New Insights from Sum Frequency Generation Vibrational Spectroscopy into … · 2016. 5. 14. ·...

Review ArticleNew Insights from Sum Frequency GenerationVibrational Spectroscopy into the Interactions of Islet AmyloidPolypeptides with Lipid Membranes

Li Fu1 Zhuguang Wang2 Victor S Batista2 and Elsa C Y Yan2

1William R Wiley Environmental Molecular Sciences Laboratory Pacific Northwest National Laboratory PO Box 999Richland WA 99352 USA2Department of Chemistry Yale University 225 Prospect Street New Haven CT 06520 USA

Correspondence should be addressed to Elsa C Y Yan elsayanyaleedu

Received 8 April 2015 Accepted 24 June 2015

Academic Editor Lucie Khemtemourian

Copyright copy 2016 Li Fu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Studies of amyloid polypeptides on membrane surfaces have gained increasing attention in recent years Several studies haverevealed thatmembranes can catalyze protein aggregation and that the early products of amyloid aggregation can disruptmembraneintegrity increasing water permeability and inducing ion cytotoxicity Nonetheless probing aggregation of amyloid proteinson membrane surfaces is challenging Surface-specific methods are required to discriminate contributions of aggregates at themembrane interface from those in the bulk phase and to characterize protein secondary structures in situ and in real time withoutthe use of perturbing spectroscopic labels Here we review the most recent applications of sum frequency generation (SFG)vibrational spectroscopy applied in conjunction with computational modeling techniques a joint experimental and computationalmethodology that has provided valuable insights into the aggregation of islet amyloid polypeptide (IAPP) on membrane surfacesThese applications show that SFG can provide detailed information about structures kinetics and orientation of IAPP duringinterfacial aggregation relevant to the molecular mechanisms of type II diabetes These recent advances demonstrate the promiseof SFG as a new approach for studying amyloid diseases at the molecular level and for the rational drug design targeting earlyaggregation products on membrane surfaces

1 Introduction

Amyloid aggregates formed by misfolded intrinsically dis-ordered proteins are implicated in many diseases [1] Herewe focus on human islet amyloid polypeptides (hIAPPs)that aggregate into parallel 120573-sheets upon the interactionwith membrane surfaces [2ndash4] The resulting aggregates aredetrimental to pancreas 120573-cells leading to the onset of typeII diabetes [5ndash7] since they disrupt the membrane integrity[8ndash13] increasing the membrane permeability to water andions [14] Understanding surface-specific biomolecular inter-actions between hIAPP aggregates and lipid membranes atthe molecular level is therefore crucial for revealing themolecular factors controlling amyloidogenesis

Previous studies have relied mainly on bulk detec-tion techniques including circular dichroism (CD) [15]

NMR [16ndash18] EPR [19 20] 2D-IR [21ndash23] AIR-FTIR[24ndash26] fluorescence spectroscopy [27 28] Raman spec-troscopy [29] and infrared reflection absorption spec-troscopy (IRRAS) [30 31] However many specific questionsconcerning the interfacial region of interactions betweenmembranes and proteins remain unclear Some of the out-standing questions are as follows Do the amyloid aggregatesstart forming at membrane surfaces or in the bulk solutionHow do the aggregates orient on membrane surfaces Arethe aggregation products parallel or antiparallel 120573-sheetsDoes the orientation of the aggregate affect the integrityof cell membranes What is the kinetics of misfolding atmembrane surfaces Is it different from misfolding in thebulk solution Do molecular inhibitors affect aggregation onmembrane surfaces Recent developments in nonlinear sumfrequency generation (SFG) vibrational spectroscopy have

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2016 Article ID 7293063 17 pageshttpdxdoiorg10115520167293063

2 Journal of Diabetes Research

demonstrated SFG as an intrinsically surface-selective tech-nique with submonolayer sensitivity and label-free detectioncapability thus showing great promise to address the abovequestions shedding light on the role of themembrane duringthe aggregation of hIAPP and other amyloid proteins [32 33]

During the last three decades we have witnessed theemergence and fast development of nonlinear optical spec-troscopic techniques among which SFG vibrational spec-troscopy has gained tremendous attention Unique advan-tages of SFG include surface selectivity submonolayer sen-sitivity chiral-selectivity phase-sensitivity and label-freedetection capabilities [34ndash36] which make SFG a promisingtool for structural characterization of interfaces including awide range of applications in material science [37] charac-terization of polymers [38 39] and catalytic systems [40 41]Recently applications have been extended to environmental[42ndash44] and biological systems [32 33 45ndash52] with unprece-dented discoveries beyond the capabilities of conventionaltools For example SFG has been used to study a varietyof atmospherically significant systems at the vaporaqueousinterface to elucidate the organization and reactions inaerosols that contain inorganicorganic compounds [42ndash44] small molecules [42] and fatty acids [42] as solutesFurthermore SFG has been applied to probe the interfacialstructures orientation and kinetics of biologically relevantmolecules at interfaces such as proteins [32 33 45ndash47]DNAs [48 49] and lipids [45 50ndash52] providing insights intothe functions of these molecules at biological interfaces andfacilitating further research into biomedical science and engi-neeringNowadays SFG is established as a valuable techniqueto understand physical chemical and biological processesat the molecular level [53] In particular applications tointerfacial biological systems span across important researchtopics in membrane biophysics [54] surface self-assembly[55 56] peptides at cell surfaces [57 58] DNA hybridization[48 59] and adsorption [60] and amyloid interactions withmembrane surfaces [61ndash63]

This review focuses on the recent development and appli-cation of SFG for probing hIAPP interacting with lipid mem-branes and discusses the implications of hIAPPmembraneinteractions in the studies of type II diabetes [14 61 62 6465] The review also summarizes the basic theoretical back-ground and experimental methods of SFG supplementedwith a brief discussion about the application of SFG to otheramyloidogenic proteins and concludes with an outlook ofSFG in applications to systems of interest in biological andmedical sciences

2 The SFG Method

21 Basic Principles of SFG Sum frequency generation (SFG)vibrational spectroscopy applies two laser beams that interactwith the system of interest simultaneously [34ndash36 66ndash69]one with visible (eg 532 nm) or near-infrared (eg 800 nm)frequency 120596vis and the other with infrared (IR) frequency 120596IR(Scheme 1) When the visible and infrared pulses overlap intime and space light with the sum of the two frequencies120596SFG = 120596vis + 120596IR is generated by the interaction of the

SFG120596vis + 120596IR = 120596sum

120596vis

120596vis

120596IR120596IR 120596sum

120596sum

Scheme 1 The second-order optical process of sum frequencygeneration vibrational spectroscopy

incident beams with the system at the interface Fixing120596vis and scanning or dispersing IR frequencies 120596IR over arange the SFG spectra are recorded by monitoring the SFGintensity as a function of 120596SFG with a monochromator anda CCD The SFG signal is dramatically enhanced when 120596IRis in resonance with a vibrational frequency of a moleculeat an interface thus showing a peak in the spectrum TheSFG peaks typically exhibit homogeneous and inhomoge-neous broadening leading to various line-shapes due to theconstructive or destructive interference with neighboringbands modulated by changes in the molecular orientation asinduced by interactions with the surface and the surroundingenvironment [70] Thus the SFG spectra contain detailedstructural information reported in terms of line-shape peakposition and polarization dependence Extracting that struc-tural information from the SFG spectra however requiresrigorous theoretical modeling and computer simulationsHence the combination of SFG and computational modelingcan be used as a label-free and in situ analytical methodologyfor effective characterization of systems at interfaces In thefollowing sections wewill illustrate the applications of SFG tothe studies of IAPP at membrane surfaces [14 61 62 64 65]

22 Surface-Specificity Monolayer Sensitivity and Polariza-tion Dependence of SFG Spectroscopy As a nonlinear opticaltechnique SFG measures the second-order susceptibility120594(2) that gives intrinsic surface selectivity [33 34 71ndash74]The second-order susceptibility 120594(2) is the direct product ofthe complex conjugate of the Raman polarizability derivativematrix (the same as the original matrix when derivativevalues are real numbers) and the IR dipole derivative vectorand thus is a second-order tensorThe generated electric fieldof SFG signal is related to the electric field of the two incidentlaser beams 119864vis and 119864IR via the tensor elements 120594(2)

119894119895119896

119864119894SFG prop sum119895119896

120594(2)119894119895119896119864119895

vis119864119896

IR (1)

where 119894 119895 and 119896 specify the direction of the Cartesiancomponent of the optical fields and can be denoted by 119909 119910and 119911 in the laboratory coordinates

Journal of Diabetes Research 3

The tensor elements of bulk phases with inversionsymmetry (eg gas solution and amorphous solid) areisotropically averaged to zero so long as the frequency ofthe visible beam is not in resonance with an electronicexcitation [33 73 74] This is because molecules rotatefreely and diffuse adopting random orientations In contrastmolecules at surfaces give prominent SFG signals sincethey have ordered alignment across the surface region andthereby their tensor elements are nonzero Furthermore thesecond-order susceptibility is proportional to the square ofthe molecular density at surfaces and thus is sensitive tothe change of molecular coverage enabling SFG to detecta monolayer of molecules at an interface The monolayersensitivity is critical for the characterization of biologicalsamples that are difficult to purify in large quantities Asa result of such unique surface-specificity and monolayersensitivity the SFG method is free from contributions ofthe bulk medium and is thus an ideal optical method toprobe membrane surfaces and their interactions with otherbiomolecules

SFG spectroscopy has the intrinsic sensitivity to chiralstructures because the measured second-order susceptibilitytensor elements 120594(2)

119894119895119896are three-dimensional When the three

indices 119894 119895 and 119896 are distinct from one another (ie 119894 = 119895 =119896) the susceptibility tensor captures the features of the chiralCartesian coordinate systemwith three distinct axes and thuscomprises information about the chirality at interfaces [33]Therefore surface chirality can be directly measured through120594(2)119894119895119896 (119894 =119895 =119896)

making chiral SFG spectroscopy highly sensitiveand easier for interpretation compared tomore conventionalchiroptical methods such as circular dichroism Ramanoptical activity and optical rotation dispersion which requirehigher-order couplings between electronic and magneticdipoles Due to its high chiral sensitivity SFG could providepreviously unattainable molecular information about proteinand biomolecules in chiral supramolecular and hierarchicstructures at surfaces [74 75]

SFG measurements can be modulated by various polar-ization settings For a particular experiment one can mod-ulate the incident visible and infrared beams and detect theSFG beam in either s- or p-polarization [66 69] or evenmixed polarizations [48 72 76] With the use of only s- orp-polarization there are in total 2 times 2 times 2 = 8 polarizationsettings for experimental geometries including ssp ppp spspss spp psp pps and sss where the first second and thirdindices indicate the polarization of the SFG visible andinfrared beams respectively (Scheme 2) The psp spp andpps polarization settings are chiral-selective and thus can beused to probe the chiral SFG spectra as discussed previously[33] The others are achiral polarization settings that aresensitive to different vibrational modes Altogether chiraland achiral SFG spectroscopy can provide a comprehen-sive analysis of vibrational modes of chiral or nonchiralmolecules at interfaces In more advanced measurementsone can even determine the absolute orientation ofmoleculesat interfaces by performing a global analysis of variouspolarization-modulated spectra [77] With these capabilitiesSFG can report on structures and orientations of molecules

s-SFG

s-visp-IR

x

x

y

y

y

z

z

xy-plane

xz-plane

Scheme 2 The 119904119904119901 polarization setting in an SFG experiment 119904-polarized SFG 119904-polarized visible and 119901-polarized IR beams Theprojection electric field of 119901-polarized and 119904-polarized light onto thelaboratory coordinates

and proteins at surfaces offering a methodology to addressmechanistic questions on amyloid aggregation that wouldotherwise be difficult to tackle by using more conventionalmethods

23 SFG Experiments The setup of the SFG spectrometer hasbeen extensively described [77ndash79] Currently scanning andbroad-bandwidth SFG spectrometers are most commonlyused Scanning SFG spectrometers use picosecond pulsed IRand visible beams and scan the IR frequency stepwise at afixed visible frequency [80] A typical broad-bandwidth SFGspectrometer consists of a femtosecond (sim100 fs) pulsed IRbeam and a picosecond (2ndash100 ps) pulsed visible beam [81]Because the femtosecond IR pulse includes a wide frequencyrange of more than 200 cmminus1 in the mid-IR region broad-bandwidth SFG spectrometers can acquire spectra by oneshot without scanning the IR frequencyThe one-shot schemeallows for monitoring the kinetics of protein conformationalchanges at interfaces [55 82] In this review we focus onrecent studies of hIAPP aggregates at membrane surfacesbased on broad bandwidth SFG spectroscopy [14 61 62 6465]

