Tensile Mechanical Measurements on Individual Collagen Fibrils
1
2611-Pos Board B597 Resolving Desmosomal Cadherin Interactions at the Single Molecule Level Sabyasachi Rakshit, Molly Lowndes, Kristine Manibog, W. James Nelson, Sanjeevi Sivasankar. Desmocollin and Desmoglein are essential Ca 2þ dependent cell adhesion pro- teins that mediate the integrity and functional organization of tissues in multi- cellular organisms; however, the molecular interactions that mediate their binding are not understood. It is currently believed that desmosomal cadherin cis-dimers are required to mediate adhesion, evidence for the role of cis-dimers is lacking. Furthermore, it is unclear if desmosomal cadherins interact via ho- mophilic or heterophilic binding and the molecular mechanism by which des- mosomal cadherins enhance their bond-strength. To resolve these questions we used Dynamic Force Spectroscopy with an Atomic Force Microscope to mea- sure the homophilic and heterophilic binding of desmosomal cadherin mono- mers and cis-dimers at the single molecule level. Our measurements showed that desmosomal cadherin monomers alone mediate Ca 2þ dependent adhesion, cis-dimers are not essential for adhesion. Furthermore, while desmocollin and desmoglein participate in Ca 2þ dependent heterophilic binding, we could not measure Ca 2þ dependent homophilic interactions between these cadherins. Dy- namic Force Spectroscopy revealed that desmosomal cadherin interactions oc- cur via a double barrier interaction potential; at low loading rates the outer barrier serves as the dominant impedance to unbinding while at higher loading rates the inner barrier serves as the primary kinetic barrier. These experiments resolve the molecular details of desmosomal cadherin binding and suggest a mechanism by which these essential cell adhesion molecules enhance bond strength and lifetime in the presence of force. 2612-Pos Board B598 Propagation of Load Through Proteins via Steered Molecular Dynamics: Effect on Fluctuation Dynamics and Architecture Steven M. Kreuzer, Chia-Cheng Liu, Esfandiar A. Khatiblou, Joel D. Marquez, Tess J. Moon. Mechanical load in the form of externally applied forces and/or moments are known to regulate biochemical activity in proteins through induced conforma- tional changes exposing cryptic binding sites, altered kinetics, and, in extreme cases, unfolding. It is unknown, however, how load propagates through protein structure from remotely applied forces to local regions: do loads manifest as changes in fluctuations, architecture or both? Single molecule force spectros- copy (SMFS) studies have demonstrated protein unfolding with nanometer- and piconewton-level resolution, yet provide little information about the intra-protein pathways of load. SMFS studies of Green Flourescent Protein (GFP) have demonstrated the anisotropy of deformation, providing an ideal test system for studying the structural response of proteins to load. To explore the manifestation of load in proteins via simulation of a controlled experiment, constant-force steered molecular dynamics (SMD) are used to generate proba- bility density functions (PDFs) at atomic-level resolution describing the struc- ture of GFP when subjected to simple, sub-unfolding, externally applied loads along the vectors used in SMFS studies. Here the focus is on describing protein structure (both vibrational and architectural) with increasing forces; the exper- imental unfolding values provide a top-force validation of the simulation re- sults. Total data include PDFs for both equilibrium and non-equilibrium loaded scenarios permitting comparison for the effect of loads. Data are ana- lyzed for principal components (via PCA) to determine fluctuation dynamics and changes in structure architecture along the spectrum of applied loads. PCA data allow comparison of both fluctuation amplitude and direction through the power spectra of mode covariance overlap. Results indicate areas of in- creased strain under load, predicting locations of unfolding via increased strain and demonstrating the anisotropic pathways of load at sub-unfolding loads. Intra-molecular constitutive properties are calculated as derivatives of free energy. 