shape determinants in the bac-terial carcinogen H. pylori, not all of which share the same curvature localiza-tion preferences.

2626-Pos Board B642Uncovering Hidden Dynamics in Live-Cell Single Molecule Data withBayesian StatisticsJosh D. Karslake1, Lucas Demey2, Victor DiRita2, Julie S. Biteen3.1Biophysics, University of Michigan, Ann Arbor, MI, USA, 2Microbiology &Molecular Genetics, Michigan State University, East Lansing, MI, USA,3Chemistry, University of Michigan, Ann Arbor, MI, USA.Single-molecule imaging techniques localize and track individual moleculesinside living cells with nanometer precision and millisecond timing; this capa-bility has allowed researchers to investigate many open questions acrossbiology. However, single-molecule image analysis is fundamentally limitedby a priori model selection, parameter unidentifiability, and other supervisorybiases. To address these issues, we have developed an analysis framework forSingle-Particle Tracking data based on nonparametric Bayesian inference. Byencoding any information we have about the system into the ‘‘prior’’, itera-tively determining the maximal parameter values and data selection by the

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likelihood, and allowing the model to shrink and expand dynamically, we usethe data itself to learn about the system and uncover properties or dynamics thatwould be hidden by other methods.This method offers a flexible and simple way to allow the data to dictatethe appropriate model structure instead of having the experimenter impose arigid structure beforehand. Here we first validate this method by determining(a) the number of diffusive populations, (b) the average diffusion coefficientfor each term, (c) the population weight fraction for each term, and (d) the ratesof conversion from one term to another in simulations of heterogeneousdiffusion. We then extend our investigations to simple experimental systems,and finally we apply our algorithm to determine the model and dynamics ofthe membrane-bound keystone virulence protein TcpP in live Vibrio choleraecells.

2627-Pos Board B643Investigating the Heteromerization of Metabotropic Glutamate Receptorsusing a Novel Single Molecule Imaging MethodAlexander L. Van Slyke1, Avtar Singth2, Nitya Deshmukh3,Paul J. Kammermeier4, Warren R. Zipfel5.1Field of Biophysics, Cornell University, Ithaca, NY, USA, 2Broad Institute,Caimbridge, MA, USA, 3Cornell University, Ithaca, NY, USA, 4Departmentof Pharmacology and Physiology, University of Rochester Medical Center,Rochester, NY, USA, 5Department of Biomedical Engineering, CornellUniversity, Ithaca, NY, USA.Metabotropic Glutamate Receptors (mGluRs) are a type of G-Protein CoupledReceptor (GPCR) which bind glutamate as a neurotransmitter and are found inneurons throughout the brain and peripheral tissues. mGluRs are known tofunction as stable homodimers, but recent evidence suggests that the formationof heteromeric complexes may also occur. Knowledge of the stoichiometry ofthese complexes in vivo is crucial for the development of pharmacologicalcompounds designed to act on them. Here we investigate the complexes formedby mGluRs using various optical methods including Single Protein RecoveryAfter Dilution (SPReAD), a novel single molecule imaging technique wehave developed which utilizes VSV-G mediated cell fusion in order to dilutethe concentration of fluorescently tagged molecules of interest. This enablesin vivo imaging without introducing harsh detergents or altering the level ofexpression.

2628-Pos Board B644Coordination ofMolecularMotors during Long-Distance Axonal TransportBianxiao Cui, Luke Kaplan, Praveen Chowdary.Chemistry, Stanford University, Stanford, CA, USA.Long-distance transport of vesicular cargoes are essential for the function andsurvival of neurons. Defects in this transport process are linked to a range ofneurodegenerative disease such as Alzheimer’s disease and Huntingtin’s dis-ease. Physically stalling the cargoes would be one of the most direct meansto perturb a cargo transport process, which, however, are technically chal-lenging in live cells. We engineered optogenetic and magnetic forces that spe-cifically stall a population of axonal cargoes that contain magnetic or opticalnanoparticle probes at the trapping area. Using a combination of force manip-ulation and high resolution microscopy methods, we show that mechanicaltugs-of-war and intracellular motor regulation are complimentary features ofthe axonal transport process.

2629-Pos Board B645Quantitative Ultra-Fast FlimMarcelle Koenig, Rhys Dowler, Paja Reisch, Ben Kraemer, Sandra Orthaus,Marcus Sackrow, Matthias Patting, Tino Roehlicke, Hans-Juergen Rahn,Michael Wahl, Felix Koberling, Rainer Erdmann.PicoQuant GmbH, Berlin, Germany.Increasing the speed of Fluorescence Lifetime Imaging (FLIM) is essential tocementing its importance as a tool in the Life Sciences. This technique isalready well established, but imaging dynamic processes requires shorter acqui-sition times. Our novel rapidFLIM approach dramatically reduces the acquisi-tion time through a combination of fast scanning, hybrid photomultiplierdetectors which are capable of handling very high count rates, and TCSPCmodules with ultra short dead times. With the new FLIMbee fast scanningadd-on for the MicroTime 200, this technique can be used with our microscopyplatform as well as being offered as an upgrade kit for conventional Laser Scan-ning Microscopes (LSMs). With this hardware combination, excellent photonstatistics can be achieved in significantly shorter time spans, allowing fast pro-cesses to be measured with the high resolution achieveable in confocal micro-scopy. Depending on the image size, rapidFLIM allows imaging at a rate ofseveral frames per second, enabling dynamic processes, such as protein inter-actions, FRET dynamics, or chemical reactions to be imaged in a time-resolved manner. With these high frame rates, FLIM can also be used on highly

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mobile species such as cell organelles and for other live cell imaging applica-tions. Recently, we have further pushed the limits of this method by systemat-ically reducing the effects of decay distortions at very high count rates,allowing quantitative data analysis to be performed even at count rates above10 Mcps. This technique has been applied to quantitatively analyze FRET mea-surements using fluorescent proteins.

2630-Pos Board B646Single-Molecule Protein Identification Through Peptide Chain Barcodingand Optical ReadoutMingjie Dai1,2, James MacDonald2, Fred Vigneault2, Erik Hernandez2,Darren Yang2,3, Wesley Wong2,3, Peng Yin1,2.1Department of Systems Biology, Harvard Medical School, Boston, MA,USA, 2Wyss Institute, Harvard University, Boston, MA, USA, 3Program inCellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA,USA.A method for single-molecule protein identification and post-translationalmodification (PTM) mapping could potentially allow high-sensitivity detectionand profiling of the cellular and circulatory proteome, and helps to better char-acterise cell types and differentiation states, analyse cellular regulatory pro-grams and dynamics changes. Current workhorse approach of massspectrometry based proteomic methods rely on digestion of proteins into shortpeptide fragments, exhibit biased fragment ionisation and have limited detec-tion sensitivity. Combined with recent development in super-resolution sin-gle-molecule fluorescence microscopy methods, we are developing a novelmethod for single-molecule protein identification through peptide chain bar-coding and optical readout. In particular, we will apply previously developedDNA-PAINT (point accumulation for imaging in nanoscale topography) andDMI (discrete molecular imaging) fluorescence super-resolution methods toprotein molecules that are residue-specifically labelled with oligonucleotideprobes. Using a combination of high-resolution (<5 nm) single-moleculeimaging and blinking kinetics analysis, we present bioinformatics analysisdemonstrating discrimination and identification power of our method, as wellas high-efficiency residue labelling and single-molecule optical identificationof short model proteins.

2631-Pos Board B647Multiple Emitter Fitting and Structured Background Detection usingReversible Jump Markov Chain Monte CarloMohamadreza Fazel, Marjolein B.M. Meddens, Michael J. Wester,Keith A. Lidke.Physics and Astronomy, University of New Mexico, ABQ, NM, USA.In single molecule based super-resolution imaging, high labeling density or thedesire for greater data collection speed can lead to clusters of overlapping PSFsin the raw super-resolution image data. Multi-emitter fitting algorithms canidentify and localize the particles in those dense regions of the data. In thiswork, we advance the state-of-the art in multi-emitter fitting by employingReversible Jump Markov Chain Monte Carlo (RJMCMC). Markov ChainMonte Carlo (MCMC) can be used to find the posterior distribution of a setof parameters, which in the multi-emitter fitting problem consists of the particlepositions, intensities and a local background. RJMCMC takes this conceptfurther and allows jumps between parameter spaces, in this case allowing theaddition or subtraction of emitters in the model. We allow four mechanismsto make jumps between spaces of various models, called birth, death, mergeand split. Birth (death) allows the addition (deletion) of an emitter anywherein the sample. Split allows the possibility that an existing particle can be actu-ally two or more neighboring particles. Merge allows the chance of two adja-cent particles join into a single particle. Another jump is embedded in thealgorithm to classify the found particles as background and signal particles.This jump employs the input intensity priors for signal and background tocalculate the probability if a particle belong to the signal or background.

2632-Pos Board B648Light Field LC-PolScopeMai Tran, Rudolf Oldenbourg.Bell Center, Marine Biological Laboratory, Woods Hole, MA, USA.Despite significant progress in polarized light microscopy, measuring the threedimensional anisotropy is still an important challenge in the analysis of aniso-tropic materials. In birefringent samples, traditional imaging methods don’treveal the 3D orientation (inclination and azimuth) of the optic axis and thebirefringence as an intrinsic material property. In samples that emit polarizedfluorescence, it is generally not possible to discern the inclination angle ofthe aligned fluorophores. To overcome these problems, the Oldenbourg Labhas introduced a new imaging method, the Light Field LC-PolScope, whichis a combination of light field imaging and LC-PolScope technology. In ourposter, we have used the Light Field LC-PolScope for acquiring images of

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simple and biological relevant test specimens for developing processingmethods to reconstruct the birefringence of juvenile shells of the Atlantichard clam and the polarized fluorescence of the labeled cell membrane of seaurchin eggs. We established acquisition and processing methods to generate3-dimensional maps of position and orientation of these optically anisotropicstructures, based on polarized light field images and compare results to moretraditional imaging methods. Our work represents the first successful attemptto generate such maps at a lateral resolution of 2mm and angular steps of 9�in terms of the azimuth and inclination angles of the optic axis. The maps ofclam shell birefringence provide structural insights into the early mineraliza-tion during juvenile clam shell development.

2633-Pos Board B649FLIM-FRET of Chromatin in Live Cells using Two DNA-Binding DyesSimone Pelicci1,2, Michele Oneto1, Melody Di Bona1,2, Alberto Diaspro1,2,Luca Lanzano1.1Nanoscopy, Istituto Italiano Tecnologia, Genova, Italy, 2Department ofPhysics, University of Genoa, Genova, Italy.The structural state of DNA in mammalian nucleus of living cells corre-sponds to varying levels of chromatin organization, ranging from the nucle-osomes to higher order structures. This varying degree of hierarchicalcompaction is integral to genome functions, allowing or preventing accessto several genetic nuclear factors (epigenetic, replicative, and transcrip-tional) [Misteli T, Cell (2007)]. However, how the higher-order chromatinstructures are formed and then behave in various cellular processes in livecells remains unclear.Forster Resonance Energy Transfer (FRET) is a process that can occur betweenan excited donor and an acceptor molecule when the two fluorophores arewithin 10nm [Forster, Naturforsch (1949)]. Fluorescence lifetime imaging(FLIM)-based detection of FRET between histone molecules tagged with fluo-rescent proteins has been used to study the nanoscale chromatin compaction inlive cells and small organisms, providing an in vivo assay where the level ofFRET is proportional to the level of chromatin compaction [Lleres et al, JCell Biol, (2009); Lleres et al, Cell Reports, (2017)].Here we test a FLIM-FRET assay based on a pair of DNA-binding dyes. Wemeasure FRET between two DNA-binding dyes (e.g. Hoechst 33342 andSyto Green 13) in live cells using frequency-domain FLIM and the phasor anal-ysis. We investigate if this FRET assay can be sensitive to the different degreesof chromatin compaction in interphase nuclei. In particular, we investigate howthe FRET level depends on the concentration of the two fluorophores and deter-mine a strategy to correct the values of FRET efficiency.

2634-Pos Board B650Locked Expansion Microscopy to in Situ Analyze Microbial CommunitiesYoungbin Lim1, Margarita Khariton1, Samuel Bray1, Katharine Ng1,Anthony Shiver1, Kerwyn C. Huang1,2, Bo Wang1,3.1Department of Bioengineering, Stanford, Stanford, CA, USA, 2Departmentof Microbiology and Immunology, Stanford University, Stanford, CA, USA,3Department of Developmental Biology, Stanford University, Stanford, CA,USA.There is a global race in microbiome research to map spatial organization ofmicrobial species in densely packed communities, but progress is limited byoptical imaging resolution and our ability in characterizing the diversity ofmicrobial species within a population in situ. Here we provide a newsuper-resolution imaging method, based on recently developed expansionmicroscopy (ExM), to address this knowledge gap. Expansion microscopyis an optical imaging technique by physically expanding tissues anchoredto a hydrogel. The current method relies on a polyelectrolyte hydrogelthat is expanded by electrostatic repulsion; as a result, the hydrogel shrinksin any ionic buffers. This technical limitation precludes the possibility of us-ing most anti-photobleaching systems (which typically require buffering)and thereby restricting the application of ExM in thick tissues with weakfluorescence signals, as these demand long imaging times. Here, we devel-oped a novel method, locked ExM, in which the first expanded polyelectro-lyte hydrogel is embedded in a second interpenetrating hydrogel mesh thatexpands through entropic forces. The second interpenetrating mesh retainsthe size of expanded tissues even in buffers with extreme ionic strengths.This method has allowed us to use anti-photobleaching systems in ExM toachieve sub-diffraction-limit resolution with high photostability. We haveapplied this method to image host-associated microbial communities, and,to our surprise, found that the expansion ratios of microbial species dependon their specific cell wall structures. We demonstrated the utilities of lockedExM to visualize and analyze spatial distributions of different microbial spe-cies within gut microbiota in a variety of organisms (worm, mouse, andhuman).