Chiral and achiral SFG spectra can be selectively collectedusing different polarization settings Typically one can usepsp spp and pps to probe the chiral SFG spectra and sspsps and ppp to probe the achiral SFG spectra [33] Whenperforming the chiral and achiral SFG measurements onecan control the polarization of the beams using appropriatewave-plates and polarizers Chiral SFG is particularly usefulfor probing biomolecules because most secondary structuresare chiral such as 120572-helices and 120573-sheets These chiralmacromolecular structures are expected to show a strongchiroptical response in chiral SFG while the solvent andother molecules lacking macroscopic chiral structures aresilent in the chiral SFG spectra Thus chiral SFG spectra ofbiomacromolecules are less affected or distorted by signalsfrom solvent or other achiral molecules simplifying thespectral analysis and interpretation

The choice of surface platform is another important factorfor efficiently probing biomolecules at interfaces For studies


800nm 800nm 800nmSFG SFG SFG

IR IR IR

hIAPP

DPPG lipid

hIAPPintermediate

hIAPPaggregates

Scheme 3 Illustration of adsorption of hIAPP on a lipid monolayer and the SFG experiment for probing the hIAPP aggregations at thelipidwater interface Adapted from [62] with permission Copyright 2010 American Chemical Society

of hIAPP there are two applicable surface platforms thatallow for probing the lipidaqueous interfacial region [83]One surface platform is made by spreading a lipid monolayeronto the water surface [31] while the other is made byfabricating a supported lipid bilayer on a solid substrateusing the Langmuir-Blodgett (LB)Langmuir-Schaefer (LS)method [51] Both platforms have been widely used asmimics of biomembranes Here we will focus on the hIAPPstudies that have been carried out using the platform of lipidmonolayer Briefly the experimental procedure (Scheme 3)[62] starts with adding hIAPP to an aqueous buffer and thenspreading the lipid monolayer at the airwater interface ThehIAPP will adsorb at the interface and interact with the lipidmembrane SFG spectroscopy can then be used to monitorlipid-induced conformational changes in hIAPP in situ andin real time at the interface

3 SFG Probes the Early Stages of hIAPPAggregation at Membrane Interfaces

The early stages of hIAPP aggregation at interfaces involvehIAPP-membrane interactions associated with the path-ogenic mechanism of type II diabetes [6 84 85] Howeverit has been challenging to probe how hIAPP adsorbs ontothe interface and whether hIAPP undergoes structural andorientation changes that might induce toxicity to pancreatic120573-cells Previous studies of the early stages of hIAPP aggre-gation mainly focused on bulk detection using methods suchas CD NMR 2D-IR and fluorescence spectroscopy that arenot surface-sensitive or require spectroscopic labeling [9]

To overcome these challenges Winter and coworkersapplied infrared reflection absorption spectroscopy (IRRAS)and found 120573-sheets structures formed in the process ofhIAPP aggregation at the airwater interface with a negativelycharged lipid [31] Inspired by the earlier IRRAS work ourgroup used SFG spectroscopy to probe hIAPP aggregationat membrane surfaces in situ and in real time monitoringthe amide I and N-H stretching vibrational modes [6162] Protein structures including 120572-helices and 120573-sheetsare formed by hydrogen bonding interactions between theamide and N-H groups along the protein backbone Thusthe vibrational frequency and line-shape of the amide I and

N-H stretching modes are sensitive markers for distinguish-ing protein secondary structures [86 87] In conventionalvibrational studies the O-H bending and O-H stretching ofwater overlap with the amide I and N-H stretching modesmasking the characteristic bands of secondary structuresthe peak assignment and characterization of the proteinsecondary structure In contrast chiral SFG provides high-quality vibrational spectra revealing conformational changespreviously undetectable by using conventional methodssince it probes only the interfacial molecules without anysignificant vibrational background fromwater solvent Belowis a summary of such vibrational SFG studies in the amide Iand N-H stretching regions probing the early aggregation ofhIAPP upon the interaction with membrane surfaces

31 hIAPP Aggregation at Interfaces Probed by Amide ISFG Signals First our group has focused on experimentsfor both human IAPP (hIAPP) and rat IAPP (rIAPP) thatprobed the amide I region in both achiral and chiral SFGspectra [32 33 61 62 65 88] The two peptides are differentby only six amino acids Remarkably the rIAPP does notaggregate into amyloids making it an ideal control systemfor SFG studies The achiral SFG spectra (Figure 1) werecollected in the absence and presence of the negativelycharged lipid dipalmitoylphosphoglycerol (DPPG) In theabsence of DPPG both hIAPP and rIAPP show amide I peaksat 1650 cmminus1 suggesting the presence of both peptides atthe airwater interface Moreover the spectra do not changeover 10 hours suggesting no noticeable structural changesIn contrast with a DPPGmonolayer and after incubation for10 hours the amide I band of hIAPP changes dramaticallyin terms of both the peak position and the line-shape Incontrast the spectra of rIAPP remained unchanged Since thefrequency of the amide I band varies with protein secondarystructures the different spectroscopic responses from hIAPPand rIAPP indicate that hIAPP exhibits structural changesupon interaction with a DPPG monolayer while rIAPP doesnot Figure 1 shows similar results for experiments in H2Oand D2O suggesting that isotopic effects are negligible onhIAPP aggregation

A closer look at the spectral change of hIAPP incubatedwith DPPG after 10 hours shows that the amide I peak posi-tion is blue-shifted by 10 cmminus1 from sim1650 to sim1660 cmminus1


SFG

inte

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(au

)

hIAPP without DPPG 0hhIAPP without DPPG 10hhIAPP with DPPG 10h

16501600 17501700

Wavenumber (cmminus1)

(a) AirD2O


SFG

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(au

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16501600 17501700


(b) AirH2O

SFG

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rIAPP without DPPG 0hrIAPP without DPPG 10hrIAPP with DPPG 10h

1700 17501600 1650


(c) AirD2O


SFG

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(d) AirH2O

Figure 1 The ssp (achiral) SFG spectra of IAPPs Human IAPP without DPPG (119905 = 0 h and 119905 = 10 h) and with DPPG at 119905 = 10 h at the (a)airD2O and (b) airH2O interfaces rat IAPP without DPPG (119905 = 0 h and 119905 = 10 h) and with DPPG at 119905 = 10 h at the (c) airD2O and (d)airH2O interfaces Adapted from [62] with permission Copyright 2010 American Chemical Society

and there is an additional peak at 1750 cmminus1 correspondingto the carbonyl stretch of the DPPG lipid [87] Nonethelessit is still challenging to specify what structural changesare involved at the lipidaqueous interface To address thisquestion we applied chiral SFG

The chiral SFG measurements (Figure 2) show moreinteresting phenomena Without the DPPG lipid neitherhIAPP nor rIAPP shows detectable chiral SFG signal in theamide I region The lack of signal is not surprising since thenative structures of hIAPP and rIAPP are disordered and donot adopt any chiral conformationHowever after incubatingwith DPPG for 10 hours hIAPP shows a strong chiral SFGsignal at 1622 cmminus1 with a shoulder at 1660 cmminus1 The low-frequency amide I band at 1622 cmminus1 and a shoulder-peak

at 1660 cmminus1 are characteristic of parallel 120573-sheets [86 87]Thus these results indicate that hIAPP formsparallel120573-sheetsupon the interaction with DPPG at the lipidwater interface

Altogether the achiral and chiral SFG studies of hIAPP inthe amide I region demonstrate that both hIAPP and rIAPPcan adsorb onto the airwater interface Furthermore hIAPPundergoes structural changes from disordered structures toparallel 120573-sheets upon the interaction with the surface ofa lipid monolayer Moreover as a control system rIAPPremains unchanged with and without lipid at the surfacerevealing clear differences in the behavior of hIAPP andrIAPP at the lipidaqueous interface Detailed analyses ofthese spectral data have provided structural kinetic andorientation information discussed in the following sections


SFG

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(au

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170016501600


(a) AirD2O


SFG

inte

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(au

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170016501600


(b) AirH2O

SFG

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170016501600


(c) AirD2O


SFG

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(au

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170016501600


(d) AirH2O

Figure 2 The psp (chiral) SFG spectra of IAPPs Human IAPP without DPPG (119905 = 0 h and 119905 = 10 h) and with DPPG at 119905 = 10 h at the (a)airD2O and (b) airH2O interfaces rat IAPP without DPPG (119905 = 0 h and 119905 = 10 h) and with DPPG at 119905 = 10 h at the (c) airD2O and (d)airH2O interfaces Adapted from [62] with permission Copyright 2010 American Chemical Society

32 SFG Allows for Kinetic Studies of hIAPP Misfolding at theEarly Stage The chiral and achiral SFG spectra have beencollected over time (Figure 3) [61 62] to explore the kineticsof aggregation of hIAPP at the interface Figure 3(a) showsthat during the aggregation process the achiral amide I bandof hIAPP gradually shifts to higher frequency with increasedintensity suggesting conformational changes in hIAPP Theincrease in intensity reflects more ordered structures ofhIAPP aggregates because SFG signals are sensitive to theordering of themolecules at interfaces Figure 3(b) shows thatthe chiral amide I signal at 1622 cmminus1 starts emerging afterthree hours and keeps increasing with an appearance of ashoulder-peak at 1660 cmminus1 This result not only suggests theformation of parallel 120573-sheets but also confirms the orderingof hIAPP during the aggregation process

The successful use of the amide I band to probe thekinetics of 120573-sheet formation in hIAPP inspired us to useSFG signals from the peptide backbone in other vibrationalregions such as N-H stretching (amide A) [61] We probedthe chiral N-H stretching signals of hIAPP during theaggregation process of hIAPP under the same experimentalconditions and observed unique spectral features The chiralSFG spectra in Figure 3(c) show that initially there are no N-H stretching signals but a peak at 3280 cmminus1 gradually buildsup and reaches a maximum value after roughly three hoursof interaction with DPPG and then slowly vanishes after10 hours This transient chiral N-H stretching signal clearlyreveals an intermediate in the hIAPP aggregation processTo investigate further the structure of this intermediate weobtained the chiral N-H stretching spectra of several model


SFG

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(au

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1700 175016501600

5min30min135min

308min335min572min


ssp hIAPP + DPPG H2O

(a)

SFG

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1600 17001650

18min200min346min

437min462min554min


psp hIAPP + DPPG H2O

(b)

SFG

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559min

448min

226min

181min

158min

86min

15min

3285 cmminus1

32003100 3300


hIAPP + DPPG

(c)

Figure 3 Kinetics of human IAPP aggregates at the lipidH2O interface probed by time-dependent SFG spectra in the amide I region using(a) ssp (achiral) and (b) psp (chiral) polarization in the presence of DPPG and (c) in the N-H stretching region using psp (chiral) polarizationin the presence of DPPG Adapted from [61 62] with permission Copyright 2010 and 2011 American Chemical Society

proteins in 120572-helical structures We concluded that the chiralSFG N-H stretching mode at 3280 cmminus1 is due to 120572-helicalstructures [61] A combination of the results of kinetic studiesusing the amide I (Figure 3(b)) and N-H stretching bands(Figure 3(c)) reveals an important finding the appearanceof the N-H stretching peak reaches a maximum and startsto disappear prior to the accumulation of the chiral amide Isignal (Figure 4(a)) providing a molecular picture of hIAPPmisfolding at membrane surfaces where hIAPP initiallyadsorbs to the membrane surface as a random coil and thenforms 120572-helical intermediates which subsequently convertinto parallel 120573-sheet aggregates

As the first kinetic study using chiral and achiral SFGto probe conformational changes of proteins at interfacesin situ and in real time the above studies demonstrateSFG as a method of high selectivity and sensitivity notonly for characterizing protein secondary structures butalso for studying the kinetics of conformational changes atinterfaces The N-H stretching and amide I bands are twowell-separated vibrational regions that can be used syner-gistically for revealing aspects of the molecular mechanismof aggregation at interfaces The kinetic data obtained at thelipidwater interface by SFG spectroscopy can be comparedto measurements in the bulk solution based on conventionalphysical methods yielding a better understanding of the rolethat the membrane surface plays in the amyloid aggrega-tion process This methodology is expected to find applica-tions in testing the efficacy of drug candidates that inhibit

the aggregation of hIAPP at lipidwater interfaces as illus-trated in the following section

4 Inhibition of the hIAPP Aggregation atMembrane Surface

Several studies have shown that the aggregation of hIAPPis associated with the disruption of membrane integrityand death of pancreas cells [9 11 12] Hence the searchfor inhibitors of hIAPP aggregate formation has been astrategy explored for drug development [77 89] Previouslymost of the screening of drug candidates inhibiting hIAPPaggregation has been tested in the bulk aqueous solutionHowever inhibitors that work in the bulk may have lowefficacy on membrane surfaces Bonn and coworkers appliedSFG to address this issue [64] and have confirmed thatthe surface indeed plays an important role in reducing theinhibition of hIAPP aggregation by drug candidates