2613-Pos Board B599 Naturally Occurring Osmolytes Modulate the Nano-Mechanical Proper- ties of Polycystic Kidney Disease (PKD) Domains Liang Ma, Meixiang Xu, Andres Oberhauser. Polycystin-1 is a large transmembrane protein, which, when mutated, cause au- tosomal dominant polycystic kidney disease, one of the most common life- threatening genetic diseases which is a leading cause of kidney failure. PC1 is a large membrane protein that is expressed along the renal tubule and ex- posed to a wide range of concentrations of urea. Urea is known as a common denaturing osmolyte that affects protein function by destabilizing their struc- ture. On the other hand, it is known that the native conformation of proteins can be stabilized by protecting osmolytes which are found in the mammalian kidney. PC1 has an unusually long ectodomain with a multimodular structure including 16 Ig-like polycystic kidney disease (PKD) domains. Here we used single-molecule force spectroscopy to directly study the effects of several nat- urally occurring osmolytes on the mechanical properties of PKD domains. This experimental approach more closely mimics the conditions found in vivo. We show that upon increasing the concentration of urea there is a remarkable de- crease in the mechanical stability of human PKD domains. We found that pro- tecting osmolytes such as sorbitol and TMAO can counteract the denaturing effect of urea. Moreover, we found that the refolding rate of a structurally ho- mologous archaeal PKD domain is significantly slowed down in urea, and this effect was counteracted by sorbitol. Our results demonstrate that naturally oc- curring osmolytes can have profound effects on the mechanical unfolding and refolding pathways of PKD domains. Based on these findings, we hypothesize that osmolytes such as urea or sorbitol may modulate PC1 mechanical proper- ties and may lead to changes in the activation of the associated Polycystin-2 channel or other intracellular events mediated by PC1. This work was funded by NIH grant R01DK073394 and the PKD Foundation (grant 116a2r). 2614-Pos Board B600 Titin Domains as Mechanical Reporters of UNC-45 Mediated Myosin Folding Andres Oberhauser, Christian Kaiser, Liang Ma, Henry Epstein. Myosins are protein machines in which allosteric cross-talk between ATP- binding and hydrolysis and the movement of actin filaments requires precise changes in the polypeptide fold of the motor domain. UNC-45, a member of the UCS family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly for myosin into muscle thick filaments in vivo. However the molecular mechanisms by which UNC-45 interacts with my- osin to promote proper folding of the myosin head domain are not known. We have devised a novel approach to analyze the mechanical refolding of the my- osin motor domain at the single molecule level utilizing AFM techniques. By chemically coupling an I27 titin polyprotein to the motor domain of myosin, we introduced a ‘‘molecular reporter’’ into the motor domain, providing a specific attachment point and a well characterized mechanical fingerprint in the AFM measurements. This approach enabled us to study the folding of the motor do- main and directly observe the effect of the chaperone UNC-45. The motor do- main misfolded after mechanical unfolding, whereas the myosin rod refolded independently of the motor domain. The misfolded motor domain recruited the otherwise robustly folding I27 modules into a misfolded state. Thus, the I27 domains function as a folding sensor. UNC-45 prevents the detrimental in- teraction between the motor domain and the I27 modules, indicating that it bound to and stabilized the non-native myosin motor domain. Using truncated versions of UNC-45, we localized this protective effect to the C-terminal UCS domain of UNC-45. Our approach enables the study, for the first time, the chap- erone effects of UNC-45 on myosin folding in mechanistic detail. This work was funded by NIH grants R01DK073394 (AFO), R01AR050051 (HFE), the Muscular Dystrophy Association and the Cecil and Ida Green En- dowment (HFE). 2615-Pos Board B601 Tensile Mechanical Measurements on Individual Collagen Fibrils Rene B. Svensson, Tue Hassenkam, Colin A. Grant, S. Peter Magnusson. Introduction: There is a hierarchical order in tendon ranging from the collagen molecule to the whole tendon. We have developed a method using the atomic force micro- scope (AFM) together with a novel data acquisition technique to quantify the sub-failure mechanical properties of individual collagen fibrils. Here we use this method to investigate how the saline environment affects tensile force transmission. Methods: Human patellar tendon tissue was obtained from a healthy young male. Six fascicles and six fibrils were mechanically tested sub-failure to determine modulus and rela- tive energy dissipation in PBS solutions of 0.02, 0.15 and 1 M concentration as well as two HEPES buffers containing NaCl or NaClþCaCl 2 . Mechanical testing was per- formed to 4% strain. Results: The tensile properties of collagen fibrils are highly resistant to changes in their saline environment. The only consistent response to environment com- position was a minor (<5%) increase in relative energy dissipation in 1 M PBS. This is in sharp contrast to previous reports of more than 100% increase in radial compressive modulus of fibrils in 1 M salt solution, and more than 100% increase in energy dissipation of AFM pulling experiments in the pres- ence of Ca 2þ . 482a Tuesday, March 8, 2011
