DNA贝尔金路由器设置

ChiralSumFrequencyGenerationSpectroscopyforCharacterizingProteinSecondaryStructuresatInterfacesLiFu,JianLiu,andElsaC.
Y.
Yan*DepartmentofChemistry,YaleUniversity,225ProspectStreet,NewHaven,Connecticut06520,UnitedStatesbSSupportingInformationABSTRACT:Insituandreal-timecharacterizationofproteinsecondarystructuresatinterfacesisimportantinbiologicalandbioengineeringsciences,yetremainstechni-callychallenging.
Inthisstudy,weusedchiralsumfrequencygeneration(SFG)spectroscopytoestablishasetofvibra-tionalopticalmarkersforcharacterizingproteinsecondarystructuresatinterfaces.
WediscoveredthattheNHstretchesalongthepeptidebackbonesofR-helicescanbedetectedinchiralSFGspectra.
WefurtherobservedthatthechiralvibrationalsignaturesoftheNHstretchtogetherwiththepeptideamideIareuniquetoR-helix,β-sheet,andrandomcoilatinterfaces.
Usingthesechiralvibrationalsignatures,westudiedtheaggregationofhumanisletamyloidpolypeptide(hIAPP),whichisimplicatedintypeIIdiabetes.
WeobservedinsituandinrealtimethemisfoldingofhIAPPfromrandomcoilstoR-helicesandthenβ-sheetsuponinteractionwithalipidwaterinterface.
OurndingsshowthatchiralSFGspectroscopyisapower-fultooltofollowchangesinproteinconformationsatinterfacesandidentifyinterfacialproteinsecondarystruc-turesthateludeconventionaltechniques.
Insituandrealtimecharacterizationofproteinsecondarystructuresatinterfacesisimportantforunderstandingthebiologicalfunctionofproteinsanddevelopingbiomaterialsandbiosensors.
However,thelackofsurface-sensitiveandlabel-freetechniquesthatcanunambiguouslydierentiatesecondarystructuresatinterfacesposeschallenges.
Consequently,pro-blemsthatrequireidenticationofsecondarystructuresattheinterfacesofcomplexproteinsystemshavenotbeenfullyexplored.
Forexample,theaggregationofamyloidproteinsintoβ-sheetstructuresisassociatedwithmanydiseases,suchasParkinson's,Alzheimer's,andpriondiseases.
1Althoughrecentndingsrevealtheimportanceoflipidmembranesincatalyzingtheaggregationprocess,2,3theaggregationpathwayofamyloidproteinsonmembranesurfacesisnotfullyunderstood.
Here,weintroducedaconceptofusingsecond-orderchiralvibrationalopticalmarkers,providedbysumfrequencygenera-tion(SFG)spectroscopy,todistinguishproteinsecondarystructuresatinterfaces.
WestudiedchiralvibrationalstructuresofpeptidebackbonesandobservedcharacteristicNHstretchandamideIsignaturesthatareuniquetorandomcoils,R-helices,andβ-sheetsatinterfaces.
Usingthesesignatures,wemonitoredtheaggregationofhumanamyloidpolypeptide(hIAPP)atalipidwaterinterfaceandobservedtheconversionofhIAPPfromarandomcoiltoanR-helixandthentoaβ-sheetinrealtime.
Wefoundthattheopticalmarkersarefreefrombackgroundofsolventsandachiralsolutesattheinterfaces.
TheyareinthespectralregionsoftheamideIandNHstretchwidelyseparatedat16001700cm1and31003350cm1,respectively.
Hence,theSFGvibrationalmarkersarerelativelyopticallycleanandbackground-free.
Thus,theyareusefulforcharacterizinginterfacialproteinsecondarystructuresthathavebeendiculttoresolveusingconventionaltechniques.
Conventionaltechniquesforcharacterizingproteinsecondarystructuresatinterfaceshavelimitations.
Althoughcirculardichro-ism(CD)isoftenusedtocharacterizesecondarystructures,4itlackssurfacesensitivity.
Surfaceplasmonresonancecandetectadsorptionofproteinsonsurfaces,5butitisnotsensitivetosecondarystructures.
TheX-rayscatteringmethodcanbeusedtoprobetheorderedproteinstructures,butitisnotsuitedforkineticstudiesinsitu.
Ramanandinfrared(IR)spectroscopycanprovideinformationaboutsecondarystructurebyprobingamideIvibrationalbands.
69However,amideIvibrationalbandsoverlapwiththebendingmodesofwater.
Thus,deuteratedwatermustbeused.
Moreover,theamideIbandsofvarioussecondarystructuresareclusteredinthespectralregionof16201680cm1andtheamideIbandsofR-helicesandrandomcoilsarebothapproximatelyat1650cm1,whichmakescharacterizationofcomplexproteinsystemsextremelydicult.