41 EGCG Shows Less Inhibition of hIAPP Fibril For-mation at Interfaces Bonn and coworkers studied (minus)-epigallocatechin gallate (EGCG) as an inhibitor for hIAPPaggregation and compared its effects in the bulk solutionand on membrane surfaces [64] EGCG is a natural productfound in green tea and belongs to a class of inhibitorscontaining polyphenols Previous studies showed that EGCGcan effectively inhibit themisfolding and fibrillation of hIAPP


120572-helix 120573-sheet aggregates

Chiral N-HChiral amide I

1 2 3 4 5 6 7 8 90 10Time (h)

00

05

10

SFG

inte

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(au

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(a)

1 2 3

Random coil120572-helical

intermediates120573-sheet

aggregates

(b)

Figure 4 The misfolding pathway of hIAPP on membrane surfaces (a) Chiral SFG intensities of the chiral N-H stretching (3280 cmminus1)and amide I signals (1620 cmminus1) as a function of time from triplicate experiments (b) The model mechanism on hIAPP aggregation at themembrane surface adsorption of disordered hIAPP onto membrane leads to formation of 120572-helical intermediates which are then convertedto 120573-sheet aggregates Adapted from [61] with permission Copyright 2011 American Chemical Society

and even disaggregate 120573-sheet-rich amyloids in the bulksolution [90 91] However the inhibitive effect of hIAPP atthe interface remains unclear

Bonn and coworkers combined achiral SFG spectroscopywith thioflavin T (ThT) fluorescence and atomic forcemicroscopy (AFM) for bulk analysis to examine the effectof EGCG on inhibiting hIAPP aggregation at the lipidwaterinterface [64] They used the amide I band of hIAPP in theachiral SFG spectra to monitor the kinetics of the formationof 120573-sheet fibrils In the absence of EGCG at the lipidwaterinterface the time-dependent SFG spectra (Figure 5(d))show blue-shifts in peak position and increasing intensitiesof amide I band similar to the observations made by Fu et al(Figure 3(a)) In the presence of EGCG blue-shifts in peakpositions can still be observed (Figure 5(e)) indicating theformation of 120573-sheets This is confirmed by the AFM imagesof the sample at interfaces transferred onto mica The AFMimages show the formation of fibrils on the film made fromhIAPP at the airwater interface with lipid in the presenceof EGCG after incubating for sim17 hours (Figure 5(c)) Theextent of fibril formation is similar to that in the absenceof EGCG (Figure 5(b)) The results at the interface elicit thehypothesis that EGCG has a reduced inhibitive effect onhIAPP aggregation at the lipidwater interface

To test this hypothesis a comparison is made for theinhibitive effect of EGCG on hIAPP aggregation at theinterface versus in the bulkThe amount of120573-sheets formed atthe lipidwater interface in both the presence and the absenceof EGCG is quantitatively estimated by the deconvolutionof each time-dependent spectrum using the Lorentzian line-shape fittingThe time-dependent120573-sheet component decon-voluted from the SFG spectra is plotted in Figure 6(a) (and ◻ curves) along with the time-dependent fluorescenceintensity from hIAPP aggregates in the bulk (times and + curves)The flat + curve in Figure 6(a) suggests a lack of amyloidfibril formation in the bulk in the presence of EGCG whichis further confirmed by the AFM image in the presence

(Figure 6(c)) of EGCG Therefore both the fluorescence andthe AFM results indicate a strong inhibition of EGCG onhIAPP aggregation in the bulk On the other hand the120573-sheet component deconvoluted from the SFG spectra isstill increasing with time at the lipidwater interface inthe presence of EGCG (Figure 6(a) ◻ curve) The abovecomparison demonstrates that the inhibitive effect of EGCGon hIAPP is indeed reduced at the interface

42 Consideration of Membrane Effects for Drug Design Thereduced efficacy of EGCG as an inhibitor at membranesurfaces may be due to the role of the surface in controllingthe structure orientation and dynamics of the aggregationprocess The proposed inhibitory mechanism for EGCGinvolves the binding of the phenol groups to the hydrophobicaromatic side chains of hIAPP In the bulk both the inhibitorand the hIAPP diffuse freely Thus the inhibitor may bindto the hIAPP aromatic groups more effectively At theamphipathic membrane-water interface however hIAPP isanchored with a specific orientation leaving the hydrophobic120573-strands pointing towards each other buried inside themembrane phase while the hydrophilic side chains makecontact with water solvent and the lipid polar head groupsThis specific orientation suppresses free diffusion of hIAPPand makes it difficult for EGCG to bind On the other handthe small EGCG molecules with multiple phenol groups aremore soluble in the bulk potentially leading to low surfacepopulation which further reduce its efficacy

The SFG study demonstrates that the effect of the surfacecan be a critical factor to be considered in the drug designfor type II diabetes The pathogenic origin of type II diabeteshas been proposed to be linked to the hIAPP aggregationprocess and the disruption of membrane integrity [5] There-fore it is important to screen drug candidates that mightaffect aggregatemembrane interactions by changing thehIAPP interfacial orientation conformation or dynamicsThe resulting conformational changes could be probed by


(a) (b)

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14min82min153min230min


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(d) (e)

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Figure 5 Kinetics study using AFM and SFG measurements during hIAPP aggregation at the phospholipid interface in the presence orabsence of EGCG AFM images of hIAPP with lipid for (a) 10 and (b) 1020min in the absence of EGCG and (c) AFM image after hIAPPaggregation in the presence of EGCG for 1020min SFG spectra of hIAPP with phospholipid in the amid I region (d) in the absence of and(e) in the presence of EGCG Adapted from [64] with permission Copyright 2012 American Chemical Society

SFG as described in the following section focused on theorientation of aggregates at the lipidwater interface

5 Orientation of hIAPP Aggregates at LipidMembrane Surfaces

Understanding at the molecular level whether hIAPP aggre-gates disrupt cell membranes could provide valuable insightsinto the pathogenic mechanisms of type II diabetes Disrup-tion due to a specific orientation of the hIAPP aggregatesadsorbed on the membrane surface might increase themembrane permeability to water and ions However deter-mining the interfacial orientation of complex biomoleculesby conventional methods is challenging In particular com-plex hIAPP aggregates in the form of pleated parallel120573-sheets pose significant challenges for both theory and

experiments [92ndash94] We have combined molecular dynam-ics and divide-and-conquer ab initio quantum chemistrycalculations of hyperpolarizability derivatives with respect tonormal modes to simulate the chiral SFG spectroscopy ofhIAPP [65] Our simulations found that the hIAPP aggregatesare neither parallel nor perpendicular to the membranesurface but rather inserted into the lipid membrane tiltedat an angle of about 45∘ relative to the surface normal Theresulting orientation optimizes amphiphilic interactions byexposing hydrophilic domains of the aggregate to the aqueousphase and hydrophobic parts to the lipids Such ldquodetergent-likerdquo orientation is expected to cause significant disruptionof the cell membrane

This section describes the theoretical and experimentalanalyses of chiral SFG spectra necessary to retrieve theorientation of the hIAPP aggregate at the interface


Bulk minus EGCG (ThT)Bulk + EGCG (ThT)Lipid interface minus EGCG (SFG)Lipid interface + EGCG (SFG)

03

04

05

06

07

08

09

SFG120573

-she

et (a

u)

10

15

20

25

ThT

fluor

esce

nce i

nten

sity

(au

)

800400 1200 16000

Time (min)

(a)

(b)

50nm

(c)

Figure 6 Bonn and coworkers compared the inhibitory effect of EGCG on hIAPP fibrillation in the bulk and at the phospholipid interface(a) In the bulk the formation of hIAPP amyloid fibrils was measured using a ThT fluorescence assay The sigmoidal increase in fluorescencesignal in the absence of EGCG (times) indicates the formation of hIAPP fibrils and the low fluorescence signal in the presence of EGCG at a1 1 molar ratio (+) suggests the inhibition of fibril formation At the phospholipid interface in absence () and presence (◻) of EGCG SFGspectra measure the formation of 120573-sheets (b) AFM also shows hIAPP fibrils formed in the absence of EGCG and (c) EGCG completelyinhibits fibril formation of hIAPP Adapted from [64] with permission Copyright 2012 American Chemical Society

The chiral SFG spectrum of the aggregated hIAPP atthe membrane surface shows a dominant peak at 1620 cmminus1with a shoulder-peak at 1660 cmminus1 in the amide I region(Figure 7(b)) These two peaks correspond to the antisym-metric (119861-mode) and symmetric (119860-mode) bands of parallel120573-sheets Detailed analyses of the molecular symmetry andvibrational coupling indicate that their relative ratio ofintensities (119868119861119868119860) is correlated with the orientation of the 120573-sheet

119868119861119860 =

10038161003816100381610038161003816100381610038161003816100381610038161003816

120594(2)119901119904119901119861

120594(2)119901119904119901119860

10038161003816100381610038161003816100381610038161003816100381610038161003816

2

=100381610038161003816100381610038161003816100381610038161003816⟨tan2120595⟩

120573119887119888119886119861

120573119886119888119887119860+ (1 minus ⟨tan2120595⟩)

120573119887119886119888119861

120573119886119888119887119860

100381610038161003816100381610038161003816100381610038161003816

2

(2)

where 120595 is the angle between the 120573-strand and the interfaceas defined in Figure 7(a) The hyperpolarizability elements(120573119886119888119887119860 120573119887119888119886119861 120573119887119886119888119861) provide the specific molecular propertyof the hIAPP aggregates with 119886 119887 119888 referring to the Cartesiancoordinates in the molecular frame of the hIAPP parallel 120573-sheet where 119887 is the direction parallel to the 120573-strand and 119888points to the axis of propagation of intermolecular 120573-sheets(Figure 7(a)) From (2) it is clear that the orientation angle 120595can be determined as the value thatmatches the experimentalratio of SFG intensities for the 119861 and 119860 bands (119868119861119860) Theintensity ratio is measured to be 48 from the fitted amideI spectrum (Figure 7(b)) Consequently knowing the valuesof three hyperpolarizability elements (120573119886119888119887119860 120573119887119888119886119861 120573119887119886119888119861) in(2) allows the determination of the average orientation (120595)determined by SFG spectroscopy

We have applied a divide-and-conquer approach thatfragments the hIAPP aggregate into domains amenable to

quantum chemistry calculations and computes the hyper-polarizability elements of the constituent fragments at thedensity functional theory (DFT) level Specifically the NMRstructure [18] was divided into 16 tripeptide pairs in the 120573-sheet region and the hyperpolarizability of each tripeptidepair was calculated by DFT (Figure 7(c)) The overall hyper-polarizability of hIAPP aggregates is then integrated fromthe hyperpolarizability elements of the individual tripeptidepairs The plot of the intensity ratio of the amide I peaks asa function of the orientation of the parallel 120573-sheet showsthat the orientation 120595 = 45ndash48∘ has the best agreement withexperimental data suggesting that the hIAPP aggregates ori-ent with the 120573-strand at 120595 asymp 45∘ from the surface The tiltedorientation of hIAPP 120573-sheet aggregates at the lipidwaterinterface suggests that significant disruption might be causedon the lipid membrane These findings are supported bymolecular dynamics simulations that analyzed the orien-tation and stability of hIAPP aggregates at DPPGwaterinterfaces [14]The simulation shows that hIAPP gets insertedinto lipid monolayers at about 120595 = 40∘ and in lipid bilayers ata tilted angle of120595 = 60∘ as shown in Figure 8 leading towaterpermeation and Na+ percolation through the membranesupporting the hypothesis of ion-cytotoxicity for islet 120573-cells[14]

These molecular dynamics simulations support theunique capabilities of chiral SFG spectroscopy for probingthe orientation of 120573-sheet amyloid aggregates providingfundamental insights that should be particularly relevantfor understanding amyloid diseases at the molecular levelThe combination of computational modeling and SFG spec-troscopy thus provides a valuable methodology to iden-tify potential noncompetitive inhibitors that might changethe conformation and orientation of hIAPP aggregates and


a

a

b

b

c

c

hIAPP

hIAPP

x

y

z

120579

120601

120595

(a)

B

R

NH

HN

O R

O

R

NH

HN

O R

O

A mode

B mode

psp

A000

005

010

SFG

inte

nsity

(au

)

1640 16801600Wavenumber (cmminus1)

(b)

811

19

23

24

8

2837

N N N

O

N

H

H

H

N N N

O

O

N

O

H

H

H

O

O

H

H

O

OR1

R1

R2

R2

R3

R3

R4

R4

K C N T A T C A Q HK C N T A T C A A L H

SSNNF

SSNNF

GAILSTY

LL

TNVGSN SGAILSTY TNVGSN S

FFN

R NARQT

TVVL

(c)

0

5

10

15

20

25

45 225135 315 36027090 1800

120595 (∘)

|120594(2)

pspB120594

(2)

pspA|2

(d)

120595 = 30∘

120595 = 48∘

120595 = 132∘

120595 = 150∘

Exp

Ipsp

(au

)