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2635-Pos Board B651High-Dimensional mRNA and Protein Content Measurements in SingleCells with Single-Molecule SensitivityDaniel M. Kalb1, Samantha Hiroshini Adikari2, Pulak Nath3,Elizabeth Hong-Geller2, James H. Werner1.1MPA-CINT, Los Alamos National Laboratory, Los Alamos, NM, USA,2Bioscience, Los Alamos National Laboratory, Los Alamos, NM, USA,3Physics, Los Alamos National Laboratory, Los Alamos, NM, USA.Although there are well established methodologies to measure and modelthe relationship between mRNA and protein content in bulk, new tech-niques are necessary to measure the fundamental expression of these com-pounds at the single-cell level with single-molecule sensitivity. Examiningthe heterogeneity of mRNA and protein expression at the single-cell levelcan lead to fundamental information about the cellular response to externalstimuli, including the sensitivity, timing, and regulatory interactions ofthese genes. Our initial results demonstrate the ability to simultaneouslyquantify up to five genes of interest in single cells using conventionalwide-field imaging and labeling methods. Single-molecule mRNA contentis measured by single-molecule fluorescence in-situ hybridization (smFISH)while protein content is quantified though the use of antibody probes. Fullsystem automation of the 3D microscope scan and custom image analysisroutines allows hundreds of individual cells to be automatically segmentedand the mRNA-protein content to be digitally counted. To mimic immuneresponse to bacterial infection, THP-1 cells are stimulated with lipopolysac-charide (LPS) and up to five genes of interest (e.g. IL1b, TNF-a, TLR2) aresimultaneously monitored to examine the distribution of single-cellresponse to this pathogenic stimulus. Despite these initial successes, it ishighly desirable to increase the number of genes simultaneously monitored.To this end we are designing a new microfluidic platform to precisely trapand lyse single cells within an analysis chamber for high-dimensional geneanalysis. Within this diffusion limited chamber, the mRNA and proteinprobes are spatially printed (using ink jet and dip-pen technology) to createspecific capture and fluorescent detection regions. Spatially arraying thedetection regions, combined with super-resolution fluorescent read-out,should enable multi-parameter analysis beyond the �5 color fluorescentlimit, allowing tens-to-hundreds of genes to be simultaneously analyzedin each single cell.

2636-Pos Board B652AMATLAB-based Instrument Control Package for Fluorescence ImagingSandeep Pallikkuth, Marjolein Meddens, Mohamad Fazel, Hanieh Farsibaf,Farzin Farzam, Michael Wester, Keith Lidke.Physics and Astronomy, University of New Mexico, Albuquerque, NM,USA.For the last few decades fluorescence imaging has been a vital tool that haslead to cutting-edge progress in physical and biological research. Whilewell-established fluorescence imaging methods are performed on commer-cial mass-produced microscopy systems, exciting new applications often de-mand custom and specialized design of microscopy systems. Hardwarecomponents needed for advancing such promising experimental develop-ments are often readily available, however, inexpensive software packagessuitable for design and control of custom microscopy systems are limitedin availability.We present here our freely available MATLAB Instrument Control (MIC) soft-ware package, a collection of programs written in MATLAB that facilitatesautomated data collection of complex, multi-component microscopy systems.MIC uses object-oriented programming where a class defines the capabilitiesof each instrument. Each instrument class inherits from a common MICabstract class and therefore has a basic common interface. Common componenttypes (e.g. lasers, camera, shutters, scanners etc.) have their own further ab-stract sub-classes to give common interfaces and to facilitate the developmentof control classes for specific new instruments. Export methods, unit tests andgraphical user interfaces are provided for each of the instrument components.Control of multi-component instruments can be by scripting or creating func-tions or classes that make uses of individual components. Use of the MATLABenvironment allows immediate access to data and image analysis even duringdata collection. Proficient MATLAB users can easily extend or modify any ofthe control classes.As an example, we also present a readily usable class for control and datacollection for a dSTORM based super-resolution imaging microscopy system.An easy-to-use graphical user interface for this class provides control of mul-tiple excitation lasers and camera parameters. A list of supported componentswill be available on the poster. Find MIC at github.com/LidkeLab/matlab-in-strument-control.

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2637-Pos Board B653Scattering of Evanescent Illumination by Sample Inhomogeneities in TIRFMicroscopy: A Theoretical StudyJeremy J. Axelrod1, Daniel Axelrod2.1Physics, University of California, Berkeley, Berkeley, CA, USA, 2Physics,Biophysics, Pharmacology, University of Michigan, Ann Arbor, MI, USA.In TIRF microscopy, the sample resides near a surface in an evanescentoptical field that, ideally, decreases in intensity with distance in a pureexponential fashion. In practice, multiple surfaces and imperfections inthe optical system and refractive index (RI) inhomogeneities in the sam-ple (often living cells) produce propagating scattered light which degradesthe exponential purity. Without altering the sample, RI inhomogeneitiescannot be avoided: how severe is the consequent optical degradation?Starting from Maxwell’s Equations, we derive a first-order perturbativeapproximation of the electric field strength of light scattered by sampleRI inhomogeneities under coherent evanescent field illumination. At first,a laterally sinusoidal variation in RI is assumed, leading to closed-formexplicit theoretical expressions. These show that for most conditions,the scattered intensity is less than 10% of the evanescent intensity atthe TIR surface. However, there are clear resonances for certain incidenceangles, wavelengths, polarizations, incident directions, and RI periodicityspacing and phases for which the scattering can be much larger. In thenext step, the sinusoidal assumption is generalized. Any hypothetical RIpattern (such as might be found in a cell cultures sample) is expressedas a spectrum of sinusoidal waves (by Fourier transform), and theabove-calculated scattered field from each sinusoidal component is inte-grated over that spectrum to produce the final result. This procedure isan approximation because it: expands only to first-order; ignores multiplescattering and reflections off the TIR surface; assumes that the RI varia-tions in the z-direction are insignificant within the depth of the evanescentfield; calculates the scattered field intensity only at the substrate surface;and considers only scattering of excitation light, not emission light, Sup-ported by NIH R01-170553 (DA and R.W. Holz), and NSF GraduateResearch Fellowship (JJA).

2638-Pos Board B654Adaptive Optics in Deep Tissue MicroscopySimon W. Leemans, Alexander Dvornikov, Enrico Gratton.Biomedical Engineering, UC Irvine, Irvine, CA, USA.Multiphoton fluorescence microscopy is a powerful technique to visualize theform and function of the cells in tissue at sub-micron scales. To diminish fluo-rescence emission losses due to light scattering in the sample, our lab hasdeveloped a transmission fluorescence microscope to recover more of theemitted fluorescence from biological samples. Multiphoton fluorescence issensitive to the peak power of the light, so the efficiency of the process im-proves when pulse duration is decreased and the focal volume is minimized.The DIVER (Deep Imaging Via Emission Recovery) microscope uses a pulsecompressor that pre-compensates for dispersion in the excitation path to mini-mize pulse duration. Spatial variations in the index of refraction in tissuedistort the focal volume, resulting in decreased resolution as imaging depthis increased. We compensate for spatial variations in index by conjugatingthe wavefront of the excitation light to the optical aberrations that are intro-duced by the tissue. Using a guide star approach, we can deduce how tissuein the excitation path is affecting the image quality. In microscopy, guide starsare created by using fluorescent beads that are smaller than the diffractionlimit of the system. The distortion to the wavefront in the imaging systemis measured indirectly using a trial and error approach to determine thetype and amplitude of orthogonal aberrations present in the image. Theoptimal configuration of the deformable mirror is obtained by maximizingthe nonlinear fluorescence in the sample, yielding images with higher resolu-tion and contrast. The use of adaptive optics in the DIVER microscope facil-itates accurate and meaningful three-dimensional imaging and modeling oftissue. This work is supported by NIH P41-GM103540 and NIH P50-GM076516 grants and the National Science Foundation through the Integra-tive Graduate Education and Research Traineeship (IGERT) Program(DGE-1144901).

2639-Pos Board B655Quantitative Image Restoration in Bright Field MicroscopyBraulio Gutierrez-Medina.IPICYT, San Luis Potosi, Mexico.Current fluorescence-based microscopy methods enable the localization ofspecific targets with exquisite sensitivity, albeit with important limitationssuch as photobleaching, phototoxicity, and spectral bleed-through. To helpminimize these potential constraints, non-fluorescence microscopy modalities

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must be used whenever appropriate, e.g. during evaluation of cellularmorphology. Bright field (BF) optical microscopy has traditionally being re-garded as a poor method to observe unstained biological samples due tointrinsic low image contrast. To address this issue, we introduce quantitativeimage restoration in bright field (QRBF), an image processing techniquewhere out-of-focus BF images of unstained, isolated cells are digitally decon-volved using a point spread function modeled from theory. We validate ourprocedure by comparing results of QRBF with corresponding reference im-ages of bacteria observed under fluorescence, showing that QRBF faithfullyrecovers shape and enables quantify size of individual cells. Furthermore,we demonstrate that effective restoration is achieved even from a single inputimage (2D-QRBF). To demonstrate the applicability of our methodology, weuse 2D-QRBF in a high-throughput image cytometer that evaluates shapechanges in Escherichia coli cells during hyperosmotic shock, finding size het-erogeneity. Altogether, digital image restoration in BF recovers cellular shapeaccurately, provides robust conditions for segmentation, and enables expediteprocessing--all without requiring additional components to the standardmicroscope.

2640-Pos Board B656Primed Green-to-Red Photoconversion of Fluorescent Proteins Occurs viaa Triplet StateKarin Nienhaus1, Manuel A. Mohr2, Andrei Yu Kobitski1,Lluc Rullan Sabater2, Christopher J. Obara3, Jennifer Lippincott-Schwartz3,G. Ulrich Nienhaus1, Periklis Pantazis2.1Applied Physics, Karlsruhe Institute of Technology, Karlsruhe, Germany,2Eidgenossische Technische Hochschule (ETH) Z€urich, Basel, Switzerland,3Janelia Research Campus, Ashburn, VA, USA.Fluorescence microscopy plays a prominent role in life sciences researchbecause it allows researchers to obtain quantitative, spatially and temporallyresolved information on biological processes, both in vitro and in vivo. A va-riety of elegant microscope designs have been devised that circumvent theAbbe resolution limit, including STED/RESOLFT and single moleculelocalization-based techniques. Genetically encoded fluorescent proteins (FPs)of the green fluorescent protein (GFP) family play a key role in optical nano-scopy because they can be employed as minimally perturbing, endogenousmarkers in live-cell/tissue/organism experiments.Green-to-red photoconvertible fluorescent proteins (pcFPs) such as EosFPare powerful tools for super-resolution photoactivation localization micro-scopy (PALM). They can be photoconverted from the green-emitting tothe red-emitting state by �400-nm illumination. Recently, an alternativemethod termed primed conversion has been discovered. It employs dualwavelength illumination with blue and far-red/near-infrared light and workswith pcFP variants having a serine or threonine at amino acid sequence po-sition 69. Initial photoactivation with blue light forms an intermediate‘‘primed’’ state that has been identified as a triplet dark state. Excitationof this intermediate into higher triplet states by far-red/near-infrared lightwith subsequent reverse intersystem crossing generates the red-emitting spe-cies. These pcFPs exhibit excellent properties for primed PALM, ultimatelyallowing a new dual PALM modality that does not occupy additional spec-tral space.

2641-Pos Board B657AiryScan Comprehensive Super-Resolution Correlation AnalysisLorenzo Scipioni1,2, Alberto Diaspro2,3, Luca Lanzano2, Enrico Gratton1.1Laboratory for Fluorescence Dynamics, Irvine, CA, USA, 2Nanoscopy,Istituto Italiano di Tecnologia, Genova, Italy, 3Physics, University of Genoa,Genova, Italy.AiryScan is a detector array developed by Zeiss that consists in 32 GaAs PMTdetectors arranged in a hexagonal pattern. The development of single point de-tector arrays is recently increasing also thanks to the possibility of achievingsuper-resolution without signal loss by means of Image Scanning Microscopy(ISM, Sheppard et al. 2013). Furthermore, super-resolution techniques appliedto fluorescence correlation techniques have shown to be effective in retrievinginformation that is not accessible with diffraction-limited methods, giving newvaluable tools to biological research. Being essentially a high-speed nano-cam-era, AiryScan allows, in principle, for the implementation of most of the state-of-the-art correlation techniques at super-resolution in a single analysis. In thiswork we will present the parallel implementation of fluorescence correlationspectroscopy (FCS, Digman et al. 2011), pair-correlation function (pCF, Hindeet al. 2010) and connectivity analysis, image-derived mean square displace-ment (iMSD, Di Rienzo et al. 2014), number and brightness (N&B, Digmanet al. 2008) and diffusion law analysis (Wawrezinieck et al. 2005) in thesame single-spot dataset in the cellular environment. This pipeline ultimatelyreturns information about diffusion, concentration, environment structure and

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accessibility, spatiotemporal confinement and oligomerization states from asingle analysis of a few seconds in living cells.The combination of fast sam-pling speed (down to 1.23 ms per frame) and super-resolution capability opensthe doors to the investigation of spatiotemporal dynamics that are not acces-sible with any other method, at present. We will demonstrate the possibilityand the advantages of having access to several methods in a single acquisitionand will show several examples of application in live cells, for instance byanalyzing EGFP diffusion in NIH-3T3 cells.