Transcript of Tensile Mechanical Measurements on Individual Collagen Fibrils
482a Tuesday, March 8, 2011
2611-Pos Board B597Resolving Desmosomal Cadherin Interactions at the Single Molecule LevelSabyasachi Rakshit, Molly Lowndes, Kristine Manibog, W. James Nelson,Sanjeevi Sivasankar.Desmocollin and Desmoglein are essential Ca2þ dependent cell adhesion pro-teins that mediate the integrity and functional organization of tissues in multi-cellular organisms; however, the molecular interactions that mediate theirbinding are not understood. It is currently believed that desmosomal cadherincis-dimers are required to mediate adhesion, evidence for the role of cis-dimersis lacking. Furthermore, it is unclear if desmosomal cadherins interact via ho-mophilic or heterophilic binding and the molecular mechanism by which des-mosomal cadherins enhance their bond-strength. To resolve these questions weused Dynamic Force Spectroscopy with an Atomic Force Microscope to mea-sure the homophilic and heterophilic binding of desmosomal cadherin mono-mers and cis-dimers at the single molecule level. Our measurements showedthat desmosomal cadherin monomers alone mediate Ca2þ dependent adhesion,cis-dimers are not essential for adhesion. Furthermore, while desmocollin anddesmoglein participate in Ca2þ dependent heterophilic binding, we could notmeasure Ca2þ dependent homophilic interactions between these cadherins. Dy-namic Force Spectroscopy revealed that desmosomal cadherin interactions oc-cur via a double barrier interaction potential; at low loading rates the outerbarrier serves as the dominant impedance to unbinding while at higher loadingrates the inner barrier serves as the primary kinetic barrier. These experimentsresolve the molecular details of desmosomal cadherin binding and suggesta mechanism by which these essential cell adhesion molecules enhance bondstrength and lifetime in the presence of force.
2612-Pos Board B598Propagation of Load Through Proteins via Steered Molecular Dynamics:Effect on Fluctuation Dynamics and ArchitectureSteven M. Kreuzer, Chia-Cheng Liu, Esfandiar A. Khatiblou,Joel D. Marquez, Tess J. Moon.Mechanical load in the form of externally applied forces and/or moments areknown to regulate biochemical activity in proteins through induced conforma-tional changes exposing cryptic binding sites, altered kinetics, and, in extremecases, unfolding. It is unknown, however, how load propagates through proteinstructure from remotely applied forces to local regions: do loads manifest aschanges in fluctuations, architecture or both? Single molecule force spectros-copy (SMFS) studies have demonstrated protein unfolding with nanometer-and piconewton-level resolution, yet provide little information about theintra-protein pathways of load. SMFS studies of Green Flourescent Protein(GFP) have demonstrated the anisotropy of deformation, providing an idealtest system for studying the structural response of proteins to load. To explorethe manifestation of load in proteins via simulation of a controlled experiment,constant-force steered molecular dynamics (SMD) are used to generate proba-bility density functions (PDFs) at atomic-level resolution describing the struc-ture of GFP when subjected to simple, sub-unfolding, externally applied loadsalong the vectors used in SMFS studies. Here the focus is on describing proteinstructure (both vibrational and architectural) with increasing forces; the exper-imental unfolding values provide a top-force validation of the simulation re-sults. Total data include PDFs for both equilibrium and non-equilibriumloaded scenarios permitting comparison for the effect of loads. Data are ana-lyzed for principal components (via PCA) to determine fluctuation dynamicsand changes in structure architecture along the spectrum of applied loads.PCA data allow comparison of both fluctuation amplitude and direction throughthe power spectra of mode covariance overlap. Results indicate areas of in-creased strain under load, predicting locations of unfolding via increased strainand demonstrating the anisotropic pathways of load at sub-unfolding loads.Intra-molecular constitutive properties are calculated as derivatives of freeenergy.