1600 16501550 1700Wavenumber (cmminus1)

(e)

Figure 7 Continued


5

10

15

20

25

30

35

27

His18

Arg1148

∘

(f)

Figure 7 Determination of the orientation of human IAPP aggregates at the lipidwater interface (a) Definition of three orientation angles(120601 120579 120595) for Euler transformation from the laboratory to the molecular frame (b) psp (chiral) SFG spectrum of hIAPP aggregates in theamide I region 119860 and 119861 denote the characteristic peaks for amide I symmetric and antisymmetric modes (c) Scheme showing the divide-and-conquer method for simulations of the SFG spectra from calculations of hyperpolarizability derivatives with respect to normal modedisplacements in human IAPP aggregates (d) Relationship between the intensity ratio of the 119861mode to the119860mode and orientation angle 120595The blue curve is obtained analytically from (1) and the red curve is obtained numerically (e) Chiral SFG spectra of human IAPP aggregatessimulated for various orientations at the interface (f) Visualized orientation of the human IAPP aggregates at the lipidaqueous interfaceAdapted from [65] with permission Copyright 2012 Elsevier

consequently reduce their toxicity as implicated in amyloiddiseases

6 Perspectives and Challenges of SFG inBiological and Medical Applications

In summary we have reviewed the application of SFGspectroscopy to study the amyloidogenesis of hIAPP inter-acting with membrane surfaces [14 61 62 64 65] Wehave shown that SFG can reveal structural and dynamicinformation characterizing the formation of aggregates insitu and in real time by using different polarization settingsand probing in different vibrational regions The versatilityof SFG experiments also provides ample information thatallows for elucidating the orientation of hIAPP aggregates atthe waterlipid interface With the capabilities of probing andcharacterizing structure orientation and dynamics studiesbased on SFG spectroscopy can provide valuable insightsintomembraneprotein interactions that are critical to a widerange of pathological diseases including amyloidogenesisand cytotoxicity to pancreas 120573-cells leading to the onset oftype II diabetes Based on these findings potential drugcandidates that specifically target the early aggregation ofhIAPP at the membranewater interface are currently beingproposed and tested by using SFG spectroscopy to guide therational design of drugs for treatment of type II diabetes

Studies of other amyloid diseases such as AlzheimerrsquosParkinsonrsquos Huntingtonrsquos and Prion diseases could alsobenefit from SFG techniques In fact Luo and coworkers havealready applied SFG to study the membrane-mediated struc-tural change of prion protein fragments and characterized theconcentration dependence of structures and orientation for

prion oligomers [95] In addition Weidner and coworkershave performed SFG studies to investigate oligomerization oflysozyme at membrane surfaces where they simultaneouslymonitored conformational states of lysozyme and the orga-nization of lipid molecules in contact with aqueous buffer atvarious values of pH [63]

It is foreseeable that SFG spectroscopy can be extendedto a wider range of studies critical for the mechanisticunderstanding of amyloid diseases and drug developmentPotential applications include studies of the interactions ofamyloid proteins with components in cell membranes (egcholesterol membrane protein and glycolipid) [96] otherrelevant biomolecules (eg insulin [97] and sphingolipid)and potential drug candidates (eg small aromatic organicmolecule and peptide analogue of amyloid protein) Thoseapplications could provide valuable insights into amyloido-genic intermediates during the onset of membrane diseases

Given the potential applications of SFG in the investi-gation of amyloidogenesis two major challenges remain inorder to develop SFG into a general biophysical tool forbiological and biomedical research These challenges includethe development of efficient methods for acquisition of high-quality spectroscopic data and unequivocal interpretationof the spectra On one hand experimentalists in the SFGfield have been striving to improve the instrumentationfor SFG spectroscopy to enhance the quality of the dataBroad bandwidth and even ultrabroad bandwidth SFG spec-troscopy have been developed to cover a wider vibrationalfrequency range with one-shot measurement [56] short-ening the time for spectral acquisition and rendering itpossible to monitor kinetics of structural and orientationalchanges High-resolution SFG spectroscopy can characterize


5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268

nlocal(z) (nmminus3)

(d)

Figure 8 Bilayer thickness around embedded hIAPP for (a) trimer and (b) tetramer aggregates as described by molecular dynamicssimulations Color key bilayer thickness (in nm) is mapped to the corresponding colors (c) Water channel formed by the hIAPP trimerin the DPPG bilayer showing water molecules (red and white) and Na+ ions (blue vdW spheres) percolating through the bilayer (d) Averageparticle density of water and Na+ ions within the membrane Adapted from [14] with permission Copyright 2013 by the Biophysical Society

surfaces and interfaces with unprecedented subwavenum-ber (lt1 cmminus1) resolution [98] providing detailed molecu-lar information necessary to understand structurefunctionrelations in physical and biological processes [99ndash101] Inaddition heterodyne-detected SFG spectroscopy has beendeveloped to enhance the signal level of SFG spectra [102ndash107] Moreover 2-dimensional SFG spectroscopy and SFGmicroscopy have emerged as complementary techniquesfor the characterization of couplings and interactions inbiomolecules [108ndash110] On the other hand theoretical meth-ods for modeling SFG spectroscopy are critical for theinterpretation of the SFG data Methodologies that combine

the essence of traditional theories of vibrational spectroscopyand the power of high-performance computing continueto be improved for more efficient calculations [111 112]that provided rigorous first-principle interpretations of theexperimental data in terms of structureorientation relationsof biological systems at interfaces Efforts have been focusedon accurate computations of molecular hyperpolarizabili-ties for different secondary structures of proteins criticalfor extracting information about molecular orientation atmembrane surfaces These advancements made by strongcollaborations between experimentalists and theoreticians inthe field of SFG spectroscopy are expected to continue to


produce valuable insights into a broad range of problems inchemical biological and biomedical sciences

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

Elsa C Y Yan is the recipient of the Starter GrantAward Spectroscopy Society of Pittsburgh the NationalScience Foundation Grant (CHE 1213362) and the NationalInstitutes of Health Grant (1R56DK105381-01) Victor SBatista acknowledges high performance computing timefromNERSC and support from theNSFGrant CHE-1213742

References

[1] H J Dyson and P E Wright ldquoIntrinsically unstructuredproteins and their functionsrdquo Nature Reviews Molecular CellBiology vol 6 no 3 pp 197ndash208 2005

[2] C Goldsbury K Goldie J Pellaud et al ldquoAmyloid fibrilformation from full-length and fragments of amylinrdquo Journalof Structural Biology vol 130 no 2-3 pp 352ndash362 2000

[3] A V Kajava U Aebi and A C Steven ldquoThe parallel super-pleated beta-structure as a model for amyloid fibrils of humanamylinrdquo Journal of Molecular Biology vol 348 no 2 pp 247ndash252 2005

[4] J D Knight J A Hebda and A D Miranker ldquoConservedand cooperative assembly of membrane-bound 120572-helical statesof islet amyloid polypeptiderdquo Biochemistry vol 45 no 31 pp9496ndash9508 2006

[5] J Janson R H Ashley D Harrison S McIntyre and P CButler ldquoThe mechanism of islet amyloid polypeptide toxicityis membrane disruption by intermediate-sized toxic amyloidparticlesrdquo Diabetes vol 48 no 3 pp 491ndash498 1999

[6] J W M Hoppener B Ahren and C J M Lips ldquoIslet amyloidand type 2 diabetes mellitusrdquo The New England Journal ofMedicine vol 343 no 6 pp 411ndash419 2000

[7] J W M Hoppener and C J M Lips ldquoRole of islet amyloid intype 2 diabetes mellitusrdquo International Journal of Biochemistryand Cell Biology vol 38 no 5-6 pp 726ndash736 2006

[8] S A Jayasinghe andR Langen ldquoLipidmembranesmodulate thestructure of islet amyloid polypeptiderdquoBiochemistry vol 44 no36 pp 12113ndash12119 2005

[9] S A Jayasinghe and R Langen ldquoMembrane interaction of isletamyloid polypeptiderdquo Biochimica et Biophysica Acta (BBA)mdashBiomembranes vol 1768 no 8 pp 2002ndash2009 2007

[10] J A Hebda and A D Miranker ldquoThe interplay of catalysisand toxicity by amyloid intermediates on lipid bilayers insightsfrom type II diabetesrdquo Annual Review of Biophysics vol 38 no1 pp 125ndash152 2009

[11] M F M Engel ldquoMembrane permeabilization by Islet AmyloidPolypeptiderdquo Chemistry and Physics of Lipids vol 160 no 1 pp1ndash10 2009

[12] M F M Engel L Khemtemourian C C Kleijer et al ldquoMem-brane damage by human islet amyloid polypeptide throughfibril growth at the membranerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 105 no16 pp 6033ndash6038 2008

[13] J D Knight and A D Miranker ldquoPhospholipid catalysis ofdiabetic amyloid assemblyrdquo Journal of Molecular Biology vol341 no 5 pp 1175ndash1187 2004

[14] C Poojari D Xiao V S Batista and B Strodel ldquoMembranepermeation induced by aggregates of human islet amyloidpolypeptidesrdquo Biophysical Journal vol 105 no 10 pp 2323ndash2332 2013

[15] R Kayed J Bernhagen N Greenfield et al ldquoConformationaltransitions of islet amyloid polypeptide (IAPP) in amyloidformation in vitrordquo Journal of Molecular Biology vol 287 no4 pp 781ndash796 1999

[16] J T Nielsen M Bjerring M D Jeppesen et al ldquoUnique identi-fication of supramolecular structures in amyloid fibrils by solid-state NMR spectroscopyrdquo Angewandte Chemie InternationalEdition vol 48 no 12 pp 2118ndash2121 2009

[17] JMadine E Jack P G Stockley S E Radford L C Serpell andD A Middleton ldquoStructural insights into the polymorphism ofamyloid-like fibrils formed by region 20-29 of amylin revealedby solid-state NMR and X-ray fiber diffractionrdquo Journal of theAmerican Chemical Society vol 130 no 45 pp 14990ndash150012008

[18] S Luca W-M Yau R Leapman and R Tycko ldquoPeptide con-formation and supramolecular organization in amylin fibrilsconstraints from solid-state NMRrdquo Biochemistry vol 46 no 47pp 13505ndash13522 2007

[19] S A Jayasinghe and R Langen ldquoIdentifying structural featuresof fibrillar islet amyloid polypeptide using site-directed spinlabelingrdquo The Journal of Biological Chemistry vol 279 no 46pp 48420ndash48425 2004

[20] M Apostolidou S Jayasinghe and R Langen ldquoStructure ofmembrane-bound IAPP studied by EPR spectroscopyrdquoBiophys-ical Journal vol 88 p 422A 2005

[21] Y L Ling D B Strasfeld S-H Shim D P Raleigh and M TZanni ldquoTwo-dimensional infrared spectroscopy provides evi-dence of an intermediate in the membrane-catalyzed assemblyof diabetic amyloidrdquo The Journal of Physical Chemistry B vol113 no 8 pp 2498ndash2505 2009

[22] S-H Shim R Gupta Y L Ling D B Strasfeld D P Raleighand M T Zanni ldquoTwo-dimensional IR spectroscopy andisotope labeling defines the pathway of amyloid formationwith residue-specific resolutionrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no16 pp 6614ndash6619 2009

[23] S-H Shim D B Strasfeld Y L Ling and M T ZannildquoAutomated 2D IR spectroscopy using a mid-IR pulse shaperand application of this technology to the human islet amyloidpolypeptiderdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 36 pp 14197ndash142022007

[24] R Sarroukh E Goormaghtigh J-M Ruysschaert and VRaussens ldquoATR-FTIR A lsquorejuvenatedrsquo tool to investigate amy-loid proteinsrdquo Biochimica et Biophysica ActamdashBiomembranesvol 1828 no 10 pp 2328ndash2338 2013

[25] E T A S Jaikaran C E Higham L C Serpell et alldquoIdentification of a novel human islet amyloid polypeptide 120573-sheet domain and factors influencing fibrillogenesisrdquo Journal ofMolecular Biology vol 308 no 3 pp 515ndash525 2001

[26] D F Moriarty and D P Raleigh ldquoEffects of sequential prolinesubstitutions on amyloid formation by human amylin20minus29rdquoBiochemistry vol 38 no 6 pp 1811ndash1818 1999


[27] T-L Lau E E Ambroggio D J Tew et al ldquoAmyloid-120573 peptidedisruption of lipid membranes and the effect of metal ionsrdquoJournal of Molecular Biology vol 356 no 3 pp 759ndash770 2006

[28] J C Lee R Langen P A Hummel H B Gray and J RWinkler ldquo120572-synuclein structures from fluorescence energy-transfer kinetics implications for the role of the protein inParkinsonrsquos diseaserdquo Proceedings of the National Academy ofSciences of the United States of America vol 101 no 47 pp16466ndash16471 2004