Posters: Optical Microscopy and SuperresolutionImaging: Applications to Cellular Molecules II

2642-Pos Board B658Cytosolic Assembly Among Bacterial Type 3 Secretion System ProteinsRevealed by High-Throughput Single-Molecule TrackingJulian Rocha1, Charles Richardson1, Mingxing Zhang1, Andreas Diepold2,Andreas Gahlmann1.1Chemistry, University of Virginia, Charlottesville, PA, USA,2Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg,Germany.In bacterial type 3 secretion, substrate proteins are actively transported fromthe bacterial cytoplasm into the cytoplasm of host cells by a largemembrane-embedded machinery called the injectisome. Injectisomes trans-port secretion substrates in response to specific environmental signals, butthe molecular details by which the cytosolic secretion substrates are selectedand transported through the type 3 secretion pathway remain unclear. Secre-tion activity and substrate selectivity are thought to be controlled by so-calledsorting platform proteins that reversibly bind to and dissociate from the cyto-plasmic side of membrane-embedded injectisomes. To determine whether andhow sorting platform proteins interact with each other in the cytosol prior totype 3 secretion system (T3SS) association, we measured the diffusive prop-erties of thousands of individual sorting platform proteins using high-throughput 3D single-molecule tracking microscopy. The obtained single-molecule trajectories reveal that the central sorting platform proteins SctQand SctL exist in several diffusive states in the cytosol of living Yersiniaenterocolitica, suggesting the formation of higher-order oligomeric proteincomplexes. We further found that formation of complexes is dependent onthe presence of other type 3 secretion system proteins and that their popula-tion fractions change upon chemical activation of the type 3 secretionpathway. These results suggest that cytosolic assembly of sorting platformproteins is important for the functional regulation of type 3 secretion. Byresolving individual diffusive states, our findings provide the first diffusivestate-resolved insights into the dynamic regulatory network that interfaces sta-tionary membrane-embedded injectisomes with the soluble cytosolic compo-nents of the T3SS.

2643-Pos Board B659Effect of Epithelial-Mesenchymal Transition on EGFR DynamicsRevealed by Single-Particle TrackingYen-Liang Liu1, Chao-Kai Chou2, Mirae Kim1, Rohan Vasisht1, Cong Liu1,Evan P. Perillo1, Hannah Horng3, Mien-Chie Hung2,4, Andrew K. Dunn1,Tim Yeh1.1Department of Biomedical Engineering, The University of Texas at Austin,Austin, TX, USA, 2Department of Molecular and Cellular Oncology, TheUniversity of Texas MD Anderson Cancer Center, Houston, TX, USA,3Department of Bioengineering, The University of Maryland, College Park,MD, USA, 4Center for Molecular Medicine and Graduate Institute of CancerBiology, China Medical University, Taichung City, Taiwan.Whereas derailed receptor trafficking is a hallmark of tumorigenesis andincreased metastatic potential of cancer cells, receptor dynamics were neverused to differentiate between metastatic and non-tumorigenic cells. Usingsingle-particle tracking techniques, we have studied the dynamics ofepidermal growth factor receptor (EGFR) in three breast cell lines (benignMCF10A, non-invasive MCF7, and highly-invasive MDA-MB-231) andshown a clear evidence that increased EGFR diffusivity and larger confine-ment size in the membrane are correlated with enhanced metastatic potentialin these cell lines. Specifically, the diffusivity of EGFR is 20% and 30%higher in MDA-MB-231 cells than in MCF7 cells and MCF10A cells, respec-tively. The size of compartments in MDA-MB-231 cells is nearly twice aslarge as those in MCF7 cells and MCF10A cells. As Epithelial-mesenchymal transition (EMT) often promotes metastasis through cytoskel-eton modifications that weaken cell cohesion and facilitate cell mobility, wehypothesize that EMT also alters receptor dynamics in the plasma membrane.Indeed, after inducing EMT in these three breast cancer cell lines, EGFR dif-fusivities significantly increase for MCF10A and MCF7 cells. In contrast, the

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EGFR diffusivity of MDA-MB-231 cells remains the same. During EMT, thechanges in cell morphology, cell polarity, motility, and cell-cell adhesion areaccompanied by the reorganization of actin filaments. As reorganization ofactin filaments also modifies plasma membrane structures through a familyof actin-binding proteins, we believe this is how the receptor dynamics changein cells going through EMT. Receptor dynamics, as revealed by single-particle tracking, can thus serve as an indicator to monitor the metastasisprocess.

2644-Pos Board B660Nanoscale Dynamics and Nuclear Envelope Organization of the MuscularDystrophy Related Protein EmerinAnthony M. Fernandez1, Markville Bautista2, Fabien Pinaud1.1MCB, University of Southern California, Los Angeles, CA, USA,2Chemistry, University of Southern California, Los Angeles, CA, USA.Emerin is a protein expressed in all somatic tissues and is part of a largergroup of nuclear envelope transmembrane proteins (NETs) responsible for awide variety of cellular functions including maintenance of nuclear shape, dif-ferentiation, nucleo-cytoskeleton mechanical signaling, and genome organiza-tion. Mutations in emerin result in Emery-Dreifuss Muscular Dystrophy(EDMD), a laminopathy which manifests in muscle degeneration, cardiac fail-ure, and early death. The mechanotransducing functions of emerin and thereasons why its mutations cause altered nuclear mechanics and muscle diseaseremain unclear. To address the pathogenesis of EDMD at the nanoscale level,we performed single molecule tracking and three-dimensional super resolu-tion microscopy of wildtype emerin and its clinically relevant mutations inhuman cells. We found that emerin diffuses in four distinct subpopulationsassociated with the endoplasmic reticulum, outer nuclear membrane, andtwo separate inner nuclear membrane populations. We have also shown thatemerin organizes into two distinct diffraction-limited nanocluster populationsat the nuclear periphery, and that this organization is disrupted by mutationsthat cause EDMD. The data also suggest that a complex interplay between thelamin A network, actin filaments, and emerin-emerin interactions dictate thedynamics and spatial distribution of emerin at the nuclear envelope. To studyhow mechanical strain on the nucleus influences the normal and pathogenicorganization of emerin, we pursued a microcontact printing strategy whichprovides exogenous control of nuclear architecture combined with nanometeraccuracy single molecule microscopy. By subjecting cells to varying nuclearstrain, we found that the mechanosensing functions of emerin are coupled toits molecular dynamics and nanoscale organization. Together, these singlemolecule and cell micropatterning experiments reveal the adaptive biophysi-cal properties of emerin and help to elucidate the nanoscale perturbations thatoccur in muscular dystrophy.

2645-Pos Board B661Variable-Angle Total Internal Reflection Fluorescence Microscopy:Exploring Integrin-mediated AdhesionDalia El Arawi1, Cyrille V�ezy1, Monique Dontenwill2, Maxime Lehmann2,Rodolphe Jaffiol1.1Laboratory of Nanotechnology, Instrumentation and Optics (LNIO),University of Technology of Troyes, Troyes, France, 2Laboratory ofBiophotonics and Pharmacology (LBP), University of Strasbourg, Illkirch,France.Cell adhesion plays a global role in cell communication and regulation. Dur-ing adhesion, cells interact with each other and with their environment,through a broad range of specific and nonspecific interactions. The specificcell binding is regulated by lock-and-key mechanisms, related to the presenceof transmembrane proteins, such as integrins. These integrins can specificallyrecognize ligands and are the keystone of focal adhesion complexes (FA).Cell adhesion also results from the synergy between long-range and short-range cell-substrate nonspecific interactions, mediated for example, bycellular glycocalyx. Therefore, our team recently proposed a real-time imag-ing technique to probe simultaneously the specific and the nonspecific aspectsof adhesion process by measuring distances from the substrate to the plasmamembrane, and to quantify adhesion strength at the single cell level. Thistechnique, called variable-angle total internal reflection fluorescence micro-scopy (va-TIRFM), allows us to map the membrane-substrate separation dis-tance, with a nanometric axial resolution. Va-TIRFM can also be used toextract the profile of potential energy related to membrane/substrate interac-tions, which reflects an energy of adhesion. Recently we used va-TIRFM tostudy live glioblastoma metastatic cells in adhesion on fibronectin. By block-ing the adhesion signaling pathways using very well-known alpha-5 beta-1and alpha-v beta-3 integrin antagonists, we showed a change in FA organiza-tion from local points to adhesion plaques, which can take up to 50 percent ofcell surface, followed by glycocalyx remodeling. Surprisingly these

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modifications were accompanied by a decrease of membrane/substrate dis-tance and energy of adhesion.

2646-Pos Board B662Superresolution Microscopy of the T Cell Receptor in the ImmunologicalSynapseFlorian Baumgart1, Benedikt K. Rossboth1, Andreas M. Arnold1,Mario Brameshuber1, Haisen Ta2, Ren�e Platzer3, Johannes B. Huppa3,Gerhard J. Sch€utz1.1Applied Physics/ Biophysics, Vienna Universty of Technology, Vienna,Austria, 2Biophysical Chemistry, Max Planck Institute, Gottingen, Germany,3Institute for Hygiene and Applied Immunology, Medical University ofVienna, Vienna, Austria.Single molecule localization-based superresolution microscopy has been usedto report the presence of protein nanoclusters at the T cell plasma membrane.In particular, the T cell receptor (TCR) complex has been suggested to benon-randomly organized in resting T cells (1, 2), which has led to a varietyof models for specific antigen recognition and T cell activation. We have pre-viously found that characteristic blinking and resulting overcounting of fluores-cent proteins and organic dyes may be easily misinterpreted as molecularclusters. As a possible solution to the problem, we developed a method todiscriminate between true protein clusters and multiple counts of the samedye molecules (3).Here, we applied this method to study the organization of the TCR complex inthe plasma membrane of resting and activated T cells. While there was clearevidence for the formation of clusters upon activation, no indication of non-homogenous distribution could be obtained under resting conditions. In inde-pendent experiments, we confirmed our observations with STED microscopy,yielding similar results. Overall, our data do not support the view of proteinnanoclustering being a general ordering principle at the T cell plasmamembrane.(1) Lillemeier et al. Nat Immunol. 2010 Jan;11(1):90-6.(2) Pageon et al. Proc Natl Acad Sci U S A. 2016 Sep 13;113(37).(3) Baumgart et al. Nat Meth. 2016;13(8):661-4.

2647-Pos Board B663Study of Tumor Cellular Damage Induced by Photosensitizing MoleculesMarco Cozzolino1,2, Luca Pesce1,2, Michele Oneto1, Chiara Montali3,Paolo Bianchini1,4, Stefania Abbruzzetti3, Cristiano Viappiani3,Alberto Diaspro1,2.1Nanoscopy, Istituto Italiano Tecnologia, genoa, Italy, 2Physics, Universityof Genoa, Genoa, Italy, 3Mathematical, Physical and Computer Sciences,University of Parma, Parma, Italy, 4Dibris, University of Genoa, Genoa, Italy.Photodynamic therapy (PDT) is a tumor treatment that uses a combination ofotherwise non-toxic chemicals, termed photosensitizers (PSs) and visible lightto produce reactive oxygen species capable of destroying tumor cells [Dough-erty TJ (1993)][Agostinis P. et al. (2011)].PS molecules are often insoluble in water and form aggregates, which impairstheir photophysical properties, generally resulting in a quenching of the produc-tive excited states. To overcome this limit, PSs carriers have been devised[Bechet D. et al. (2008) ][Konan, Y. N. et al. (2002)].Among them, proteins offer several potential advantages. Hydrophobic PSmolecules spontaneously bind to internal hydrophobic cavities of the propersize, preserving their monomeric, photoactive (both photosensitizing and fluo-rescent) state. [Sharman, W. M. et al. (2004)].Here we identify the uptake of Hypericin and Zinc Phthalocyanine (eitherdelivered alone or using Apomyoglobin as a carrier) in living cells directlyfrom fluorescent emission using various microscopy techniques such as spin-ning disk, light sheet, and two-photon microscopy.We aim to investigate whichcellular components are involved in the damage by the photosensitized ROS,and to understand which are the differences in the accumulation, cellular dam-age, and general efficacy between these PSs and their respective constructs withApomyoglobin.

2648-Pos Board B664MannanMolecular Sub-structures Control Nanoscale Glucan Exposure inCandidaMatthew S. Graus1, Michael Wester2, Douglas W. Lowman3,David L. Williams4, Michael D. Kruppa5, Jesse M. Young6, Harry C. Pappas1,Keith A. Lidke7, Aaron K. Neumann1.1Pathology, School of Medicine, University of New Mexico, Albuquerque,NM, USA, 2Department of Mathematics and Statistics, University of NewMexico, Albuquerque, NM, USA, 3AppRidge International, LLC, Telford,TN, USA, 4Surgery, Quillen College of Medicine, East Tennessee StateUniversity, Johnson City, TN, USA, 5Biomedical Sciences, Quillen Collegeof Medicine, East Tennessee State University, Johnson City, TN, USA,

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6Molecular Genetics and Microbiology, School of Medicine, University ofNew Mexico, Johnson City, TN, USA, 7Physics and Astronomy, Universityof New Mexico, Albuquerque, NM, USA.N-linked mannans (N-mannans) in the cell wall of Candida albicans arethought to mask b-(1,3)-glucan from recognition by Dectin-1, contributingto innate immune evasion. Lateral cell wall exposures of glucan on C.albicans are predominantly single receptor-ligand interaction sites and arerestricted to nanoscale geometries. Candida species exhibit a range of basalglucan exposures and their mannans also vary in size and complexity at themolecular level. We used super resolution fluorescence imaging and a seriesof protein mannosylation mutants in C. albicans and C. glabrata to investigatethe role of specific N-mannan features in regulating the nanoscale geometry ofglucan exposure. Decreasing acid labile mannan abundance and a-(1,6)-mannan backbone length correlated most strongly with increased densityand nanoscopic size of glucan exposures in C. albicans and C. glabrata,respectively. Additionally, a C. albicans clinical isolate with high glucanexposure produced similarly perturbed N-mannan structures and exhibitedsimilar changes to nanoscopic glucan exposure geometry. We conclude thatacid labile N-mannan controls glucan exposure geometry at the nanoscale.Furthermore, variations in glucan nanoexposure characteristics are clinicallyrelevant and are likely to impact the nature of the pathogenic surface pre-sented to innate immunocytes at dimensions relevant to receptor engagement,aggregation and signaling.