2613-Pos Board B599Naturally Occurring Osmolytes Modulate the Nano-Mechanical Proper-ties of Polycystic Kidney Disease (PKD) DomainsLiang Ma, Meixiang Xu, Andres Oberhauser.Polycystin-1 is a large transmembrane protein, which, when mutated, cause au-tosomal dominant polycystic kidney disease, one of the most common life-threatening genetic diseases which is a leading cause of kidney failure. PC1is a large membrane protein that is expressed along the renal tubule and ex-posed to a wide range of concentrations of urea. Urea is known as a commondenaturing osmolyte that affects protein function by destabilizing their struc-ture. On the other hand, it is known that the native conformation of proteinscan be stabilized by protecting osmolytes which are found in the mammaliankidney. PC1 has an unusually long ectodomain with a multimodular structureincluding 16 Ig-like polycystic kidney disease (PKD) domains. Here we used
single-molecule force spectroscopy to directly study the effects of several nat-urally occurring osmolytes on the mechanical properties of PKD domains. Thisexperimental approach more closely mimics the conditions found in vivo. Weshow that upon increasing the concentration of urea there is a remarkable de-crease in the mechanical stability of human PKD domains. We found that pro-tecting osmolytes such as sorbitol and TMAO can counteract the denaturingeffect of urea. Moreover, we found that the refolding rate of a structurally ho-mologous archaeal PKD domain is significantly slowed down in urea, and thiseffect was counteracted by sorbitol. Our results demonstrate that naturally oc-curring osmolytes can have profound effects on the mechanical unfolding andrefolding pathways of PKD domains. Based on these findings, we hypothesizethat osmolytes such as urea or sorbitol may modulate PC1 mechanical proper-ties and may lead to changes in the activation of the associated Polycystin-2channel or other intracellular events mediated by PC1.This work was funded by NIH grant R01DK073394 and the PKD Foundation(grant 116a2r).
2614-Pos Board B600Titin Domains as Mechanical Reporters of UNC-45 Mediated MyosinFoldingAndres Oberhauser, Christian Kaiser, Liang Ma, Henry Epstein.Myosins are protein machines in which allosteric cross-talk between ATP-binding and hydrolysis and the movement of actin filaments requires precisechanges in the polypeptide fold of the motor domain. UNC-45, a member ofthe UCS family of proteins, acts as a chaperone for myosin and is essentialfor proper folding and assembly for myosin into muscle thick filaments invivo. However the molecular mechanisms by which UNC-45 interacts with my-osin to promote proper folding of the myosin head domain are not known. Wehave devised a novel approach to analyze the mechanical refolding of the my-osin motor domain at the single molecule level utilizing AFM techniques. Bychemically coupling an I27 titin polyprotein to the motor domain of myosin, weintroduced a ‘‘molecular reporter’’ into the motor domain, providing a specificattachment point and a well characterized mechanical fingerprint in the AFMmeasurements. This approach enabled us to study the folding of the motor do-main and directly observe the effect of the chaperone UNC-45. The motor do-main misfolded after mechanical unfolding, whereas the myosin rod refoldedindependently of the motor domain. The misfolded motor domain recruitedthe otherwise robustly folding I27 modules into a misfolded state. Thus, theI27 domains function as a folding sensor. UNC-45 prevents the detrimental in-teraction between the motor domain and the I27 modules, indicating that itbound to and stabilized the non-native myosin motor domain. Using truncatedversions of UNC-45, we localized this protective effect to the C-terminal UCSdomain of UNC-45. Our approach enables the study, for the first time, the chap-erone effects of UNC-45 on myosin folding in mechanistic detail.This work was funded by NIH grants R01DK073394 (AFO), R01AR050051(HFE), the Muscular Dystrophy Association and the Cecil and Ida Green En-dowment (HFE).
2615-Pos Board B601Tensile Mechanical Measurements on Individual Collagen FibrilsRene B. Svensson, Tue Hassenkam, Colin A. Grant, S. Peter Magnusson.Introduction:There is a hierarchical order in tendon ranging from the collagen molecule tothe whole tendon. We have developed a method using the atomic force micro-scope (AFM) together with a novel data acquisition technique to quantify thesub-failure mechanical properties of individual collagen fibrils. Here we usethis method to investigate how the saline environment affects tensile forcetransmission.
Methods:Human patellar tendon tissue was obtainedfrom a healthy young male. Six fasciclesand six fibrils were mechanically testedsub-failure to determine modulus and rela-tive energy dissipation in PBS solutions of0.02, 0.15 and 1M concentration as well astwo HEPES buffers containing NaCl orNaClþCaCl2. Mechanical testing was per-formed to 4% strain.Results: The tensile properties of collagen fibrils are highly resistant to changes intheir saline environment. The only consistent response to environment com-position was a minor (<5%) increase in relative energy dissipation in 1 MPBS. This is in sharp contrast to previous reports of more than 100% increasein radial compressive modulus of fibrils in 1 M salt solution, and more than100% increase in energy dissipation of AFM pulling experiments in the pres-ence of Ca2þ.
![Provided by the author(s) and University College Dublin ... · [1]. The tissue-specific orientations of collagen fibrils give rise to different functional and mechanical properties](https://static.fdocuments.us/doc/165x107/5f961b1ddcb03c00ae5bc27f/provided-by-the-authors-and-university-college-dublin-1-the-tissue-specific.jpg)