[29] A A Profit V Felsen J Chinwong E-R E Mojica and RZ B Desamero ldquoEvidence of 120587-stacking interactions in theself-assembly of hIAPP22-29rdquo Proteins Structure Function andBioinformatics vol 81 no 4 pp 690ndash703 2013

[30] S Li M Micic J Orbulescu J D Whyte and R M LeblancldquoHuman islet amyloid polypeptide at the air-aqueous interfacea Langmuir monolayer approachrdquo Journal of the Royal SocietyInterface vol 9 no 76 pp 3118ndash3128 2012

[31] D H J Lopes A Meister A Gohlke A Hauser A Blume andR Winter ldquoMechanism of islet amyloid polypeptide fibrillationat lipid interfaces studied by infrared reflection absorptionspectroscopyrdquo Biophysical Journal vol 93 no 9 pp 3132ndash31412007

[32] E C Yan Z Wang and L Fu ldquoProteins at interfaces probed bychiral vibrational sum frequency generation spectroscopyrdquoTheJournal of Physical Chemistry B vol 119 no 7 pp 2769ndash27852015

[33] E C Y Yan L Fu Z Wang and W Liu ldquoBiological macro-molecules at interfaces probed by chiral vibrational sum fre-quency generation spectroscopyrdquoChemical Reviews vol 114 pp8471ndash8498 2014

[34] Y R Shen ldquoSurface properties probed by second-harmonic andsum-frequency generationrdquo Nature vol 337 no 6207 pp 519ndash525 1989

[35] K B Eisenthal ldquoLiquid interfaces probed by second-harmonicand sum-frequency spectroscopyrdquo Chemical Reviews vol 96no 4 pp 1343ndash1360 1996

[36] G L Richmond ldquoMolecular bonding and interactions ataqueous surfaces as probed by vibrational sum frequencyspectroscopyrdquo Chemical Reviews vol 102 no 8 pp 2693ndash27242002

[37] M J Shultz C Schnitzer D Simonelli and S Baldelli ldquoSumfrequency generation spectroscopy of the aqueous interfaceionic and soluble molecular solutionsrdquo International Reviews inPhysical Chemistry vol 19 no 1 pp 123ndash153 2000

[38] Z Chen Y R Shen and G A Somorjai ldquoStudies of polymersurfaces by sum frequency generation vibrational spectroscopyrdquoAnnual Review of Physical Chemistry vol 53 pp 437ndash465 2002

[39] J Wang M L Clarke X Chen M A Even W C Johnsonand Z Chen ldquoMolecular studies on protein conformationsat polymerliquid interfaces using sum frequency generationvibrational spectroscopyrdquo Surface Science vol 587 no 1-2 pp1ndash11 2005

[40] X C Su P S Cremer Y R Shen and G A Somorjai ldquoHigh-pressure CO oxidation on Pt(111) monitored with infrared-visible sum frequency generation (SFG)rdquo Journal of the Ameri-can Chemical Society vol 119 no 17 pp 3994ndash4000 1997

[41] X Zhuang P B Miranda D Kim and Y R Shen ldquoMappingmolecular orientation and conformation at interfaces by surfacenonlinear opticsrdquo Physical Review BmdashCondensed Matter andMaterials Physics vol 59 no 19 pp 12632ndash12640 1999

[42] A M Jubb W Hua and H C Allen ldquoEnvironmental chem-istry at vaporwater interfaces insights from vibrational sum

frequency generation spectroscopyrdquo Annual review of physicalchemistry vol 63 pp 107ndash130 2012

[43] G Y Stokes E H Chen A M Buchbinder W F PaxtonA Keeley and F M Geiger ldquoAtmospheric heterogeneousstereochemistryrdquo Journal of the American Chemical Society vol131 no 38 pp 13733ndash13737 2009

[44] I S Martinez M D Peterson C J Ebben et al ldquoOn molecularchirality within naturally occurring secondary organic aerosolparticles from the central Amazon Basinrdquo Physical ChemistryChemical Physics vol 13 no 26 pp 12114ndash12122 2011

[45] X Y ChenM L Clarke JWang and Z Chen ldquoSum frequencygeneration vibrational spectroscopy studies on molecular con-formation and orientation of biological molecules at interfacesrdquoInternational Journal of Modern Physics B vol 19 no 4 pp 691ndash713 2005

[46] X Chen J Wang C B Kristalyn and Z Chen ldquoReal-timestructural investigation of a lipid bilayer during its interactionwith melittin using sum frequency generation vibrational spec-troscopyrdquo Biophysical Journal vol 93 no 3 pp 866ndash875 2007

[47] J Wang X Y Chen M L Clarke and Z Chen ldquoDetection ofchiral sum frequency generation vibrational spectra of proteinsand peptides at interfaces in siturdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no14 pp 4978ndash4983 2005

[48] G Y Stokes J M Gibbs-Davis F C Boman et al ldquoMakinglsquosensersquo of DNArdquo Journal of the American Chemical Society vol129 no 24 pp 7492ndash7493 2007

[49] S R Walter and F M Geiger ldquoDNA on stage showcasingoligonucleotides at surfaces and interfaces with second har-monic and vibrational sum frequency generationrdquo Journal ofPhysical Chemistry Letters vol 1 no 1 pp 9ndash15 2010

[50] J Liu and J C Conboy ldquoDirect measurement of the transbi-layermovement of phospholipids by sum-frequency vibrationalspectroscopyrdquo Journal of the American Chemical Society vol126 no 27 pp 8376ndash8377 2004

[51] J Liu and J C Conboy ldquoStructure of a gel phase lipidbilayer prepared by the Langmuir-Blodgett Langmuir-Schaefermethod characterized by sum-frequency vibrational spec-troscopyrdquo Langmuir vol 21 no 20 pp 9091ndash9097 2005

[52] T C Anglin J Liu and J C Conboy ldquoFacile lipid flip-flop ina phospholipid bilayer induced by gramicidin A measured bysum-frequency vibrational spectroscopyrdquo Biophysical Journalvol 92 no 1 pp L01ndashL03 2007

[53] C S Tian and Y R Shen ldquoRecent progress on sum-frequencyspectroscopyrdquo Surface Science Reports vol 69 no 2-3 pp 105ndash131 2014

[54] L Qiao A Ge M Osawa and S Ye ldquoStructure and stabilitystudies ofmixedmonolayers of saturated and unsaturated phos-pholipids under low-level ozonerdquo Physical Chemistry ChemicalPhysics vol 15 no 41 pp 17775ndash17785 2013

[55] Z Wang L Fu and E C Y Yan ldquoCndashH stretch for probingkinetics of self-assembly into macromolecular chiral structuresat interfaces by chiral sum frequency generation spectroscopyrdquoLangmuir vol 29 no 12 pp 4077ndash4083 2013

[56] KMeister S Strazdaite A LDeVries et al ldquoObservation of ice-like water layers at an aqueous protein surfacerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 111 no 50 pp 17732ndash17736 2014

[57] X Chen and Z Chen ldquoSFG studies on interactions betweenantimicrobial peptides and supported lipid bilayersrdquoBiochimicaet Biophysica Acta (BBA)mdashBiomembranes vol 1758 no 9 pp1257ndash1273 2006


[58] Y Rao S J J Kwok J Lombardi N J Turro andK B EisenthalldquoLabel-free probe of HIV-1 TAT peptide binding to mimeticmembranesrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 111 no 35 pp 12684ndash126882014

[59] Z G Li C N Weeraman M S Azam E Osman and J MGibbs-Davis ldquoThe thermal reorganization ofDNA immobilizedat the silicabuffer interface a vibrational sum frequency gener-ation investigationrdquo Physical Chemistry Chemical Physics vol17 pp 12452ndash12457 2015

[60] G W H Wurpel M Sovago and M Bonn ldquoSensitive probingof DNA binding to a cationic lipid monolayerrdquo Journal of theAmerican Chemical Society vol 129 no 27 pp 8420ndash8421 2007

[61] L Fu J Liu and E C Y Yan ldquoChiral sum frequency generationspectroscopy for characterizing protein secondary structures atinterfacesrdquo Journal of the American Chemical Society vol 133no 21 pp 8094ndash8097 2011

[62] L Fu G Ma and E C Y Yan ldquoIn situ misfolding of humanislet amyloid polypeptide at interfaces probed by vibrationalsum frequency generationrdquo Journal of the American ChemicalSociety vol 132 no 15 pp 5405ndash5412 2010

[63] I I Rzeznicka R Pandey M Schleeger M Bonn and TWeidner ldquoFormation of lysozyme oligomers at model cellmembranes monitored with sum frequency generation spec-troscopyrdquo Langmuir vol 30 no 26 pp 7736ndash7744 2014

[64] M F M Engel C C Vandenakker M Schleeger K P VelikovG H Koenderink and M Bonn ldquoThe polyphenol EGCGinhibits amyloid formation less efficiently at phospholipid inter-faces than in bulk solutionrdquo Journal of the American ChemicalSociety vol 134 no 36 pp 14781ndash14788 2012

[65] D Xiao L Fu J Liu V S Batista and E C Y Yan ldquoAmphiphilicadsorption of human islet amyloid polypeptide aggregates tolipidaqueous interfacesrdquo Journal of Molecular Biology vol 421no 4-5 pp 537ndash547 2012

[66] A G Lambert P B Davies and D J Neivandt ldquoImple-menting the theory of sum frequency generation vibrationalspectroscopy a tutorial reviewrdquo Applied Spectroscopy Reviewsvol 40 no 2 pp 103ndash145 2005

[67] H-FWangW Gan R Lu Y Rao and B-HWu ldquoQuantitativespectral and orientational analysis in surface sum frequencygeneration vibrational spectroscopy (SFG-VS)rdquo InternationalReviews in Physical Chemistry vol 24 no 2 pp 191ndash256 2005

[68] Y-R Shen The Principles of Nonlinear Optics Wiley-Interscience New York NY USA 1984

[69] R W Boyed Nonlinear Optics Academic Press New York NYUSA 2003

[70] H-F Wang L Velarde W Gan and L Fu ldquoQuantitative sum-frequency generation vibrational spectroscopy of molecularsurfaces and interfaces lineshape polarization and orienta-tionrdquoAnnual Review of Physical Chemistry vol 66 pp 189ndash2162015

[71] C K Lin L Yang M Hayashi et al ldquoTheory and applicationsof sum-frequency generationsrdquo Journal of the Chinese ChemicalSociety vol 61 no 1 pp 77ndash92 2014

[72] M A Belkin and Y R Shen ldquoNon-linear optical spectroscopyas a novel probe for molecular chiralityrdquo International Reviewsin Physical Chemistry vol 24 no 2 pp 257ndash299 2005

[73] G J Simpson ldquoMolecular origins of the remarkable chiralsensitivity of second-order nonlinear opticrdquo ChemPhysChemvol 5 no 9 pp 1301ndash1310 2004

[74] LMHaupert andG J Simpson ldquoChirality in nonlinear opticsrdquoAnnual Review of Physical Chemistry vol 60 pp 345ndash365 2009

[75] A J Moad and G J Simpson ldquoA unified treatment of selectionrules and symmetry relations for sum-frequency and secondharmonic spectroscopiesrdquo Journal of Physical Chemistry B vol108 no 11 pp 3548ndash3562 2004

[76] J Wang M L Clarke and Z Chen ldquoPolarization mapping amethod to improve sum frequency generation spectral analy-sisrdquo Analytical Chemistry vol 76 no 8 pp 2159ndash2167 2004

[77] J A Hebda I Saraogi M Magzoub A D Hamilton and AD Miranker ldquoA peptidomimetic approach to targeting pre-amyloidogenic states in type II diabetesrdquoChemistry and Biologyvol 16 no 9 pp 943ndash950 2009

[78] D K Hore J L King F GMoore D S AlaviM Y Hamamotoand G L Richmond ldquoTi Sapphire-based picosecond visible-infrared sum-frequency spectroscopy from 900ndash3100 cmminus1rdquoApplied Spectroscopy vol 58 no 12 pp 1377ndash1384 2004

[79] E L Hommel G Ma and H C Allen ldquoBroadband vibrationalsum frequency generation spectroscopy of a liquid surfacerdquoAnalytical Sciences vol 17 no 11 pp 1325ndash1329 2001

[80] J P Smith and V Hinson-Smith ldquoProduct review SFG comingof agerdquo Analytical Chemistry vol 76 pp 287Andash290A 2004

[81] G Ma and H C Allen ldquoSurface studies of aqueous methanolsolutions by vibrational broad bandwidth sum frequency gen-eration spectroscopyrdquo Journal of Physical Chemistry B vol 107no 26 pp 6343ndash6349 2003

[82] L Fu D Q Xiao Z G Wang V S Batista and E C Y YanldquoChiral sum frequency generation for in situ probing protonexchange in antiparallel beta-sheets at interfacesrdquo Journal of theAmerican Chemical Society vol 135 no 9 pp 3592ndash3598 2013