2649-Pos Board B665STED Nanoscopy of the Centrosome Linker Reveals a CEP68-Organized,Periodic Rootletin Network Anchored to a C-Nap1 Ring at CentriolesRifka Vlijm1, Xue Li2, Marko Panic2, Diana R€uthnick2, Shoji Hata2,Frank Herrmannsdorfer3, Thomas Kuner3, Mike Heilemann3,Johann Engelhardt1, Stefan W. Hell1,4, Elmar Schiebel2.1Optical nanoscopy, Max Planck Institute for Medical Research, Heidelberg,Germany, 2Deutsches Krebsforschungszentrum (DKFZ)–ZMBH Allianz,Universit€at Heidelberg, Zentrum f€ur Molekulare Biologie der Universit€atHeidelberg (ZMBH), Heidelberg, Germany, 3Department of FunctionalNeuroanatomy, Institute for Anatomy and Cell Biology, Heidelberg,Germany, 4Department of NanoBiophotonics, Max Planck Institute forBiophysical Chemistry, Gottingen, Germany.The centrosome linker proteins C-Nap1, rootletin and CEP68 connect thetwo centrosomes of a cell during interphase into one microtubule orga-nizing centre. This coupling is important for cell migration, cilia forma-tion and timing of mitotic spindle formation. Very little is known aboutthe structure of the centrosome linker. Here, we used STimulated Emis-sion Depletion (STED)microscopy to show that each C-Nap1 ring at theproximal end of the two centrioles organizes a rootletin ring and in addi-tion multiple rootletin fibres that radiate outwards from the ring into thecytoplasm. Rootletin filaments have a repeat organization of 75 nm andbind CEP68 via its C-terminal spectrin repeat containing region in 75nm intervals. CEP68 is essential in forming rootletin filaments that branchoff centrioles and modulates the thickness of rootletin fibres. Thus, thecentrosome linker consists of a vast network of repeating rootletin unitswith C-Nap1 as ring organizer and CEP68 as filament modulator. Thepunctual contact model is consistent with the biological properties ofthe centrosome linker.

2650-Pos Board B666Quantitative Microscopy Pipeline for Building a Model of the HumanCellWinfried Wiegraebe, Allen Institute for Cell Science Team.Microscopy & Image Processing Pipeline, Allen Institute for Cell Science,Seattle, WA, USA.We aim to replace the typical textbook artistic rendering of a cell with a modelcreated in silico, based on large datasets of 3D images of genome-edited livecells collected by light microscopes.We have created about 20 human induced pluripotent stem cell lines expressingendogenously EGFP tagged proteins that localize to the major cellular organ-elles. We plate and feed the cells, using a robot, in 96-well glass bottom platesand image them using spinning disk microscopy. So far, we have collected 3Dimages from more than 15,000 live cells, comprised of high replicates for eachgenome-edited cell line. We can also apply drugs externally or store the sampleovernight in an incubator. We can prescreen the sample on a slide scanner andthen image areas of interest with higher resolution and better image quality onone of the spinning disk microscopes.As a part of our workflow, we wrote software to identify the same sample arearepeatedly and on different microscopes. This will allow us to revisit the samelocation during long-term time-lapse experiments after imaging other cells. The

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software is compatible with any microscope brand and is thus vendor indepen-dent. Only a thin communication layer between our software package and themacro interface of the vendor software needs to be updated to adapt thesoftware to other systems.We are using machine learning approaches to combine this information into apredictive model for protein localization. We will present the outline of thepipeline and the components involved. We will also present our workflow qual-ity control criteria, and the methods we have developed to ensure day-to-dayconsistency between our data sets. All our procedures, tools and data are sharedon our webpage www.allencell.org.

2651-Pos Board B667Expansion Microscopy: A Tool to Investigate Hutchinson-GilfordProgeria Syndrome at Molecular LevelLuca Pesce1,2, Marco Cozzolino1,2, Luca Lanzano1, Alberto Diaspro1,3,Paolo Bianchini1,4.1Istituto Italiano Tecnologia, Genova, Italy, 2Department of Physics,University of Genoa, Genoa, Italy, 3University of Genoa, Department ofPhysics, Italy, 4University of Genoa, Dibris, Italy.Hutchinson-Gilford Progeria Syndrome (HGPS) is a lethal congenital disor-der characterized by premature aging symptom in children and early death.It is often used as model to understand the molecular mechanism of aging.This condition is caused by a single nucleotide substitution which leads acryptic splice site in the C-terminal Lamin A. The process gives rise to a path-ological protein without 50 amino-acid called LAD50, whose expression in-duces many cellular defects, as lobulated nuclei, loss of peripheralheterochromatin, accumulation of DNA damage, telomere aberration, clus-tering of nuclear pores, leading to premature cellular senescence (Schreiberet al. 2013). In addition, different forms of cancer are associated with thesecharacteristics.Expansion Microscopy (ExM) is a novel method that allows super-resolutionimaging with conventional microscopes (Chozinski et al. 2016). It consists insoaking the cells with a polymer, inducing the polymerization to form a densemeshwork throughout the cell, cross-linking the proteins to the polymer and,after digestion, rehydrating of the sample. The swelling of the polymer gelleads to a fourfold isotropic stretching of the sample, thereby increasing the dis-tance between objects that otherwise couldn’t be resolved with an ordinarymicroscope.Here, we apply ExM to visualize nanoscale alterations of nuclear organiza-tion in a cellular model of HPGS. In particular, by using cells transfectedwith LAD50-GFP we focus on nuclear blebs, i.e. protrusions from nuclearsurface whose mechanisms of formation and pathological role are stilllargely unknown (Funkhouser et al. 2013). In addition to Lamin structure,we also image the organization of heterochromatin-associated DNA inmisshapen nuclei. Finally, we discuss the combination of ExM with STEDnanoscopy (Bianchini et al. 2015) to further increase its effective spatialresolution.

2652-Pos Board B668Single Molecule Imaging of Chromatin Remodeling in Live CellsCharles A. Kenworthy1, Vincent Wong1, Patrycja Dziuba1, Luke D. Lavis2,Wei-Li Liu1, Robert H. Singer1,2, Robert A. Coleman1.1Anatomy and Structural Biology, Albert Einstein College of Medicine,Bronx, NY, USA, 2Howard Hughes Medical Institute Janelia ResearchCenter, Ashburn, VA, USA.ATP-dependent chromatin-remodeling complexes such as PBAF mediatechanges in chromatin structure, leading to dynamic regulation of transcrip-tional bursting. PBAF is targeted to genomic loci by histone acetylation.Despite extensive in vitro studies, how these chromatin remodelers rapidlybind and discriminate genomic targets in vivo remains unclear. Therefore,we sought to understand how the PBAF complex interacts with different chro-matin states using live-cell single molecule fluorescence microscopy. Dualcolor tracking revealed that PBAF binds H3.3 marked chromatin withinactively transcribed regions for faster time periods relative to binding toHP1a containing heterochromatin. Notably, elevation of histone acetylationlevels increased the frequency of PBAF revisiting to genomic foci as definedby clustered binding. Furthermore, deletion of bromodomains within PBAFreduced chromatin target search efficiency, clustered binding activity, andanchoring to the genome. These findings suggest that acetyl-lysine dependentclustered binding of PBAF to select genomic loci may facilitate rapid chro-matin remodeling in actively transcribed regions. Our work also indicatesthat the dynamics of PBAF mediated chromatin state alterations proceedat fast timescales that may fine-tune transcription regulation. We willfurther discuss our efforts to directly monitor PBAF mediated nucleosomemobilization in live cells.

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2653-Pos Board B669Studying Variations in CEACAM1 Nanoscale Organization, Structure,and DynamicsAmine Driouchi1, Christopher M. Yip2.1Biochemistry, University of Toronto, Toronto, ON, Canada, 2ChemicalEngineering, University of Toronto, Toronto, ON, Canada.CEACAMs are cell surface glycoproteins involved in homo- and hetero-philicintercellular interactions that control cellular growth, differentiation, tumouri-genesis, inflammation and infection. There is growing evidence regarding therole that CEACAM1 isoforms play in regulating interactions between variouscell types. In particular, given its potential as a tumour suppressor or immuno-modulator,CEACAM1 is being targeted in clinical trials. However, there remainclear challenges owing to observation that different CEACAM1 isoformsdisplay cell-type specific preferential expression patterns under different physi-ological conditions. Moreover, the ability of CEACAMs to form dimers andhigher-order oligomers, which are thought to impact regulation of intercellularsignals, adds considerable complexity to our understanding of its functionalrole(s). Super resolution microscopy has enabled the quantitative characteriza-tion of the nanoscale distribution of many membrane proteins, including theirability to form both micro- and nano-scale clusters. However, clustering andself-association are not necessarily the same and mapping how oligomeric stateimpact clustering and / or the oligomeric state of the proteinswithin a cluster is ofparticular interest tomany.We report here on the development and application ofa correlative STORM/homoFRET strategy to study CEACAM1 isoforms andmutants. This approach exploits eYFP for homoFRET measurements, an AlexaFluor 647 (AF647) labeled an anti-eYFP single domain antibody (nanobody) fordSTORM, andVoronoi tessalation to perform cluster analysis of dSTORMdata-set. Building on these data, we have also applied co-localization approaches tomeasure the CEACAM1 association to lipid-ordered regions, ezrin, and actin.We have further explored the use of single particle tracking (SPT) and meansquare displacement analysis (MSD) to characterize CEACAM1 dynamics asa function of oligomeric state and location. These studies have helped developa more fulsome perspective on the structure, association, and dynamics of thisimportant class of membrane proteins.

2654-Pos Board B670Single Molecule Study of the Mechanism of Attack of the Human Antimi-crobial Peptide LL-37 on E. coliYanyu Zhu, Sonisilpa Mohapatra, James Weisshaar.University of Wisconsin Madison, University of Wisconsin Madison,Madison, WI, USA.Evolutionarily conserved anti-microbial peptides (AMPs) are useful proto-types and promising drugs due to increasing antibiotic resistance. We haveused single-cell, time-resolved fluorescence microscopy and single-moleculefluorescence microscopy to study the effects of human cathelicidin LL-37on the E. coli chromosome (visualized through ParB-GFP), RNA polymerase(RNAP), ribosome and cytoplasmic protein Kaede. This enables us to corre-late in real time the motion reduction of chromosome and RNAP, the haltingof cell growth, and the permeabilization of the cytoplasmic membrane (CM)by LL-37. We have found that LL-37 could change the spatial distribution ofribosome and reduce the motion of chromosomal loci, RNAP, ribosome andKaede. For chromosome, the apparent diffusion coefficient D decreased by10 times on both long (� min) and short (� s) timescales 10 min after inject-ing 1x MIC (minimum inhibitory concentration) LL-37, larger than thosetreated with the ATP-depleting drugs NaN3 and CCCP, suggesting LL-37might have effects on chromosome beyond energy depletion. Besides, someof the sharp clear foci of ParB complex dissolved into broader but dimmerfoci. We speculate that LL-37 dissociates the ParB complex by disruptingthe protein-protein or protein-DNA interactions. The general ability of adrug to disrupt protein-DNA interactions would be a catastrophe for infectiousbacteria. It indicates that LL-37 has multi-target effects and its damage to bac-terial cells goes far beyond the membrane permeabilization. This may make itharder for bacteria to gain resistance. A comprehensive understanding of allthese effects and how they are interlinked with each other could be of medicalsignificance.

2655-Pos Board B671Spatial Distribution of H-NS in E. coli under Environmental StressNafiseh Rafiei1,2, William Navarre3, Joshua N. Milstein1,2.1Institute of Biomaterials and Biomedical Engineering, University ofToronto, Toronto, ON, Canada, 2Department of Chemical and PhysicalSciences, University of Toronto, Mississauga, ON, Canada, 3Department ofMolecular Genetics, University of Toronto, Toronto, ON, Canada.The heat-stable nucleoid structuring (H-NS) protein is an abundant DNA-binding protein in gram-negative bacteria. H-NS impacts both DNA

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condensation and globally regulates gene expression by binding to AT-richdomains of DNA. H-NS is known to play a key role in the adaptation ofbacteria to thermal and osmotic shifts in the environment, although themechanism is still poorly understood. In this study, we probed thespatial redistribution of H-NS in E. coli via super-resolved microscopy inresponse to osmotic stress and cold-shock at two phases of bacterialgrowth: exponential and stationary phase. While we saw little effect underconditions of cold-shock, we observed that in early stationary phase, underosmotic stress, H-NS markedly alters its intracellular organization, detach-ing from a tightly condensed chromosome, and moving toward the periph-ery of the cell. This response by H-NS to osmotic shock, notably, is notobserved in exponential phase despite a similar condensation of the chro-mosomal DNA. To understand this observation we have been exploringthe response of H-NS to other key regulators of the osmotic stressresponse.

2656-Pos Board B6723D Single-Molecule Tracking of Confined Diffusers: Resolving Intracel-lular Diffusive States in Living Bacterial CellsTing Yan, Julian Rocha, Alecia Marie Achimovich, Andreas Gahlmann.Chemistry, University of Virginia, Charlottesville, VA, USA.The spatial trajectory of a single protein in the cytosol of a living cell con-tains information about its molecular interactions in the native environ-ment. However, it remains challenging to resolve different intracellulardiffusive states, which could indicate the formation of biologically relevantmolecular complexes that do not form in vitro. This problem is especiallypronounced when experimentally measured single-molecule trajectories areshort, few in numbers, and spatially confined. Here, we show that intracel-lular diffusive states can be accurately resolved if sufficiently largenumbers of single-molecule trajectories are available, so that a well-sampled distribution of apparent diffusion coefficients can be analyzed.We introduce an empirical data analysis framework based on Monte Carlosimulations of confined Brownian motion that is able to recover the truediffusion coefficients of freely diffusing molecules in arbitrarily shapedconfinement volumes. To validate this framework experimentally, we usehigh-throughput 3D single-molecule tracking microscopy to measure thetrajectories of 10,000-100,000 fluorescent proteins and fluorescent fusionproteins in rod-shaped bacterial cells. The data show that the diffusiveproperties of different types of fluorescent proteins vary widely and addi-tionally depend on the bacterial species in which they are expressed. Thesefindings highlight the need for in situ control experiments, to first deter-mine the diffusive properties of the fluorescent protein label itself, beforeconclusions can be drawn about native molecular interactions of the labeledproteins.