[83] A Relini N Marano and A Gliozzi ldquoProbing the interplaybetween amyloidogenic proteins and membranes using lipidmonolayers and bilayersrdquo Advances in Colloid and InterfaceScience vol 207 no 1 pp 81ndash92 2014

[84] H Braak and E Braak ldquoNeuropathological stageing ofAlzheimer-related changesrdquo Acta Neuropathologica vol 82 no4 pp 239ndash259 1991

[85] F Chiti and C M Dobson ldquoProtein misfolding functionalamyloid and human diseaserdquo Annual Review of Biochemistryvol 75 pp 333ndash366 2006

[86] A Barth and C Zscherp ldquoWhat vibrations tell us aboutproteinsrdquoQuarterly Reviews of Biophysics vol 35 no 4 pp 369ndash430 2002

[87] L K Tamm and S A Tatulian ldquoInfrared spectroscopy ofproteins and peptides in lipid bilayersrdquo Quarterly Reviews ofBiophysics vol 30 no 4 pp 365ndash429 1997

[88] L Fu Z Wang and E C Y Yan ldquoChiral vibrational structuresof proteins at interfaces probed by sum frequency generationspectroscopyrdquo International Journal of Molecular Sciences vol12 no 12 pp 9404ndash9425 2011

[89] I Saraogi J A Hebda J Becerril L A Estroff A D Mirankerand A D Hamilton ldquoSynthetic 120572-helix mimetics as agonistsand antagonists of islet amyloid polypeptide aggregationrdquoAngewandte Chemie vol 49 no 4 pp 736ndash739 2010

[90] D E Ehrnhoefer J Bieschke A Boeddrich et al ldquoEGCGredirects amyloidogenic polypeptides into unstructured off-pathway oligomersrdquo Nature Structural and Molecular Biologyvol 15 no 6 pp 558ndash566 2008

[91] F Meng A Abedini A Plesner C B Verchere and D PRaleigh ldquoThe Flavanol (minus)-epigallocatechin 3-gallate inhibitsamyloid formation by islet amyloid polypeptide disaggregatesamyloid fibrils and protects cultured cells against IAPP-induced toxicityrdquo Biochemistry vol 49 no 37 pp 8127ndash81332010


[92] N F Dupuis C Wu J-E Shea and M T Bowers ldquoTheamyloid formation mechanism in human IAPP dimers have 120573-strandmonomer-monomer interfacesrdquo Journal of the AmericanChemical Society vol 133 no 19 pp 7240ndash7243 2011

[93] A Morriss-Andrews and J-E Shea ldquoComputational studies ofprotein aggregation methods and applicationsrdquoAnnual Reviewof Physical Chemistry vol 66 pp 643ndash666 2015

[94] G Bellesia and J-E Shea ldquoEffect of Β-sheet propensity onpeptide aggregationrdquo The Journal of Chemical Physics vol 130no 14 Article ID 145103 2009

[95] H Li S Ye F Wei S Ma and Y Luo ldquoIn situ molecular-levelinsights into the interfacial structure changes of membrane-associated prion protein fragment [118ndash135] investigated by sumfrequency generation vibrational spectroscopyrdquo Langmuir vol28 no 49 pp 16979ndash16988 2012

[96] Z Jiang M deMessieres and J C Lee ldquoMembrane remodelingby 120572-synuclein and effects on amyloid formationrdquo Journal of theAmerican Chemical Society vol 135 no 43 pp 15970ndash159732013

[97] S Li and RM Leblanc ldquoAggregation of insulin at the interfacerdquoThe Journal of Physical Chemistry B vol 118 no 5 pp 1181ndash11882014

[98] A L Mifflin L Velarde J Ho et al ldquoAccurate line shapes fromsub-1 cmminus1 resolution sum frequency generation vibrationalspectroscopy of 120572-pinene at room temperaturerdquoThe Journal ofPhysical Chemistry A vol 119 no 8 pp 1292ndash1302 2015

[99] L Velarde X-Y Zhang Z Lu A G Joly Z Wang and H-F Wang ldquoCommunication spectroscopic phase and lineshapesin high-resolution broadband sum frequency vibrational spec-troscopy resolving interfacial inhomogeneities of lsquoidenticalrsquomolecular groupsrdquo Journal of Chemical Physics vol 135 no 24Article ID 241102 2011

[100] L Velarde and H-F Wang ldquoUnified treatment and mea-surement of the spectral resolution and temporal effectsin frequency-resolved sum-frequency generation vibrationalspectroscopy (SFG-VS)rdquo Physical Chemistry Chemical Physicsvol 15 no 46 pp 19970ndash19984 2013

[101] L Fu Y Zhang Z H Wei and H F Wang ldquoIntrinsic chiralityand prochirality at airR-(+)- and S-(minus)-limonene interfacesspectral signatures with interference chiral sum-frequency gen-eration vibrational spectroscopyrdquoChirality vol 26 pp 149ndash1602014

[102] S Yamaguchi and T Tahara ldquoHeterodyne-detected electronicsum frequency generation lsquouprsquo versus lsquodownrsquo alignment ofinterfacial moleculesrdquo Journal of Chemical Physics vol 129 no10 Article ID 101102 2008

[103] I V Stiopkin H D Jayathilake A N Bordenyuk and A VBenderskii ldquoHeterodyne-detected vibrational sum frequencygeneration spectroscopyrdquo Journal of the American ChemicalSociety vol 130 no 7 pp 2271ndash2275 2008

[104] DHu Z Yang andKC Chou ldquoInteractions of polyelectrolyteswith water and ions at airwater interfaces studied by phase-sensitive sum frequency generation vibrational spectroscopyrdquoThe Journal of Physical Chemistry C vol 117 no 30 pp 15698ndash15703 2013

[105] W Hua A M Jubb and H C Allen ldquoElectric field reversal ofNa2SO4 (NH 4)2SO4 and Na2CO3 relative to CaCl2 and NaClat the airaqueous interface revealed by heterodyne detectedphase-sensitive sum frequencyrdquo Journal of Physical ChemistryLetters vol 2 no 20 pp 2515ndash2520 2011

[106] C L Anfuso R C Snoeberger III A M Ricks et al ldquoCovalentattachment of a rhenium bipyridyl CO2 reduction catalyst to

rutile TiO2rdquo Journal of the American Chemical Society vol 133no 18 pp 6922ndash6925 2011

[107] C L Anfuso D Xiao A M Ricks C F A Negre V S Batistaand T Lian ldquoOrientation of a series of CO2 reduction catalystson single crystal TiO2 probed by phase-sensitive vibrationalsum frequency generation spectroscopy (PS-VSFG)rdquo Journal ofPhysical Chemistry C vol 116 no 45 pp 24107ndash24114 2012

[108] W Xiong J E Laaser R D Mehlenbacher and M T ZannildquoAdding a dimension to the infrared spectra of interfaces usingheterodyne detected 2D sumfrequency generation (HD 2DSFG) spectroscopyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 52 pp20902ndash20907 2011

[109] J E Laaser andM T Zanni ldquoExtracting structural informationfrom the polarization dependence of one- and two-dimensionalsum frequency generation spectrardquo The Journal of PhysicalChemistry A vol 117 no 29 pp 5875ndash5890 2013

[110] J E Laaser D R Skoff J-J Ho et al ldquoTwo-dimensionalsum-frequency generation reveals structure and dynamics ofa surface-bound peptiderdquo Journal of the American ChemicalSociety vol 136 no 3 pp 956ndash962 2014

[111] H Fujisaki and J E Straub ldquoVibrational energy relaxation inproteinsrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 102 no 19 pp 6726ndash6731 2005

[112] H Fujisaki K Yagi K Hirao and J E Straub ldquoQuantumdynamics of N-methylacetamide studied by the vibrationalconfiguration interaction methodrdquo Chemical Physics Lettersvol 443 no 1ndash3 pp 6ndash11 2007


demonstrated SFG as an intrinsically surface-selective tech-nique with submonolayer sensitivity and label-free detectioncapability thus showing great promise to address the abovequestions shedding light on the role of themembrane duringthe aggregation of hIAPP and other amyloid proteins [32 33]

During the last three decades we have witnessed theemergence and fast development of nonlinear optical spec-troscopic techniques among which SFG vibrational spec-troscopy has gained tremendous attention Unique advan-tages of SFG include surface selectivity submonolayer sen-sitivity chiral-selectivity phase-sensitivity and label-freedetection capabilities [34ndash36] which make SFG a promisingtool for structural characterization of interfaces including awide range of applications in material science [37] charac-terization of polymers [38 39] and catalytic systems [40 41]Recently applications have been extended to environmental[42ndash44] and biological systems [32 33 45ndash52] with unprece-dented discoveries beyond the capabilities of conventionaltools For example SFG has been used to study a varietyof atmospherically significant systems at the vaporaqueousinterface to elucidate the organization and reactions inaerosols that contain inorganicorganic compounds [42ndash44] small molecules [42] and fatty acids [42] as solutesFurthermore SFG has been applied to probe the interfacialstructures orientation and kinetics of biologically relevantmolecules at interfaces such as proteins [32 33 45ndash47]DNAs [48 49] and lipids [45 50ndash52] providing insights intothe functions of these molecules at biological interfaces andfacilitating further research into biomedical science and engi-neeringNowadays SFG is established as a valuable techniqueto understand physical chemical and biological processesat the molecular level [53] In particular applications tointerfacial biological systems span across important researchtopics in membrane biophysics [54] surface self-assembly[55 56] peptides at cell surfaces [57 58] DNA hybridization[48 59] and adsorption [60] and amyloid interactions withmembrane surfaces [61ndash63]

This review focuses on the recent development and appli-cation of SFG for probing hIAPP interacting with lipid mem-branes and discusses the implications of hIAPPmembraneinteractions in the studies of type II diabetes [14 61 62 6465] The review also summarizes the basic theoretical back-ground and experimental methods of SFG supplementedwith a brief discussion about the application of SFG to otheramyloidogenic proteins and concludes with an outlook ofSFG in applications to systems of interest in biological andmedical sciences

2 The SFG Method

21 Basic Principles of SFG Sum frequency generation (SFG)vibrational spectroscopy applies two laser beams that interactwith the system of interest simultaneously [34ndash36 66ndash69]one with visible (eg 532 nm) or near-infrared (eg 800 nm)frequency 120596vis and the other with infrared (IR) frequency 120596IR(Scheme 1) When the visible and infrared pulses overlap intime and space light with the sum of the two frequencies120596SFG = 120596vis + 120596IR is generated by the interaction of the

SFG120596vis + 120596IR = 120596sum

120596vis

120596vis

120596IR120596IR 120596sum

120596sum

Scheme 1 The second-order optical process of sum frequencygeneration vibrational spectroscopy

incident beams with the system at the interface Fixing120596vis and scanning or dispersing IR frequencies 120596IR over arange the SFG spectra are recorded by monitoring the SFGintensity as a function of 120596SFG with a monochromator anda CCD The SFG signal is dramatically enhanced when 120596IRis in resonance with a vibrational frequency of a moleculeat an interface thus showing a peak in the spectrum TheSFG peaks typically exhibit homogeneous and inhomoge-neous broadening leading to various line-shapes due to theconstructive or destructive interference with neighboringbands modulated by changes in the molecular orientation asinduced by interactions with the surface and the surroundingenvironment [70] Thus the SFG spectra contain detailedstructural information reported in terms of line-shape peakposition and polarization dependence Extracting that struc-tural information from the SFG spectra however requiresrigorous theoretical modeling and computer simulationsHence the combination of SFG and computational modelingcan be used as a label-free and in situ analytical methodologyfor effective characterization of systems at interfaces In thefollowing sections wewill illustrate the applications of SFG tothe studies of IAPP at membrane surfaces [14 61 62 64 65]

22 Surface-Specificity Monolayer Sensitivity and Polariza-tion Dependence of SFG Spectroscopy As a nonlinear opticaltechnique SFG measures the second-order susceptibility120594(2) that gives intrinsic surface selectivity [33 34 71ndash74]The second-order susceptibility 120594(2) is the direct product ofthe complex conjugate of the Raman polarizability derivativematrix (the same as the original matrix when derivativevalues are real numbers) and the IR dipole derivative vectorand thus is a second-order tensorThe generated electric fieldof SFG signal is related to the electric field of the two incidentlaser beams 119864vis and 119864IR via the tensor elements 120594(2)

119894119895119896

119864119894SFG prop sum119895119896

120594(2)119894119895119896119864119895

vis119864119896

IR (1)

where 119894 119895 and 119896 specify the direction of the Cartesiancomponent of the optical fields and can be denoted by 119909 119910and 119911 in the laboratory coordinates








s-SFG

s-visp-IR

x

x

y

y

y

z

z

xy-plane

xz-plane








IR IR IR

hIAPP

DPPG lipid

hIAPPintermediate

hIAPPaggregates










SFG

inte

nsity

(au

)


16501600 17501700


(a) AirD2O


SFG

inte

nsity

(au

)

16501600 17501700


(b) AirH2O

SFG

inte

nsity

(au

)


1700 17501600 1650


(c) AirD2O


SFG

inte

nsity

(au

)

16501600 17501700


(d) AirH2O







SFG

inte

nsity

(au

)


170016501600


(a) AirD2O


SFG

inte

nsity

(au

)

170016501600


(b) AirH2O

SFG

inte

nsity

(au

)


170016501600


(c) AirD2O


SFG

inte

nsity

(au

)

170016501600


(d) AirH2O





SFG

inte

nsity

(au

)

1700 175016501600

5min30min135min

308min335min572min



(a)

SFG

inte

nsity

(au

)

1600 17001650

18min200min346min

437min462min554min



(b)

SFG

inte

nsity

(au

)

559min

448min

226min

181min

158min

86min

15min

3285 cmminus1

32003100 3300


hIAPP + DPPG

(c)











1 2 3 4 5 6 7 8 90 10Time (h)

00

05

10

SFG

inte

nsity

(au

)

(a)

1 2 3



aggregates

(b)









(a) (b)

50nm

(c)



0

2

4

6

8

10

12

SFG

inte

nsity

(au

)




0

2

4

6

8

10

12

SFG

inte

nsity

(au

)


(d) (e)

minusEGCG +EGCG









03

04

05

06

07

08

09

SFG120573

-she

et (a

u)

10

15

20

25

ThT

fluor

esce

nce i

nten

sity

(au

)

800400 1200 16000

Time (min)

(a)

(b)

50nm

(c)



119868119861119860 =

10038161003816100381610038161003816100381610038161003816100381610038161003816

120594(2)119901119904119901119861

120594(2)119901119904119901119860

10038161003816100381610038161003816100381610038161003816100381610038161003816

2

=100381610038161003816100381610038161003816100381610038161003816⟨tan2120595⟩

120573119887119888119886119861

120573119886119888119887119860+ (1 minus ⟨tan2120595⟩)

120573119887119886119888119861

120573119886119888119887119860

100381610038161003816100381610038161003816100381610038161003816

2

(2)






a

a

b

b

c

c

hIAPP

hIAPP

x

y

z

120579

120601

120595

(a)

B

R

NH

HN

O R

O

R

NH

HN

O R

O

A mode

B mode

psp

A000

005

010

SFG

inte

nsity

(au

)


(b)

811

19

23

24

8

2837

N N N

O

N

H

H

H

N N N

O

O

N

O

H

H

H

O

O

H

H

O

OR1

R1

R2

R2

R3

R3

R4

R4


SSNNF

SSNNF

GAILSTY

LL


FFN

R NARQT

TVVL

(c)

0

5

10

15

20

25

45 225135 315 36027090 1800

120595 (∘)

|120594(2)

pspB120594

(2)

pspA|2

(d)

120595 = 30∘

120595 = 48∘

120595 = 132∘

120595 = 150∘

Exp

Ipsp

(au

)


(e)

Figure 7 Continued


5

10

15

20

25

30

35

27

His18

Arg1148

∘

(f)










5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268


(d)








Acknowledgments


References





























































































































s-SFG

s-visp-IR

x

x

y

y

y

z

z

xy-plane

xz-plane








IR IR IR

hIAPP

DPPG lipid

hIAPPintermediate

hIAPPaggregates










SFG

inte

nsity

(au

)


16501600 17501700


(a) AirD2O


SFG

inte

nsity

(au

)

16501600 17501700


(b) AirH2O

SFG

inte

nsity

(au

)


1700 17501600 1650


(c) AirD2O


SFG

inte

nsity

(au

)

16501600 17501700


(d) AirH2O







SFG

inte

nsity

(au

)


170016501600


(a) AirD2O


SFG

inte

nsity

(au

)

170016501600


(b) AirH2O

SFG

inte

nsity

(au

)


170016501600


(c) AirD2O


SFG

inte

nsity

(au

)

170016501600


(d) AirH2O





SFG

inte

nsity

(au

)

1700 175016501600

5min30min135min

308min335min572min



(a)

SFG

inte

nsity

(au

)

1600 17001650

18min200min346min

437min462min554min



(b)

SFG

inte

nsity

(au

)

559min

448min

226min

181min

158min

86min

15min

3285 cmminus1

32003100 3300


hIAPP + DPPG

(c)











1 2 3 4 5 6 7 8 90 10Time (h)

00

05

10

SFG

inte

nsity

(au

)

(a)

1 2 3



aggregates

(b)









(a) (b)

50nm

(c)



0

2

4

6

8

10

12

SFG

inte

nsity

(au

)




0

2

4

6

8

10

12

SFG

inte

nsity

(au

)


(d) (e)

minusEGCG +EGCG









03

04

05

06

07

08

09

SFG120573

-she

et (a

u)

10

15

20

25

ThT

fluor

esce

nce i

nten

sity

(au

)

800400 1200 16000

Time (min)

(a)

(b)

50nm

(c)



119868119861119860 =

10038161003816100381610038161003816100381610038161003816100381610038161003816

120594(2)119901119904119901119861

120594(2)119901119904119901119860

10038161003816100381610038161003816100381610038161003816100381610038161003816

2

=100381610038161003816100381610038161003816100381610038161003816⟨tan2120595⟩

120573119887119888119886119861

120573119886119888119887119860+ (1 minus ⟨tan2120595⟩)

120573119887119886119888119861

120573119886119888119887119860

100381610038161003816100381610038161003816100381610038161003816

2

(2)






a

a

b

b

c

c

hIAPP

hIAPP

x

y

z

120579

120601

120595

(a)

B

R

NH

HN

O R

O

R

NH

HN

O R

O

A mode

B mode

psp

A000

005

010

SFG

inte

nsity

(au

)


(b)

811

19

23

24

8

2837

N N N

O

N

H

H

H

N N N

O

O

N

O

H

H

H

O

O

H

H

O

OR1

R1

R2

R2

R3

R3

R4

R4


SSNNF

SSNNF

GAILSTY

LL


FFN

R NARQT

TVVL

(c)

0

5

10

15

20

25

45 225135 315 36027090 1800

120595 (∘)

|120594(2)

pspB120594

(2)

pspA|2

(d)

120595 = 30∘

120595 = 48∘

120595 = 132∘

120595 = 150∘

Exp

Ipsp

(au

)


(e)

Figure 7 Continued


5

10

15

20

25

30

35

27

His18

Arg1148

∘

(f)










5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268


(d)








Acknowledgments


References
























































































































IR IR IR

hIAPP

DPPG lipid

hIAPPintermediate

hIAPPaggregates










SFG

inte

nsity

(au

)


16501600 17501700


(a) AirD2O


SFG

inte

nsity

(au

)

16501600 17501700


(b) AirH2O

SFG

inte

nsity

(au

)


1700 17501600 1650


(c) AirD2O


SFG

inte

nsity

(au

)

16501600 17501700


(d) AirH2O







SFG

inte

nsity

(au

)


170016501600


(a) AirD2O


SFG

inte

nsity

(au

)

170016501600


(b) AirH2O

SFG

inte

nsity

(au

)


170016501600


(c) AirD2O


SFG

inte

nsity

(au

)

170016501600


(d) AirH2O





SFG

inte

nsity

(au

)

1700 175016501600

5min30min135min

308min335min572min



(a)

SFG

inte

nsity

(au

)

1600 17001650

18min200min346min

437min462min554min



(b)

SFG

inte

nsity

(au

)

559min

448min

226min

181min

158min

86min

15min

3285 cmminus1

32003100 3300


hIAPP + DPPG

(c)











1 2 3 4 5 6 7 8 90 10Time (h)

00

05

10

SFG

inte

nsity

(au

)

(a)

1 2 3



aggregates

(b)









(a) (b)

50nm

(c)



0

2

4

6

8

10

12

SFG

inte

nsity

(au

)




0

2

4

6

8

10

12

SFG

inte

nsity

(au

)


(d) (e)

minusEGCG +EGCG









03

04

05

06

07

08

09

SFG120573

-she

et (a

u)

10

15

20

25

ThT

fluor

esce

nce i

nten

sity

(au

)

800400 1200 16000

Time (min)

(a)

(b)

50nm

(c)



119868119861119860 =

10038161003816100381610038161003816100381610038161003816100381610038161003816

120594(2)119901119904119901119861

120594(2)119901119904119901119860

10038161003816100381610038161003816100381610038161003816100381610038161003816

2

=100381610038161003816100381610038161003816100381610038161003816⟨tan2120595⟩

120573119887119888119886119861

120573119886119888119887119860+ (1 minus ⟨tan2120595⟩)

120573119887119886119888119861

120573119886119888119887119860

100381610038161003816100381610038161003816100381610038161003816

2

(2)






a

a

b

b

c

c

hIAPP

hIAPP

x

y

z

120579

120601

120595

(a)

B

R

NH

HN

O R

O

R

NH

HN

O R

O

A mode

B mode

psp

A000

005

010

SFG

inte

nsity

(au

)


(b)

811

19

23

24

8

2837

N N N

O

N

H

H

H

N N N

O

O

N

O

H

H

H

O

O

H

H

O

OR1

R1

R2

R2

R3

R3

R4

R4


SSNNF

SSNNF

GAILSTY

LL


FFN

R NARQT

TVVL

(c)

0

5

10

15

20

25

45 225135 315 36027090 1800

120595 (∘)

|120594(2)

pspB120594

(2)

pspA|2

(d)

120595 = 30∘

120595 = 48∘

120595 = 132∘

120595 = 150∘

Exp

Ipsp

(au

)


(e)

Figure 7 Continued


5

10

15

20

25

30

35

27

His18

Arg1148

∘

(f)










5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268


(d)








Acknowledgments


References























































































































SFG

inte

nsity

(au

)


16501600 17501700


(a) AirD2O


SFG

inte

nsity

(au

)

16501600 17501700


(b) AirH2O

SFG

inte

nsity

(au

)


1700 17501600 1650


(c) AirD2O


SFG

inte

nsity

(au

)

16501600 17501700


(d) AirH2O







SFG

inte

nsity

(au

)


170016501600


(a) AirD2O


SFG

inte

nsity

(au

)

170016501600


(b) AirH2O

SFG

inte

nsity

(au

)


170016501600


(c) AirD2O


SFG

inte

nsity

(au

)

170016501600


(d) AirH2O





SFG

inte

nsity

(au

)

1700 175016501600

5min30min135min

308min335min572min



(a)

SFG

inte

nsity

(au

)

1600 17001650

18min200min346min

437min462min554min



(b)

SFG

inte

nsity

(au

)

559min

448min

226min

181min

158min

86min

15min

3285 cmminus1

32003100 3300


hIAPP + DPPG

(c)











1 2 3 4 5 6 7 8 90 10Time (h)

00

05

10

SFG

inte

nsity

(au

)

(a)

1 2 3



aggregates

(b)









(a) (b)

50nm

(c)



0

2

4

6

8

10

12

SFG

inte

nsity

(au

)




0

2

4

6

8

10

12

SFG

inte

nsity

(au

)


(d) (e)

minusEGCG +EGCG









03

04

05

06

07

08

09

SFG120573

-she

et (a

u)

10

15

20

25

ThT

fluor

esce

nce i

nten

sity

(au

)

800400 1200 16000

Time (min)

(a)

(b)

50nm

(c)



119868119861119860 =

10038161003816100381610038161003816100381610038161003816100381610038161003816

120594(2)119901119904119901119861

120594(2)119901119904119901119860

10038161003816100381610038161003816100381610038161003816100381610038161003816

2

=100381610038161003816100381610038161003816100381610038161003816⟨tan2120595⟩

120573119887119888119886119861

120573119886119888119887119860+ (1 minus ⟨tan2120595⟩)

120573119887119886119888119861

120573119886119888119887119860

100381610038161003816100381610038161003816100381610038161003816

2

(2)






a

a

b

b

c

c

hIAPP

hIAPP

x

y

z

120579

120601

120595

(a)

B

R

NH

HN

O R

O

R

NH

HN

O R

O

A mode

B mode

psp

A000

005

010

SFG

inte

nsity

(au

)


(b)

811

19

23

24

8

2837

N N N

O

N

H

H

H

N N N

O

O

N

O

H

H

H

O

O

H

H

O

OR1

R1

R2

R2

R3

R3

R4

R4


SSNNF

SSNNF

GAILSTY

LL


FFN

R NARQT

TVVL

(c)

0

5

10

15

20

25

45 225135 315 36027090 1800

120595 (∘)

|120594(2)

pspB120594

(2)

pspA|2

(d)

120595 = 30∘

120595 = 48∘

120595 = 132∘

120595 = 150∘

Exp

Ipsp

(au

)


(e)

Figure 7 Continued


5

10

15

20

25

30

35

27

His18

Arg1148

∘

(f)