2657-Pos Board B673Single Virion Super-Resolution Microscopy Unveils Mechanistic Detailsof Env Glycoprotein Recognition by the Broadly Neutralizing HIV-1Antibodies 4E10 and 10E8Pablo Carravilla1, Edurne Rujas1, Itziar R Oar-Arteta1, Sara Insausti1,Eneko Largo1, Jakub Chojnacki2, Taylor Sicard3, Jean-Philippe Julien3,Christian Eggeling2, Nerea Huarte1, Jos�e Requejo-Isidro1, Jos�e L Nieva1.1Biofisika Institute. Department of Biochemistry and Molecular Biology,University of the Basque Country, Bilbao, Spain, 2MRCHuman ImmunologyUnit, Weatherall Institute of Molecular Medicine, University of Oxford,Oxford, United Kingdom, 3Program in Molecular Medicine, The Hospital forSick Children Research Institute, Toronto, ON, Canada.Anti-HIV broadly neutralizing antibodies (bnAb-s) 4E10 and 10E8 bind theirepitopes on the membrane proximal external region (MPER) of the Env sur-face glycoprotein. The exact mechanisms of recognition and neutralization ofthe native Env remain obscure. Both antibodies present an unusually longCDR-H3 loop that is essential for function and shown to interact with lipidbilayers. Here, using fluorescence correlation spectroscopy we characterizefirst the nature of antibody-lipid interactions and demonstrate that both, mem-brane molecular order and electrostatic surface charge, determine the behaviorand diffusivity of anti-MPER bnAb-s. Super-resolution single virus experi-ments using a collection of engineered antibodies and different virus strainsfurther demonstrate that, although 10E8 and 4E10 do not bind directly tothe viral lipid envelope, antibody-membrane electrostatic and hydrophobic in-teractions are essential for Env recognition. Moreover, we find a strong cor-relation between neutralization potency and extent of binding to Env. Theseresults shed light on the role of the hydrophobic CDR-H3 loop and delineatethe molecular features that are essential for the recognition and blocking ofthe native Env.

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2658-Pos Board B674Structure and Dynamics of the Trypanosome Plasma MembraneMarius Glogger, Markus Engstler, Susanne Fenz.Biocenter: Cell and Developmental Biology, Wuerzburg University,Wuerzburg, Germany.African trypanosomes are the causative agents of sleeping sickness in hu-mans and Nagana in livestock. During their lifecycle the single-celled par-asites adapt to the environment of the vector and the host. In thebloodstream of their host, they exhibit a dense coat of GPI-anchored variantsurface glycoproteins (VSG). This coat is a central element of the parasite’sstrategy to evade the host’s immune system. Fluidity of the VSG coat is aprerequisite of its proper shielding function. However, the diffusionbehavior of the VSGs is limited by the physical properties of their lipid ma-trix. We have recently introduced super-resolution imaging of intrinsicallyfast moving flagellates based on cyto-compatible hydrogel embedding.Building on this work we employ leaflet-specific membrane probes andsingle-molecule fluorescence microscopy to elucidate the structure anddynamics of the plasma membrane in living trypanosomes. Using lipid-anchored eYFP as a probe for the inner membrane leaflet, we found specificdomains where the fluorescence either accumulates or appears diluted ratherthan being homogenously distributed. We hypothesize that this structuringof the membrane is associated with the underlying cytoskeleton. The nextsteps include employment of a more stable fluorescent label to achieveconclusive information on the dynamic interaction of single probes withthe observed domains. Moreover, we aim to track fluorescently labeledlipids in the outer leaflet to gain insight in inter-leaflet coupling in vivo,and we plan a two-color experiment to simultaneously investigate mem-brane and VSG dynamics.

2659-Pos Board B675Bioluminescence Resonance Energy Transfer (BRET)-based Imaging ofG-Protein Coupled Receptor Signaling and TraffickingHiroyuki Kobayashi, Louis-Philippe Picard, Anne-Marie Schonegge,Michel Bouvier.Biochemistry and Molecular Medicine, IRIC, University of Montreal,Montreal, QC, Canada.Bioluminescence resonance energy transfer (BRET) is a proximity-basedassay based on the energy transfer between a luminescent donor and fluores-cent acceptor. Because BRET occurs only when the distance between thedonor and the acceptor is less than 10 nm, it is frequently used in monitoringprotein-protein interaction and conformational rearrangements in live cells.Compared with fluorescence-based assays such as FRET, the luminescence-based BRET assays have the advantage of not inducing any phototoxicityor autofluorescence. However, the use of BRET has been largely limited tospectrometric measurements because of the low luminescence output thatlimits the spatio-temporal resolution required for high quality imaging.Recent advance in detection devices with higher sensitivity and more efficientBRET probes open new opportunities for the development of BRET-basedimaging. Here we report that by selecting of proper BRET probes, configura-tions and luciferase substrates can significantly increase the signal-to-noiseratio of the BRET image. In particular, we combined long Stokes’ shift fluo-rescent proteins such as CyRFP with high signal intensity luciferase such asNanoLuc to simultaneously image protein-protein interactions and subcellu-lar re-distribution of proteins of interest with high spatio-temporal resolution.Using G-alpha and G-gamma protein subunits fused to BRET donors andacceptors we monitored both subunit dissociation and translocation uponG protein-coupled receptor stimulation. Similarly, the recruitment of beta-arrestin to GPCRs and the following endocytosis of the receptor-beta-arrestin complex could be monitored in real time using enhanced bystanderBRET (ebBRET) that is based on the energy transfer between naturally inter-acting luciferase and green fluorescent protein from Renilla sea pansy. Takentogether, these data clearly show that BRET is a valid alternative to FRET forreal time imaging in living cells.

2660-Pos Board B676Quantitative Super-Resolution Imaging Reveals Mammalian GlycocalyxDynamicsLeonhard Moeckl, Kayvon Pedram, Anish Roy, Carolyn Bertozzi,William Esco Moerner.Stanford University, Stanford, CA, USA.Despite the long-held knowledge that each mammalian cell is surrounded by aglycocalyx – a complex layer of glycoproteins, proteoglycans, and glycolipidswith a vast number of constituents –, its detailed organization has remainedenigmatic, even though its relevance in biological and medical contexts is

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undisputed. There are three main reasons for this surprising elusiveness: First,the highly amorphous nature of the glycocalyx makes many conventionalmethods, for example genetics, hard to apply; second, the huge number ofstructurally similar building units heavily impedes specific labeling; andfinally, its axial thickness is typically in the range of hundreds of nanometers,meaning that conventional microscopy fails to resolve fine structures. In ourwork, we combined highly specific labeling protocols to target fluorophores tosingle building units of glycocalyx constituents and apply quantitative super-resolution microscopy. We were able to extract for the first time specific in-sights into the organization of the mammalian glycocalyx. We found thatsialic acids and GalNAc, two prominent sugars present in the glycocalyx,both extend over its whole range with sialic acids reaching further into theextracellular space. A combination of super-resolution microscopy withflow cytometry and controlled activation of glycosylation pathways allowedus subsequently to investigate the relation between the glycocalyx thicknessand the presence of glycocalyx building units. Here, we could show that theamount of sialic acids and the glycocalyx thickness are uncoupled. This indi-cates that not only the mere presence of glycocalyx building units, but also theway they are anchored within the glycocalyx determine the physical proper-ties of the structure. Taken together, our work allows us for the first time toaccess thickness and relative positioning details about the organization ofthe mammalian glycocalyx, which should contribute to a detailed understand-ing of this crucial cellular feature.

2661-Pos Board B677Time Resolved Intensity Photobleaching - A Novel Method for StudyingProteins in Live CellsYuval Garini, Eugene Brozgol.Physics, Bar Ilan University, Ramat Gan, Israel.We present a method for exploring proteins in living cells. In a single mea-surement, the method enables to reveal a significant number of quantitativeproperties of the proteins dynamics including the diffusion coefficient, boundand free fraction, protein concentration and binding-unbinding kinetics. Themethod combines in a single measurement quantitative photobleaching, fluo-rescence correlation spectroscopy and fluorescence lifetime imaging. After la-beling the protein of interest with a fluorescent protein, we use timecorrelation single photon counting to measure the fluorescence intensity atfixed points in the sample. The information provides both the fluorescent in-tensity as a function of time for �60 seconds, the rapid intensity fluctuations(KHz) and the decay time of each arriving photon. This information is pro-cessed according to a procedure that takes the biophysical dynamic modelof a protein into account and the full kinetic behavior of the protein isrevealed.

2662-Pos Board B678The Microscopic Structure of Crunchy and Crispy JellyfishMie T. Pedersen1, Morten Christensen1, Lars Duelund1, Per L. Hansen2,Jonathan R. Brewer3, Mathias P. Clausen1.1SDU Biotechnology, University of Southern Denmark, Odense, Denmark,2University of Southern Denmark, Odense, Denmark, 3Department ofBiochemistry and Molecular Biology, University of Southern Denmark,Odense, Denmark.The empirical world of gastronomy is full of fascinating transformations offood material. This applies to the types of dishes served in high-end restau-rants as well as traditional preparations of food served around the globe.Whereas the impact of delicious food on everyday human wellbeing is indis-putable, our scientific understanding of gastronomic phenomena is surpris-ingly limited.Using tools from modern biophysics and -chemistry, we aim at gaining a betterunderstanding of the (supra-) molecular behavior of the types of biological softmatter materials that food is, and at relating the molecular properties of foodwith our sensory experience while eating.As an example, we have studied jellyfish - a food material mostly uncommonto the Western palate, but a delicacy in traditional Asian cuisine having agastronomic history of more than a thousand years. It is eaten mainly for itsinteresting crunchy mouthfeel resulting from a month-long salt preservationusing sodium chloride and alum. This preservation drastically changes thetexture of the jellyfish from being gel-like to resembling that of pickledcucumbers.We have used state-of-the-art two-photon microscopy and super-resolutionSTED microscopy to visualize the rearrangements in the filamentous networkconstituting the jellyfish mesoglea gel during the transformation from a soft gelto a crunchy texture. We further interpret our data in light of polyelectrolytetheory and a modified Flory-Higgins theory that describes ionic gel collapse

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in poor solvent to suggest an alternative preservation method. Using ethanol,we thus have created what can be classified as jellyfish chips that has a crispytexture and could be of potential gastronomic interest.

2663-Pos Board B679Molecular Mechanism of Antimicrobial Activity of Low DC VoltageAgainst E. coliVenkata Rao Krishnamurthi1, Ariel Rogers1, Janet Peifer1, Yong Wang1,2.1Department of Physics, University of Arkansas, Fayetteville, AR, USA,2Cell and Molecular Biology Program, Microelectronics-Photonics Program,University of Arkansas, Fayetteville, AR, USA.Infections caused by drug-resistant bacteria have become a global threat,which creates an urgent need of alternative strategies for combating path-ogenic bacteria. It has been known for a long time that high electric volt-ages/currents kill bacteria, while more recently, it was reported that lowelectric currents and fields also show antimicrobial activities. However,very few investigations have been carried out to understand the underlyingmechanism at the microscopic scale, and the molecular mechanism of anti-microbial activity of low electric voltages/currents is unclear. In this study,we attempt to address this knowledge gap. We observed that E. coli bacte-ria subjected to low DC voltages > 1V showed significant membrane dam-age and cell death. At lower DC voltages <=1V, we observed changes ofthe bacterial morphology: the cell-length became shorter and the bacterialost their rod-shape. This observation intrigued us to investigate how thespatial organization of MreB (an essential shape-maintaining protein inE. coli) is affected by low DC voltages, using quantitative single-molecule localization microscopy (SMLM), in which individual moleculesare localized with a spatial resolution of �20 nm. Furthermore, SMLM wasapplied to examine how low electric voltages influence the distribution ofhistone-like structuring (H-NS) protein, a universal key protein for chromo-some organization and genetic regulation. We observed spatial reorganiza-tion of the molecules in the presence of low DC voltages (<=1V). Inaddition, we varied the magnitude and treatment time of DC voltages, todetermine the quantitative dependences. It is expected that our study ofthe mechanism of low electric voltages at the molecular level will shedlight on its future applications of fighting against antibiotic resistantbacteria.

2664-Pos Board B680Determining how Pemphigus Vulgaris Impacts the Nanoscale Architectureof DesmosomesTara Urner1, Emily Bartle1, Tejeshwar Rao1, Andrew Kowalczyk2,Alexa Mattheyses1.1Cell, Developmental and Integrative Biology, University of Alabama atBirmingham, Birmingham, AL, USA, 2Cell Biology, Emory University,Atlanta, GA, USA.Desmosomes are macromolecular cell-cell junctions that confer mechanicalstrength to epithelial tissues. These complexes contain adhesive transmem-brane cadherin proteins attached to dense intracellular plaques which arecoupled to the intermediate filament cytoskeleton. In the devastating autoim-mune disease Pemphigus Vulgaris (PV), autoantibodies target the adhesivedomain of the transmembrane cadherin desmoglein 3 (Dsg3), causing severeskin and mucosal blistering. Although advances in treatments for the diseasehave improved clinical outcomes, the pathomechanisms underlying PVremain unknown. It is hypothesized that both steric hindrance of cadherinbinding by PV immunoglobulin G (IgG) and aberrant cell signaling play arole in the disease, but the mechanism driving the weakened adhesion remainsunknown. One obstacle to studying PV pathogenesis is the relative inaccessi-bility of structural information about the desmosome due to its size (approx-imately a half micron in diameter) and molecular complexity. To overcomethis obstacle we utilized two complimentary fluorescence microscopy tech-niques to study nanoscale architectural changes in desmosomes induced byPV IgG binding. The 20 nm spatial resolution of dSTORM allowed us todirectly observe the time-scale and localization of PV IgG binding. UsingdSTORM, we investigated possible nanoscale rearrangement within desmo-somal plaques in response to binding. Using fluorescence polarization micro-scopy, in combination with a Dsg3-GFP chimeric probe, we measured howPV IgG binding impacts the organization of Dsg3 in live cells. This novelapproach allowed direct visualization of how PV IgG binding disrupts Dsg3ordering within the adhesive interface, a dimension of PV pathogenesisinaccessible by other assays. We anticipate that dSTORM and order detectionvia fluorescence polarization microscopy will be powerful tools for investi-gating the disruption of desmosomal structure and function underlying thepathogenesis of PV.

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2665-Pos Board B681Super-Resolution Imaging of DNA Replisome Dynamics in Live BacillussubtilisYilai Li1, JeremyW. Schroeder2, Yi Liao3, Ziyuan Chen1, Lyle A. Simmons2,Julie S. Biteen3.1Biophysics, University of Michigan, Ann Arbor, MI, USA, 2Molecular,Cellular, and Developmental Biology, University of Michigan, Ann Arbor,MI, USA, 3Chemistry, University of Michigan, Ann Arbor, MI, USA.DNA replication happens in all living organisms and assures that the genomeis accurately copied and maintained. The replisome is the molecular machinein cells that replicates DNA, and this protein assembly includes DNA poly-merases which directly synthesize DNA by adding nucleotides. Although thebacterial replisome has been studied extensively in vitro, single-moleculemicroscopy is now providing a new perspective on the dynamics and archi-tecture of replisome components in vivo. Here we study the architecture anddynamics of several highly conserved replisome components in vivo in themodel organism Bacillus subtilis. Photoactivated localization microscopy(PALM) and single-molecule tracking enable us to localize and track everysingle protein molecule with a resolution of 20 - 40 nm. In our study, weinvestigate the dynamics of a number of replisome components underdifferent conditions, including the replicative DNA polymerases PolC andDnaE and the b-clamp loader DnaX. We study quantitatively the real-timebehavior of different replisome components during the DNA synthesis pro-cess. Surprisingly, our investigations have revealed that all of these repli-some components are highly dynamic and exchange more rapidly thanpreviously expected, and we characterize the molecular scale distributionof each replisome component as well as responses to cellular mutationsand external stimuli with a combination of single-molecule tracking, time-lapse imaging, and spatiotemporal image correlation spectroscopy. Overall,these new insights into DNA replication indicate that the activities of bacte-rial replisomal proteins may be regulated in cells by coordinating and modu-lating the dynamics of protein recruitment, binding, and unbinding at the siteof DNA synthesis.

2666-Pos Board B6823DArchitecturalReconstructionofMammalianCentrioleDistalAppendagesusing Superresolution MicroscopyT Tony Yang1, Weng Man Chong1, Zhengmin Chen1, Meng-Fu Bryan Tsou2,Jung-Chi Liao1.1Academia Sinica, Taipei, Taiwan, 2Memorial Sloan-Kettering CancerCenter, New York, NY, USA.The primary cilium is an essential microdomain of cells grown upon themother centriole/basal body serving multiple sensory roles (e.g., smell, light,force) and mediating multiple signaling activities (e.g., hedgehog, Wnt,cAMP). Distal appendages (DAPs) are nanoscale, pinwheel-like structuresprotruding from the distal end of the centriole that mediate membrane dock-ing during ciliogenesis, marking the cilia base around the ciliary gate.Despite their functional importance, the detailed architecture of DAPs re-mains largely unknown. Here, we determined a superresolved multiplex of16 centriole-distal-end components. Surprisingly, rather than pinwheels,intact DAPs exhibit a cone-shaped architecture with components filling thespace between each pinwheel blade, a new structural element we termedthe distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1,and CEP164 form the backbone of pinwheel blades, with CEP83 confinedat the root and CEP164 extending to the tip near the membrane-dockingsite. By contrast, FBF1 marks the distal end of the DAM near the ciliarymembrane. Strikingly, unlike CEP164 which is essential for ciliogenesis,FBF1 is required for ciliary gating of transmembrane proteins, revealingDAPs as an essential component of the ciliary gate. Our findings redefineboth the structure and function of DAPs.Funding sources: Ministry of Science and Technology, Taiwan (Grant No. 103-2112-M-001-039-MY3), Academia Sinica Career Development Award.

2667-Pos Board B683In Situ Imaging of Spatial Organization of Accessible Chromatin atthe Nanoscale with ATAC-see and Single-Molecule Super-ResolutionFluorescence MicroscopyMaurice Y. Lee1,2, Xingqi Chen3, Anna-Karin Gustavsson2,Howard Y. Chang3, W.E. Moerner2.1Biophysics, Stanford University, Stanford, CA, USA, 2Chemistry, StanfordUniversity, Stanford, CA, USA, 3Center for Personal Dynamic Regulomes,Stanford University, Stanford, CA, USA.The spatial organization of accessible chromatin in the nucleus tightly regu-lates DNA replication, DNA repair, and gene regulation. Recently, a new

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biochemical labeling technology for the sequencing and imaging of accessiblechromatin was developed. This method, called assay of transposase-accessible chromatin with visualization (ATAC-see), uses the hyperactiveTn5 transposase to covalently insert fluorescently-labeled DNA sequencingadapters directly into the accessible genome. Here, we combined ATAC-see with our latest super-resolution fluorescence microscopy methods, whichuse long axial range point spread functions for fiducial localization, to chartout the 2D map of how accessible chromatin is spatially organized in theHeLa cell nucleus at the nanoscale. In addition, with multi-color super-reso-lution imaging, this map of accessible chromatin can be cross-referenced withthe maps of other landmark proteins to gain a better understanding of how themany different players work together in concert to regulate gene expression inthe human nucleus.

2668-Pos Board B684Quantitative Super-Resolution Microscopy of Proteins at the SynapticLevelSilvia Scalisi1,2, Andrea Barberis3, Enrica Maria Petrini3, Alberto Diaspro1,2,Francesca Cella Zanacchi1.1Nanoscopy, Istituto Italiano Tecnologia, Genova, Italy, 2Physics, Universityof Genoa, Genova, Italy, 3Neuroscience and Brain Technologies, IstitutoItaliano Tecnologia, Genova, Italy.Single-molecule localization (SML) techniques provide a powerful tool toanswer biological questions requiring the observation of subcellular structuresat the nanoscale. Quantitative single-molecule analysis allows quantifying thenumber and distribution of molecules in several biological systems beyond thediffraction limit [1]. In the last few years, many computationalmethods employ-ing clustering analysis algorithms have been developed to extract quantitativeinformation from SML data sets. In neuroscience, quantitative SML has beenapplied to reveal density and spatial organization of synaptic proteins [2].Recently, it has been reported that under plasticity conditions, chemicallyinduced by long term potentiation (iLTP) of inhibitory synapses, GABAAreceptors are immobilized and confined at synapses in cultured hippocam-pal neurons. iLTP expression relies on the recruitment and accumulation ofthe scaffold protein gephyrin at synaptic areas [3], thereby enhancing theclustering of synaptic GABAA receptors and potentiating GABAergicsynaptic currents [4]. In our work we exploit super-resolution approaches(STORM) combined with clustering analysis to study the nanoscaledistribution of the inhibitory postsynaptic scaffold, in particular to countGABAA receptors in close proximity to gephyrin nanodomains duringiLTP.Furthermore we applied the quantitative SML approaches to study a neuronalprotein complex (formed by Protocadherin19, Negr1, FGFR2 and NCAM)involved in neurodevelopmental autism spectrum disorder [5]. We used quan-titative STORM to highlight the role of Protocadherin19 in relation to the dis-tribution of the other proteins forming the complex.References:[1] Deschout H., Nature Methods. (2014), 11, 253.[2] Specht C.G., Neuron. (2013), 79, 308.[3] Pennacchietti F., Journal of Neuuroscience. (2017) 37, 1747.[4] Petrini E.M., Nature Communication. (2014) 5, 3921.[5] Pischedda F., Molecular & Cellular Proteomics (2014), 13, 733.

2669-Pos Board B685Studying Protein Dynamics and Organization in Live Cell Membranes byImaging FCS and SOFI/SRRF AnalysesXue Wen Ng1,2, George Barbastathis2,3, Thorsten Wohland1,4.1NUS Centre for Bioimaging Sciences and Department of Chemistry,National University of Singapore, Singapore, Singapore, 2Singapore-MITAlliance for Research and Technology (SMART) Centre, Singapore,Singapore, 3Department of Mechanical Engineering, Massachusetts Instituteof Technology, Cambridge, MA, USA, 4Department of Biological Sciences,National University of Singapore, Singapore, Singapore.Super-resolution imaging based on localization of sparsely distributed fluores-cence image series has been instrumental over the past decade in understandingbiological systems at superior spatial resolution. However, their applicationsare often limited to fixed cells due to difficulties in localization of mobileparticles. Recent computational super-resolution techniques, namely super-resolution optical fluctuation imaging (SOFI) and super-resolution radialfluctuations (SRRF), had shown great promise to extract high spatial resolutionimages from living systems. SOFI performs higher-order temporal correlationanalysis on the fluorescence fluctuations of fluorophores that are able tostochastically switch between a bright and dark state independently whileSRRF conducts a spatial analysis on a diffraction-limited image stack to

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generate time series of radiality maps and subsequently implements temporalanalysis on the stack of radiality maps to produce a super-resolved image. Ifacquired sufficiently fast, i.e. with frame rates of about 1000 s�1, thesame raw image stack can be utilized to determine diffusion dynamics byautocorrelation analysis of the fluorescence fluctuations at each pixel by so-called Imaging fluorescence correlation spectroscopy (Imaging FCS). Thecombination of these analytical tools thus permits one to acquire diffusiondynamics along with super-resolution information in live samples to decipherbiological processes occurring at the molecular level. Here, we apply ImagingFCS and SOFI/SRRF analyses on time-lapse total internal reflectionfluorescence microscopy (TIRFM) or single plane illumination microscopy(SPIM) images of proteins in live cell membranes. We compare the perfor-mance of various modalities of SOFI and SRRF and evaluate the complemen-tary information from Imaging FCS to investigate the interaction of integral,inner leaflet and transmembrane proteins with the actin cytoskeleton in livecells.

2670-Pos Board B686Quantitative Super-Resolution Microscopy Detects HER2 ReorganizationFollowing Meditope-Antibody TreatmentDevin L. Wakefield, Raphael Jorand, Cindy Zer, John C. Williams,Tijana Jovanovic-Talisman.Beckman Research Institute City of Hope, Duarte, CA, USA.We combine quantitative super-resolution microscopy and meditope tech-nology to investigate the effects of antibody based ligands on the organiza-tion of cancer-specific targets. A cyclic peptide, known as a meditope, bindswithin the Fab framework of Cetuximab and meditope enabled monoclonalantibodies (memAbs). We imaged BT-474 breast cancer cells treated withmultivalent meditopes complexed with memAb trastuzumab, an antibodyspecific for human epidermal growth factor receptor 2 (HER2). We charac-terized the molecular organization of HER2 on these cells using a newlydeveloped analysis interface. Important features of this extensive interfaceinclude minimal user input, rapid analysis, statistical considerations, andcompatibility with various quantitative routines (pair-correlation, clusteringalgorithms, and image segmentation). Our results indicate that meditope ge-ometry strongly influences HER2 clustering. For divalent meditope, an in-crease in linker length (linear amino acid sequences) correlated with anincrease in detected HER2 density and number of clusters. Additionally,compared to divalent meditope, trivalent and tetravalent meditope treatmentresulted in more dense and extensively clustered HER2 detected on the cellsurface. In particular, the increase in meditope valency correlated with anincrease in the fraction of detected HER2 receptors in clusters. Our resultssuggest that multivalent meditopes effectively induce clustering ofmemAb-bound HER2 in a valency dependent manner. Ultimately, exploringthe molecular organization of receptors with our analysis interface representsan important step toward optimizing a diversity of ligands with potentialtherapeutic purposes.

2671-Pos Board B687Direct Detection of ER-mitochondrial Contacts with Fully QuantifiedFluorescence MicroscopyChristopher R. King, Jennifer Lippincott-Schwartz.Janelia Research Campus, Ashburn, VA, USA.Inter-organelle contacts between the endoplasmic reticulum and the mito-chondria are tightly regulated in cells. ER-mitochondrial contact sites havebeen painstakingly characterized through biochemical means, and yetmuch remains to be understood due to their small physical extent. As aresult, even with super-resolution imaging, these inter-organelle contact sitesare difficult to study through fluorescence colocalization measurements.Here, we have developed Fully Quantified Fluorescence Microscopy foruse with nearly any fluorescence microscope. With this, we are working tocharacterize the spatial and temporal properties of ER-mitochondrial contactsites in live cells.