5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268


(d)








Acknowledgments


References























































































































SFG

inte

nsity

(au

)


170016501600


(a) AirD2O


SFG

inte

nsity

(au

)

170016501600


(b) AirH2O

SFG

inte

nsity

(au

)


170016501600


(c) AirD2O


SFG

inte

nsity

(au

)

170016501600


(d) AirH2O





SFG

inte

nsity

(au

)

1700 175016501600

5min30min135min

308min335min572min



(a)

SFG

inte

nsity

(au

)

1600 17001650

18min200min346min

437min462min554min



(b)

SFG

inte

nsity

(au

)

559min

448min

226min

181min

158min

86min

15min

3285 cmminus1

32003100 3300


hIAPP + DPPG

(c)











1 2 3 4 5 6 7 8 90 10Time (h)

00

05

10

SFG

inte

nsity

(au

)

(a)

1 2 3



aggregates

(b)









(a) (b)

50nm

(c)



0

2

4

6

8

10

12

SFG

inte

nsity

(au

)




0

2

4

6

8

10

12

SFG

inte

nsity

(au

)


(d) (e)

minusEGCG +EGCG









03

04

05

06

07

08

09

SFG120573

-she

et (a

u)

10

15

20

25

ThT

fluor

esce

nce i

nten

sity

(au

)

800400 1200 16000

Time (min)

(a)

(b)

50nm

(c)



119868119861119860 =

10038161003816100381610038161003816100381610038161003816100381610038161003816

120594(2)119901119904119901119861

120594(2)119901119904119901119860

10038161003816100381610038161003816100381610038161003816100381610038161003816

2

=100381610038161003816100381610038161003816100381610038161003816⟨tan2120595⟩

120573119887119888119886119861

120573119886119888119887119860+ (1 minus ⟨tan2120595⟩)

120573119887119886119888119861

120573119886119888119887119860

100381610038161003816100381610038161003816100381610038161003816

2

(2)






a

a

b

b

c

c

hIAPP

hIAPP

x

y

z

120579

120601

120595

(a)

B

R

NH

HN

O R

O

R

NH

HN

O R

O

A mode

B mode

psp

A000

005

010

SFG

inte

nsity

(au

)


(b)

811

19

23

24

8

2837

N N N

O

N

H

H

H

N N N

O

O

N

O

H

H

H

O

O

H

H

O

OR1

R1

R2

R2

R3

R3

R4

R4


SSNNF

SSNNF

GAILSTY

LL


FFN

R NARQT

TVVL

(c)

0

5

10

15

20

25

45 225135 315 36027090 1800

120595 (∘)

|120594(2)

pspB120594

(2)

pspA|2

(d)

120595 = 30∘

120595 = 48∘

120595 = 132∘

120595 = 150∘

Exp

Ipsp

(au

)


(e)

Figure 7 Continued


5

10

15

20

25

30

35

27

His18

Arg1148

∘

(f)










5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268


(d)








Acknowledgments


References























































































































SFG

inte

nsity

(au

)

1700 175016501600

5min30min135min

308min335min572min



(a)

SFG

inte

nsity

(au

)

1600 17001650

18min200min346min

437min462min554min



(b)

SFG

inte

nsity

(au

)

559min

448min

226min

181min

158min

86min

15min

3285 cmminus1

32003100 3300


hIAPP + DPPG

(c)











1 2 3 4 5 6 7 8 90 10Time (h)

00

05

10

SFG

inte

nsity

(au

)

(a)

1 2 3



aggregates

(b)









(a) (b)

50nm

(c)



0

2

4

6

8

10

12

SFG

inte

nsity

(au

)




0

2

4

6

8

10

12

SFG

inte

nsity

(au

)


(d) (e)

minusEGCG +EGCG









03

04

05

06

07

08

09

SFG120573

-she

et (a

u)

10

15

20

25

ThT

fluor

esce

nce i

nten

sity

(au

)

800400 1200 16000

Time (min)

(a)

(b)

50nm

(c)



119868119861119860 =

10038161003816100381610038161003816100381610038161003816100381610038161003816

120594(2)119901119904119901119861

120594(2)119901119904119901119860

10038161003816100381610038161003816100381610038161003816100381610038161003816

2

=100381610038161003816100381610038161003816100381610038161003816⟨tan2120595⟩

120573119887119888119886119861

120573119886119888119887119860+ (1 minus ⟨tan2120595⟩)

120573119887119886119888119861

120573119886119888119887119860

100381610038161003816100381610038161003816100381610038161003816

2

(2)






a

a

b

b

c

c

hIAPP

hIAPP

x

y

z

120579

120601

120595

(a)

B

R

NH

HN

O R

O

R

NH

HN

O R

O

A mode

B mode

psp

A000

005

010

SFG

inte

nsity

(au

)


(b)

811

19

23

24

8

2837

N N N

O

N

H

H

H

N N N

O

O

N

O

H

H

H

O

O

H

H

O

OR1

R1

R2

R2

R3

R3

R4

R4


SSNNF

SSNNF

GAILSTY

LL


FFN

R NARQT

TVVL

(c)

0

5

10

15

20

25

45 225135 315 36027090 1800

120595 (∘)

|120594(2)

pspB120594

(2)

pspA|2

(d)

120595 = 30∘

120595 = 48∘

120595 = 132∘

120595 = 150∘

Exp

Ipsp

(au

)


(e)

Figure 7 Continued


5

10

15

20

25

30

35

27

His18

Arg1148

∘

(f)










5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268


(d)








Acknowledgments


References

























































































































1 2 3 4 5 6 7 8 90 10Time (h)

00

05

10

SFG

inte

nsity

(au

)

(a)

1 2 3



aggregates

(b)









(a) (b)

50nm

(c)



0

2

4

6

8

10

12

SFG

inte

nsity

(au

)




0

2

4

6

8

10

12

SFG

inte

nsity

(au

)


(d) (e)

minusEGCG +EGCG









03

04

05

06

07

08

09

SFG120573

-she

et (a

u)

10

15

20

25

ThT

fluor

esce

nce i

nten

sity

(au

)

800400 1200 16000

Time (min)

(a)

(b)

50nm

(c)



119868119861119860 =

10038161003816100381610038161003816100381610038161003816100381610038161003816

120594(2)119901119904119901119861

120594(2)119901119904119901119860

10038161003816100381610038161003816100381610038161003816100381610038161003816

2

=100381610038161003816100381610038161003816100381610038161003816⟨tan2120595⟩

120573119887119888119886119861

120573119886119888119887119860+ (1 minus ⟨tan2120595⟩)

120573119887119886119888119861

120573119886119888119887119860

100381610038161003816100381610038161003816100381610038161003816

2

(2)






a

a

b

b

c

c

hIAPP

hIAPP

x

y

z

120579

120601

120595

(a)

B

R

NH

HN

O R

O

R

NH

HN

O R

O

A mode

B mode

psp

A000

005

010

SFG

inte

nsity

(au

)


(b)

811

19

23

24

8

2837

N N N

O

N

H

H

H

N N N

O

O

N

O

H

H

H

O

O

H

H

O

OR1

R1

R2

R2

R3

R3

R4

R4


SSNNF

SSNNF

GAILSTY

LL


FFN

R NARQT

TVVL

(c)

0

5

10

15

20

25

45 225135 315 36027090 1800

120595 (∘)

|120594(2)

pspB120594

(2)

pspA|2

(d)

120595 = 30∘

120595 = 48∘

120595 = 132∘

120595 = 150∘

Exp

Ipsp

(au

)


(e)

Figure 7 Continued


5

10

15

20

25

30

35

27

His18

Arg1148

∘

(f)










5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268


(d)








Acknowledgments


References























































































































(a) (b)

50nm

(c)



0

2

4

6

8

10

12

SFG

inte

nsity

(au

)




0

2

4

6

8

10

12

SFG

inte

nsity

(au

)


(d) (e)

minusEGCG +EGCG









03

04

05

06

07

08

09

SFG120573

-she

et (a

u)

10

15

20

25

ThT

fluor

esce

nce i

nten

sity

(au

)

800400 1200 16000

Time (min)

(a)

(b)

50nm

(c)



119868119861119860 =

10038161003816100381610038161003816100381610038161003816100381610038161003816

120594(2)119901119904119901119861

120594(2)119901119904119901119860

10038161003816100381610038161003816100381610038161003816100381610038161003816

2

=100381610038161003816100381610038161003816100381610038161003816⟨tan2120595⟩

120573119887119888119886119861

120573119886119888119887119860+ (1 minus ⟨tan2120595⟩)

120573119887119886119888119861

120573119886119888119887119860

100381610038161003816100381610038161003816100381610038161003816

2

(2)






a

a

b

b

c

c

hIAPP

hIAPP

x

y

z

120579

120601

120595

(a)

B

R

NH

HN

O R

O

R

NH

HN

O R

O

A mode

B mode

psp

A000

005

010

SFG

inte

nsity

(au

)


(b)

811

19

23

24

8

2837

N N N

O

N

H

H

H

N N N

O

O

N

O

H

H

H

O

O

H

H

O

OR1

R1

R2

R2

R3

R3

R4

R4


SSNNF

SSNNF

GAILSTY

LL


FFN

R NARQT

TVVL

(c)

0

5

10

15

20

25

45 225135 315 36027090 1800

120595 (∘)

|120594(2)

pspB120594

(2)

pspA|2

(d)

120595 = 30∘

120595 = 48∘

120595 = 132∘

120595 = 150∘

Exp

Ipsp

(au

)


(e)

Figure 7 Continued


5

10

15

20

25

30

35

27

His18

Arg1148

∘

(f)










5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268


(d)








Acknowledgments


References
























































































































03

04

05

06

07

08

09

SFG120573

-she

et (a

u)

10

15

20

25

ThT

fluor

esce

nce i

nten

sity

(au

)

800400 1200 16000

Time (min)

(a)

(b)

50nm

(c)



119868119861119860 =

10038161003816100381610038161003816100381610038161003816100381610038161003816

120594(2)119901119904119901119861

120594(2)119901119904119901119860

10038161003816100381610038161003816100381610038161003816100381610038161003816

2

=100381610038161003816100381610038161003816100381610038161003816⟨tan2120595⟩

120573119887119888119886119861

120573119886119888119887119860+ (1 minus ⟨tan2120595⟩)

120573119887119886119888119861

120573119886119888119887119860

100381610038161003816100381610038161003816100381610038161003816

2

(2)






a

a

b

b

c

c

hIAPP

hIAPP

x

y

z

120579

120601

120595

(a)

B

R

NH

HN

O R

O

R

NH

HN

O R

O

A mode

B mode

psp

A000

005

010

SFG

inte

nsity

(au

)


(b)

811

19

23

24

8

2837

N N N

O

N

H

H

H

N N N

O

O

N

O

H

H

H

O

O

H

H

O

OR1

R1

R2

R2

R3

R3

R4

R4


SSNNF

SSNNF

GAILSTY

LL


FFN

R NARQT

TVVL

(c)

0

5

10

15

20

25

45 225135 315 36027090 1800

120595 (∘)

|120594(2)

pspB120594

(2)

pspA|2

(d)

120595 = 30∘

120595 = 48∘

120595 = 132∘

120595 = 150∘

Exp

Ipsp

(au

)


(e)

Figure 7 Continued


5

10

15

20

25

30

35

27

His18

Arg1148

∘

(f)










5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268


(d)








Acknowledgments


References























































































































a

a

b

b

c

c

hIAPP

hIAPP

x

y

z

120579

120601

120595

(a)

B

R

NH

HN

O R

O

R

NH

HN

O R

O

A mode

B mode

psp

A000

005

010

SFG

inte

nsity

(au

)


(b)

811

19

23

24

8

2837

N N N

O

N

H

H

H

N N N

O

O

N

O

H

H

H

O

O

H

H

O

OR1

R1

R2

R2

R3

R3

R4

R4


SSNNF

SSNNF

GAILSTY

LL


FFN

R NARQT

TVVL

(c)

0

5

10

15

20

25

45 225135 315 36027090 1800

120595 (∘)

|120594(2)

pspB120594

(2)

pspA|2

(d)

120595 = 30∘

120595 = 48∘

120595 = 132∘

120595 = 150∘

Exp

Ipsp

(au

)


(e)

Figure 7 Continued


5

10

15

20

25

30

35

27

His18

Arg1148

∘

(f)










5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268


(d)








Acknowledgments


References























































































































5

10

15

20

25

30

35

27

His18

Arg1148

∘

(f)










5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268


(d)








Acknowledgments


References























































































































5

45

4

35

3

25

2

(a)

5

45

4

35

3

25

2

(b)

(c)

WaterSodium

minus1

0

1

z (n

m)

04 268


(d)








Acknowledgments


References


























































































































Acknowledgments


References






















































































































New Insights from Sum Frequency Generation Vibrational Spectroscopy into … · 2016. 5. 14. ·...

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