Posters: Bioengineering II

2672-Pos Board B688Quantitative Characterization of Gel Electrophoresis ImagesRiccardo Ziraldo1, Massa J. Shoura2, Stephen D. Levene3.1Bioengineering, The University of Texas at Dallas, Richardson, TX, USA,2Pathology and Genetics, Stanford University, Stanford, CA, USA,3Bioengineering, Biological Sciences, and Physics, The University of Texasat Dallas, Richardson, TX, USA.Although gel electrophoresis has been an invaluable tool for the character-ization of biopolymers, many details of the underlying physical mechanisms

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that govern electrophoretic mobility as a function of molecular size andconformation remain poorly understood. In the case of circular DNA it iswell known that topological properties such as supercoiling, knotting, andcatenation can dramatically alter the mobilities of molecules identical insize (topoisomers). In the case of species that migrate as distinct, well-resolved bands, band intensity has been used as both a qualitative and quan-titative measure of the amounts of distinguishable molecular species presentin the sample. However, in cases where electrophoretic-gel patternsapproach a continuum, information about distributions of species is moreambiguous. We have developed a general software tool to fit univariate func-tions to gel-mobility profiles using minimal information from standard sam-ples to model the contribution from individual species in a sample. Inaddition to applications involving complex topologies of circular DNA,the ability to estimate the sizes of unresolved bands in a line-profile contin-uum is potentially a useful approach for the preparation of next-generation-sequencing fragment libraries.

2673-Pos Board B689Fractionation of Human Red Blood Cells Based on Intrinsic MagnetizationJeffrey Chalmers.Chemical and Biomolecular Eng, The Ohio State University, Columbus, OH,USA.Red blood cell (RBC) transfusion is clinically used to treat hemo-dynamic instability and O2 carrying deficits in patients with acute bloodloss, and patients with chronic anemia caused by bone marrow failure/suppression. Currently, cold storage of human RBCs (hRBCs) canpreserve hRBCs for a maximum of six weeks (i.e. 42 days), set bythe United States Food and Drug Administration (US FDA). However,as stored RBCs age, they undergo biochemical and biophysical changesthat are often referred to as the storage lesion, which decreases the efficacyof transfusion while increasing the risk for transfusion-associated adverseeffects. It is well known that upon transfusion of stored RBCs, there isa population of RBCs (i.e. healthy RBCs) that circulate for more than24 hours, and another smaller population (i.e. damaged RBCs) that arecleared within 24 hours post transfusion. This population of cellsdestined to be cleared quickly can be higher than 25% in units stored fora mean of 30 days. The objective of our current project is to remove agedRBCs based on hemoglobin content. Under the influence of ultra-high mag-netic fields and gradients, we have demonstrated that it is possible to frac-tionate RBCs into multiple factions based solely on difference in theintrinsic magnetization of the deoxygenated form of hemoglobin insidethe RBCs (i.e. labeless separation). We hypothesize for our currentlyfunded National Institute of Heart Lung and Blood project that healthyRBCs with higher Hb content correlate with longer half lives in transfusedanimal models than unhealthy RBCs which have lost some of theirhemoglobin.

2674-Pos Board B690Blood Clot Contraction is Reduced in Sickle Cell Disease due to IncreasedRigidity of ErythrocytesValerie Tutwiler, Rustem I. Litvinov, Anna D. Protopopova,Chandrasekaran Nagaswami, J Eric Russell, Donald L. Siegel,Carlos H. Villa, Daniel Pan, Vladimir R. Muzykantov, John W. Weisel.University of Pennsylvania, Philadelphia, PA, USA.Blood clot contraction, the volumetric shrinkage of the clot, has beenimplicated to play a role in hemostasis and thrombosis. The cellular andmolecular composition of the clot, including red blood cells (RBCs), influ-ences the extent and rate of the three phases of clot contraction. Therefore,there’s a need to examine the effects of diseases where RBCs differ fromthose of healthy individuals, such as sickle cell disease (SCD). SCD is ahypercoagulable state that contributes to vaso-occlusive events and anincreased risk of venous thromboembolism. To follow clot contraction,we used an optical tracking methodology that allows for quantitative mea-surement of clot size with time. Atomic force microscopy was used toexamine the rigidity of RBCs. Clots made from the blood of SCD patientson average had a 53% decrease in the extent of clot contraction, and con-tracted 2.4X slower during Phase 2 and 2.7X slower in Phase 3 whencompared to healthy subjects, which corresponded to a 4.5-fold increasein the rigidity of the RBCs. We hypothesized that the reduction in clotcontraction was due to increased RBC stiffness in SCD patients. Consistentwith this, the addition of naturally rigid llama ovalocytes to human plateletrich plasma and the addition of antibodies to the Wrightb epitope, whichincreases RBC rigidity, resulted in a decrease in extent of clot contractionand a 2.2-fold increase the RBC rigidity, compared to healthy untreated hu-man RBCs. These results demonstrate that clot contraction is altered in

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SCD and suggest that RBC rigidity contributes to the observed reductioninclot contraction. A better understanding of RBC deformability in theprocess of clot contraction may inform the development of more targetedtreatments for thrombosis in patients with altered RBC mechanicalproperties.

2675-Pos Board B691Mechanical Phenotyping of Acute Myeloid Leukemias for PredictingResponse to Retinoic AcidBrian Li, Junghyun Kim, Lydia L. Sohn.University of California, Berkeley, Berkeley, CA, USA.Mechano-node-pore sensing (mechano-NPS) is a new electronic techniquefor mechanical phenotyping of single cells. The technique involvesmeasuring a modulated current pulse caused by a cell transiting through,and being constricted in, a microchannel segmented by a series of widenednodes. By analyzing the modulated current pulse, we can determine a cell’ssize, propensity to be deformed, transverse deformation, and recovery timefrom deformation. Here, we use mechano-NPS to identify the mechanicalphenotypes of acute myeloid leukemia (AML) cell lines that are otherwiseindistinguishable by immunological phenotyping (e.g., CD14 and CD33expression). Specifically, we analyzed NB-4 and AP-1060 cells – two linesof similar maturity, cytogenetics, and immunophenotype, but with differentsensitivity to all-trans-retinoic acid (ATRA), a first-line therapeutic forcertain subtypes of AML. NB-4 is sensitive to ATRA in culture and willterminally differentiate and die, while AP-1060 matures and dies only athigh concentrations of ATRA. Mechanical phenotyping of NB-4 andAP-1060 cells shows that the two are clearly distinct from each other,with NB-4 cells being more deformable and taking much longer to recoverfrom deformation. These findings demonstrated the sensitivity of mechano-NPS in discerning between these two cell lines. We then screened HL-60cells – another AML line different in its maturity and cytogenetics, butwell-known to readily differentiate in ATRA. We show that HL-60 cellsare also mechanically distinct from NB-4 cells in that they are even moredeformable and recover from deformation faster. Our results suggest thatan AML’s response to ATRA is correlated with, and can be predicted by,cell mechanical phenotype. Ultimately, cell mechanical phenotyping, usedin combination with existing in vitro assays, could help guide importantclinical decisions.

2676-Pos Board B692Microfluidic Rheology to Study Effects of Cell Cycle to ViscoelasticProperties of Epithelial CellsYoungbin Kim, Junghyun Kim, Oliva Scheideler, Emma Cimenelli,Lydia L. Sohn.UC Berkeley, Berkeley, CA, USA.We introduce a microfluidic rheology platform to measure the viscoelasticproperties of cells in different cell cycle stages. While there are a growingnumber of studies on cell mechanical properties, only a few have investi-gated these properties during cell-cycle progression in which there are sig-nificant changes in the cytoskeleton. Currently, atomic force microscopy(AFM) is the preferred method for cell micro-rheology due to its high res-olution and high precision in loading frequency. However, its lowthroughput could be a critical limitation to correlate biophysical propertiesof cells with specific cell-cycle stage due to small sample size. Here, wepropose a high throughput platform that employs microfluidics and electri-cal sensing to quantify cellular viscoelastic properties at the single celllevel.Our microfluidic rheometer consists of a PDMS mold bonded to a glass sub-strate with predefined platinum electrodes. The mold consists of a microflui-dic channel that has two central features: a node-pore pair and a sinusoidal-shaped contraction channel. As a cell transits the node-pore pair, a currentpulse is recorded whose magnitude reflects cell size. As it transits thecontraction channel, a cell undergoes periodic deformation due to the peri-odically changing channel width. The magnitude and frequency of cellulardeformation is reflected in the subsequent current pulse produced.Combining these measured parameters with the fluidic conditions underwhich they were obtained, we can quantify cell storage and loss moduli.With our platform, we show that epithelial cells (MCF7 and MCF10A)have distinct viscoelastic properties that reflect their malignant status.Even within one cell population, however, we find that there is a variationin storage modulus. In this study, we focus on the origin of this variationand the specific role cell-cycle progression plays in altering whole-cellviscoelastic properties.

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2677-Pos Board B693AMarkov StateModel of the Sarcomere to Explain the Effects of DATP onCardiac ContractionKimberly J. McCabe1, Yasser Aboelkassem1, Sukriti Dewan1,Michael Regnier2, Andrew D. McCulloch1.1Bioengineering, University of California, San Diego, San Diego, CA, USA,2Bioengineering, University of Washington, San Diego, CA, USA.2-deoxy-ATP is a naturally occurring homolog of ATP. dATP is known toincrease force production, enhance crossbridge cycling speed, and accel-erate the removal of Ca2þ from the cytoplasm during cell relaxation in vi-tro as well as in animal models. At low ratios of dATP to ATP in cells,steady state force has been shown to increase nonlinearly at physiological[Ca2þ], while at higher ratios the relationship becomes linear. Thus,dATP has shown promise as a potential therapeutic because it creates sig-nificant increases in force even at low dATP/ATP ratios. The purpose ofthis study is to discover specific mechanisms by which dATP alters myosindynamics within the sarcomere using multiscale computational modeling.We have developed a comprehensive mechanistic Monte Carlo MarkovState model of rat sarcomere contraction which includes cooperativity inboth thin filament activation and crossbridge cycling through nearest-neighbor interactions due to overlapping Tropomyosin (Tm) molecules onthe actin thin filament. This novel sarcomere model was used to test the ef-fects of dATP on contractility within the sarcomere. We combined experi-mental data on the subcellular, cellular, and organ level as well asMolecular Dynamics data to parameterize and validate the model. We testedcombinations of 4 different possible kinetic transitions in which dATP ishypothesized to affect crossbridge cycling and tested various degrees of co-operativity in the system to find a likely combination of mechanisms bywhich dATP affects contraction.

2678-Pos Board B694AAV-Mediated Delivery of Ribonucleotide Reductase andMicrodystrophinRescues Function in Dystrophic MiceJason Murray, Guy Odom, Sigurast Olafsson, Stephen Hauschka,Jeffrey Chamberlain, Farid Moussavi-Harami, Michael Regnier.Physiology and Biophysics, University of Washington, Seattle, WA, USA.Duchenne muscular dystrophy (DMD) is caused by a lack of functional dystro-phin in myocytes leading to progressive muscle dysfunction, loss of mobility,and cardiomyopathy. The most common cause of death in DMD patients is nowheart failure. Our group has demonstrated that viral vector-mediated overex-pression of ribonucleotide reductase (RNR) leads to increased concentrationof 2-deoxy-ATP (dATP), which can be used by myosin and significantly in-creases performance of striated muscle. As a novel DMD combinatorial thera-peutic, we have developed an AAV-platform utilizing microdystrophin (mDys)to stabilize the sarcolemma of myocytes and RNR to improve contractile func-tion to prevent or rescue hearts from failure. In one study, mdx4cv mice wereinfused at two weeks of age with either mDys or mDysþRNR using AAV vec-tors carrying a striated muscle regulatory cassette (CK8). Four months post-injection, both groups that received mDys displayed increased specific forceproduction in the hindlimb and improved recovery from fatigue compared tocontrols. mDysþRNR-treated animals had increased levels of dATP in the ti-bialis anterior measured by HPLS/MS-MS, but no increase in specific forceproduction compared to mDys alone. There were no significant differences be-tween treated and untreated animals in contractility or calcium transients in iso-lated cardiomyocytes, or in diaphragm function in vivo. In a second study,22-24-month old mdx4cv mice were treated with either AAV6-CK8-mDys,and/or a cardiac-specific regulatory cassette driving expression of RNR. Fivemonths post-treatment, Langendorff perfusions were performed. All threegroups displayed improved systolic function, with the RNR-treated group alsoshowing improvement in several diastolic parameters. Based on these promisingpreliminary results, we will continue to investigate a combinatorial gene therapyapproach of mDys and RNR for preservation of health in young animals andrescue of function in aging models with and without dystrophin deficiency.

2679-Pos Board B695Microscopy Electroporation ProbeTayyebeh (Azita) Sberbaghi Sberbaghi, Ebrahim Ghafar-Zadeh.York University, Toronto, ON, Canada.This study presents a new approach for drug delivery into the cell layers ofZebrafish Follicle. Zebrafish follicle is used as a model of ovarian develop-ment. Zebrafish follicle consists of an oocyte surrounded by two thin layersof cells (theca and granulosa cell). The delivery of biomolecoules in to thecells in these layers using electroporation is an unmet challenge. Electropora-tion is a non-invasive method widely used for transferring molecules into cells

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for various applications including stem cell based tissue construction and genetherapy. Despite broad advantages of electroporation, no reports have beenreleased for application of drug delivery into Zebrafish follicle cells. Hereinwe reports the design, implementation of a new high throughput electropora-tion system for delivery of bio-molecules into zebrafish follicle cells of(Danio rerio) in a controllable manner. This platform consists of an arrayof 32 microfluidic chambers incorporated with the same number of address-able microelectrodes interconnected to a computer for the controlled electricalstimulation. We demonstrate and discussed the advantage of proposed methodfor gene delivery into the cells using PI and DAPI. This study will take us astep closer to deliver other types of genes into Zebrafish follicle for ovariantreatment purposes.

2680-Pos Board B696Enhancing Electrotransfection Efficiency Through Improvement inNuclear Entry of Plasmid DNALisa D. Cervia, Chun-Chi Chang, Liangli Wang, Mao Mao, Fan Yuan.Biomedical Engineering, Duke University, Durham, NC, USA.The nuclear envelope is a physical barrier to gene delivery. To understandmechanisms of nuclear entry of electrotransfected plasmid DNA (pDNA),the current study investigated effects of nuclear envelope break down(NEBD) on electrotransfection efficiency (eTE). The NEBD occurs when cellsenter M phase in cell cycle. Thus, cells used in the study were synchronized atG2-M phase through treatment with nocodazole or release from double thymi-dine block prior to electrotransfection. Results from the study showed that theeTE was greatly enhanced by the treatments while the cell viability was notsignificantly compromised. These results indicated that eTE in many applica-tions of electrotransfection depended on the percentage of cells in the M phasewithin a period after electric pulse application, during which pDNA degrada-tion was insignificant. Other attempts to enhance trafficking through the nu-clear envelop barrier included using nuclear pore dilating agents (Trans-1,2-Cyclohexanediol and Pitstop-2) and nuclear localization sequences. The nu-clear pore dilating agent was able to increase the expression levels in allcell lines tested; however, the effect was not as great as with nuclear envelopebreakdown induced by nocodazole synchronization. The nuclear localizationsequence proved ineffective at transfecting the cell lines tested, indicatingthat the sequences may be specific to certain cell lines and have more of animpact in cells that have a long division rater, and are thereby relatively diffi-cult to transfect. Data from the study also suggested that induction of transientNEBD could be a practical approach to improving efficiency ofelectrotransfection.

Posters: Biosurfaces

2681-Pos Board B697Spontaneous Reduction of Biomolecules on the Surface of Water DropletsJae Kyoo Lee1, Devleena Samanta1, Inho Nam1,2, Hong Gil Nam2,3,Richard N. Zare1.1Department of Chemistry, Stanford University, Stanford, CA, USA, 2Centerof Plant Aging Research, Institute for Basic Science, Daegu, Republic ofKorea, 3Department of New Biology, DGIST, Daegu, Republic of Korea.Reduction and oxidation reactions are a family of reactions that involves thetransfer of electrons between species. Redox reactions play a crucial role inliving organism as they are involved in photosynthesis, respiration, andmetabolism. Chemical reactions in confined environments behave differentlythan the same ones in bulk solution. We have found that chemical reactionsare significantly accelerated in micron-sized aqueous droplets (microdrop-lets) by the factor over 105 for various reactions including protein unfolding,protein-ligand binding, chlorophyll demetallation, and hydrogen-deuteriumexchange (PNAS 2015, Q Rev Biophys 2016 and 2017). We also foundthat the reactions with a high thermodynamic barrier can spontaneouslyoccur in microdroplets by lowering the entropy change at the water surface(PNAS, in press). Here we present spontaneous reduction of biomoleculesincluding pyruvate, cystine, lipoic acid, and fumarate with up to � 95 %reduction efficiency within hundreds of microseconds at the water-air inter-face on microdroplets without an added reducing agent. The reduction effi-ciency decreased as the concentration of pyruvate increased above 10 nM,suggesting the capacity of the reducing power of water microdroplet islimited to around tens of nanomoles per liter. The increase of surface-to-volume ratio of the microdroplets by generating smaller microdroplets ledto increased reduction efficiency, indicating the reduction occurs at orvery near the surface of microdroplets. The increase of O2 composition inthe surrounding gas reduced reduction efficiency, suggesting that the oxida-tion of OH- due to a strong electric field at the water surface is a probable

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mechanism of the reduction. This discovery of spontaneous reduction atthe water-air interface may provide a new biophysical mechanism of alteringmetabolomic balance without the participation of charge carriers or reducingagents/enzymes as well as elucidating the reduction of biomolecules in theprebiotic era.

2682-Pos Board B698Unravelling the Secrets of Catechol-Cation Binding SynergyGeorge Degen, Jacob Israelachvili.Chemical Engineering, University of California, Santa Barbara, SantaBarbara, CA, USA.Aqueous electrolyte solutions, including physiological fluids and seawater,limit polymer adhesion to surfaces, yet the marine mussel produces proteinglues that adhere robustly under water, even in highly adverse, such ascontaminated, conditions. Adhesive mussel foot proteins commonly containadjacent catecholic 3,4-dihydroxyphenylalanine (Dopa) and cationic lysineresidues. Previous work has shown that the pairing of these residues resultsin significantly higher adhesion energies on inorganic surfaces than eitherDopa or lysine alone. Two mechanisms for synergistic binding have been pro-posed: either lysine evicts adsorbed cations on the substrate, enabling subse-quent binding of Dopa to the substrate; or paired Dopa and lysine residuesbind simultaneously to the substrate. To evaluate the validity of these twomechanisms, a series of synthetic analogs of siderophores (bacterial iron che-lators) was synthesized. The intramolecular spacing between catecholic andcationic moieties was systematically varied across the series. Adhesionforces/energies of monolayers of siderophore analogs confined between sym-metrical mica surfaces were measured using a Surface Forces Apparatus(SFA). Our results show no statistically significant dependence of adhesionenergy on intramolecular spacing. These results support the surface cationeviction mechanism for catechol-cation synergy, and suggest that proteinsincorporating Dopa and lysine residues will display elevated adhesion en-ergies even when the groups are not directly adjacent. The knowledge thatcatechol-cation binding synergy operates even when the moieties are spatiallyseparated will guide future design of biologically inspired adhesives formedical, dental, and marine applications.

2683-Pos Board B699Role of Salts and Surfaces on ECM Constituents in Biological MediaMatt McKenzie, Aravind Rammohan.Corning Inc, Corning, NY, USA.For Cell based therapies having chemically defined surfaces with chemicallydefined media offer an advantage with respect to growing these cells. Typicalconstituents of the media are a number of different inorganic salts and ECMmimectics that can enable cell adhesion. It is critical to understand the inter-actions between the ECM mimectics, salts and the surfaces for effectivedesign of such media and the surfaces. In this work, we probe the conforma-tion of a specific ECM mimectic (ProNectin F) in solution with a change ofconcentration of commonly used monovalent (KCl, NaCl) and divalent(CaCl2) salts. Using Molecular Dynamics we find there is an effect of ion-induced conformation change on the active RGD site of Pronectin F. Thisconformational change is stronger in a CaCl2 solution compared to KCland the mixed salt solutions show a larger RGD active site angle fluctuation.We also study the interaction between the different conformations of Pronec-tin F with different salts and integrin using both MD and docking techniques.We find that the type of cation also has a significant role in altering the RGDbinding site on the integrin. Then we look into the role of the surface adhesionwith water and Pronectin F. Hydrophobic residues play an important role inhow this mimectics adheres to the surface. This adhesion process decreaseshydrogen bonding to allow for a structural change that minimizes the interac-tion between the hydrophobic residue and water to increase its interactionswith the surface.

2684-Pos Board B700Vapor-Deposited Porous Polymers for the Fabrication of Giant LipidVesiclesNareh Movsesian, Noah Malmstadt, Malancha Gupta.Chemical Engineering, University of Southern California, Los Angeles, CA,USA.Giant unilamellar vesicles (GUVs) are self-assembled biomimetic modelmembrane systems useful for examining biomembrane properties andbuilding artificial cell membranes for membrane protein incorporationand encapsulation of other biomolecules. Hydrogel-assisted rehydration isan emerging technique to form GUVs under physiological conditions athigh yields circumventing the shortcomings of traditional techniques such

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as electroformation and gentle hydration. Here, we present a new hydrogelmaterial, porous negatively charged poly (methacrylic acid-co-ethyleneglycol diacrylate) (xPMAA) for the hydrogel-assisted rehydration method.Porous xPMAA membranes are fabricated using an unconventionalsolvent-free initiated chemical vapor deposition technique and utilized ashydrogel substrates for vesicle formation. Zwitterionic and charged lipidmixtures are deposited on hydrogel membranes as thin lipid films and sub-sequently swollen in an aqueous hydration buffer. Vesicle yield and sizeare controlled by surface roughness, density and charge of the polymer.Our findings show that high hydrogel porosity and reduced electrostatic in-teractions between the polymer and the lipid are preferred for vesicle for-mation. In addition, the presence of ions in the hydration buffer affects theyield of GUV formation due to Debye screening of the polymer charge bybuffer ions.

2685-Pos Board B701Anchoring Giant Plasma Membrane Vesicles to a Surface for NovelBiosensingAomeng Cui1, Daniel E. Oseid2, Julie N.L. Albert3, Anne S. Robinson2,3.1Columbia University, New York, NY, USA, 2Tulane Brain Institute, TulaneUniversity, New Orleans, LA, USA, 3Department of Chemical andBiomolecular Engineering, Tulane University, New Orleans, LA, USA.Giant plasma membrane vesicles (GPMVs) are unilamellar bodies that can beeasily isolated from mammalian cells. SNAP-tag is a protein tag which bindscovalently with benzylguanine (BG) derivatives, and is also capable of beingfused to a protein of interest. Currently, specifically anchoring cellular mate-rial remains a challenge for bioengineering. The purpose of our project was tocreate a BG-functionalized surface to immobilize GPMVs expressing SNAP-tag fused to A2A, a G-protein coupled receptor. We harvested GPMVs fromwildtype HEK cells transfected with pSNAPf-A2A DNA through addition ofPFA/DTT solution. The surface was constructed through a series of thiol-ene click reactions and functionalized with benzylguanine. Live-cell imagingof SNAP-A2A-expressing GPMVs interacting with the surface revealed strongimmobilization compared to cells transfected with only A2A and SNAP-A2A-expressing GPMVs on a PEG-thiol functionalized surface. Horizontally-oriented storage of GPMVs immobilized on a BG-functionalized surfacemaintained the integrity of vesicles and ensured viability after two days.Modification of the surface also produced a gradient of benzylguanine func-tionalization that selectively immobilized GPMVs, though the exact interac-tion between BG and SNAP-tag should be further characterized. This noveltechnique to anchor vesicles from mammalian cells could be instrumentalfor the development of functional biosensors. Using this anchoring system,a wide variety of compounds could be quickly detected via binding to anexogenously expressed protein of interest within the same membraneenvironment.

2686-Pos Board B702Tethering Antibody on a Pegylated Liposome-Lipid Bilayer to PromoteFlexible Chain Movement for Multivalent Antibody-Antigen Interactionsand to Minimize Applied Force on Cells upon ReleasePo-Ying Yeh.Genomics Research Center, Academia Sinica, Taipei, Taiwan.The isolation of circulating tumor cells (CTCs)) is one of the most active areasof translational cancer research. To develop a surface that is non-fouling forreducing the interfering cell adhesion while being efficient in enriching andisolating CTCs of high viability is urgently needed in CTC study. In thisstudy, we construct a CTC-LIPO platform, an antibody functionalizedliposome conjugated with supported lipid bilayer microfluidics, to addressthis issue.

BPJ 8701_8

The liposome is composed of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-biotinyl (polyethyleneglycol)-2000 and cholesterol. Light scattering, zeta-potential and DSCwere used to identify liposomes with the average size of �110nm in diam-eter, neutral charges and transition temperature at 54oC. The successful sur-face construction to systematically build up supported lipid bilayer,liposome, and antibody were quantitatively monitored by quartz crystal mi-crobalance with dissipation. To evaluate the isolation efficiency, several pre-stained cancer cell lines were spiked into the CTC-LIPO platform; afterbuffer wash to removing the non-specific adsorption, air foam was injectedto disrupt the liposome construct, thus releasing the cancer cells for subse-quent evaluation. The isolation and release efficiency are 80% and 65%,where 80% of released cells maintain viability and can proliferate andform cell colonies after seeding for 5 days. Our results indicated that abiocompatible CTC capture surface is critical for ensuring CTC maintenanceand proliferation.Keywords: liposome, lipid bilayer, circulating tumor cells, microfluidics

2687-Pos Board B703CLK-Peptides as Superior Surface Stabilizers for Silver Nano Structures:Role of Peptide Chain Length and Applications in NanomedicineHoracio Poblete1, Manuel Manuel Ahumada2, Erik Jacques3,Cristina Andronic2, Jeffrey Comer4, Emilio Alarcon3.1Center for Bioinformatics andMolecular Simulations, Universidad de Talca,Talca, Chile, 2Bio-Nanomaterials Chemistry and Engineering Laboratory,Division of Cardiac Surgery, University of Ottawa Heart Institute, Ottawa,ON, Canada, 3aBio-Nanomaterials Chemistry and Engineering Laboratory,Division of Cardiac Surgery, University of Ottawa Heart Institute, Ottawa,ON, Canada, 4Deparment of Anatomy and Physiology, Kansas StateUniversity, Manhattan, KS, USA.Three new collagen mimetic peptides containing the CLK motif as anchoringarms were tested for silver nanoparticle surface stabilization. Our experimentaland molecular dynamic data indicate that peptide length has an important effectnot only in the resulting nanosilver’s colloidal stability but also in the biologicalperformance of the composite.

2688-Pos Board B704Quantitation of Surface-Conjugated DNA Density for Single-MoleculeApplicationsTheodore Yu1, Yuchen Liang2, Stephen D. Levene1, Walter Hu2.1Bioengineering, University of Texas at Dallas, Richardson, TX, USA,2Electrical Engineering, University of Texas at Dallas, Richardson, TX, USA.Technologies based on spectroscopic methods currently dominate the next-generation DNA-sequencing (NGS) market. Semiconductor-based NGS ap-proaches still have potential as a cost-effective technology, but requiredevelopment to improve accuracy. In particular, semiconductor-basedsequencing has relied on bead amplification and pH sensitivity. Instead ofrelying exclusively on transient pH changes, we are developing methodolo-gies that exploit nanowire field-effect transistor (NWFET) properties todetect polymerase-catalyzed extension of a DNA primer. Gold is an attrac-tive substrate for NWFET detection, as it resists the formation of oxideswhich adversely impact FET sensitivity and provides convenient surfacechemistry for DNA derivatization. Reliable determination of the surface den-sity of ssDNA primers conjugated to gold surfaces remains a challenge. Weare using a combination of confocal microscopy, atomic-force microscopy(AFM) and scanning electron microscopy (SEM) to quantify primer den-sities. The goal of these studies is to provide accurate and reliable primerquantitation in the high surface-density regime approaching a target valueof 1011 cm�2.

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