BothRamanandIRspectroscopylacksurfaceselectivityandoftenrequiremetalsubstratestoenhancesurfacesignal5,10orreectiongeometrytosuppressbulksignals.
11Two-dimensionalIRspectroscopyhasbeenusedtoidentifysecondarystructures.
1215However,itcanonlybeappliedtoproteinsinbulksolution,notatinterfaces.
Sincethelate1980s,SFGspectroscopyhasbeendevelopedintoapowerfulsurface-selectivetechniquetoobtainstructuralanddynamicinformationinphysicalandmaterialsciences,1620suchasprobingchemicalphysicsofsmallmoleculesatinter-faces.
2125Itisasecond-ordercoherentopticaltechnique,requiringspatialandtemporaloverlapofanIRbeamandavisiblebeamattheinterface,toproducevibrationalopticalsignals.
SFGissurface-selectiveduetotheintrinsicasymmetryoftheinterfacesuchthatsecond-orderpolarizationinducedatinterfacialmol-eculescanaddupcoherentlytoproducesurfaceselectivesignals.
16Simpsonetal.
derivedachiralSFGtheorytoshowthatsignalscanbegeneratedevenfromachiralmoleculesthatarearrangedinmacromolecularchiralarchitectures.
26SuchsignalsReceived:February19,2011ThisisanopenaccessarticlepublishedunderanACSAuthorChoiceLicense,whichpermitscopyingandredistributionofthearticleoranyadaptationsfornon-commercialpurposes.
havebeenobservedexperimentallyusingbiomolecules,includ-ingtheCHstretchesofDNAandtheamideIofproteins.
2730InspiredbythechiralSFGtheoryanddetectionofthechiralamideIsignalfromβ-sheets,28,30werealizedthattheNHgroupsalongchiralpeptidebackbonesofvarioussecondarystructurescouldalsobechiral-SFGactive.
Totestthisidea,weprobedboththeNHstretchandamideIregionsofmodelpeptidesandproteins.
WeobservedthatchiralNHstretchandamideIarehighlyuniquetotheR-helixandtheβ-sheet.
Weproposeusingthesesignalsasopticalmarkerstocharacterizeproteinsecondarystructuresatinterfaces,similartotheuseofCDsignalstoidentifyproteinsecondarystructuresinbulksolution.
Toestablishthechiralvibrationalopticalmarkers,weobtainedtheSFGspectraofmodelpeptidesandproteins(Figure1A).
Forβ-sheets,weusedhIAPP,a37-aminoacidpeptidehormonesecretedbyhumanpancreaticβ-cells.
Thispeptideformsparallelβ-sheetsattheairwaterinterfaceinthepresenceofnegativelychargedlipids.
31ForR-helices,wechosethreemodelsystems:(1)LKR14,a14-aminoacidpeptidewiththesequence(LKKLLKL)2;32(2)pH-lowinsertionpeptide(pHLIP),a36-aminoacidpeptidederivedfromhelix3ofbacteriorhodopsin;33and(3)bovinerhodopsin,a7-R-helicaltransmembraneGprotein-coupledreceptor.
34Acombinationoftechniques,includingCDspectros-copy,IRspectroscopy,andX-rayphotoelectronspectroscopy,hasshownthatLKR14andpHLIPcanformR-helicesinamphiphilicenvironments.
32,33,35,36RhodopsincanformastablemonolayerattheairwaterinterfacewhensurfacepressureiscarefullycontrolledinaLangmuirtrough.
37,38Asacontrol,weusedratisletamyloidpolypeptide(rIAPP),whichdiersfromhIAPPbysixaminoacids.
39Itisrelativelyunstructuredinsolutionanddoesnotformβ-sheetstructureseveninthepresenceoflipids.
3ToobtaintheSFGspectra,wedissolvedthepeptidesinaqueoussolutionandprobedthemattheairwaterinterface(SupportingInformation).
ForthehIAPPexperiments,wead-dedlipidmoleculestoinducetheformationofamyloidattheairwaterinterface.
Fortherhodopsinexperiments,weexpres-sedandpuriedrecombinantbovinerhodopsin40andmadeamonolayerofrhodopsinattheairwaterinterface,asdescribedbyLavoieetal.
38Weusedourbroad-bandwidthSFGspectro-meter41toobtainthechiralandachiralSFGspectrumofeachpeptideandproteininboththeNHstretchandamideIreg-ions.
Weusedpsp(p-polarizedSFG,s-polarizedvisible,andp-polarizedinfrared)polarizationfortheacquisitionofchiralSFGspectra(Figure1)andssppolarizationfortheacquisitionofachiralspectra(FigureS1).
Inthepresenceofthenegativelychargedlipiddipalmitoylpho-sphoglycerol(DPPG),hIAPPaggregatesintoaparallelβ-sheetstructureattheairwaterinterface.
ThehIAPPaggregateshowsapeakat1622cm1andashoulderat1660cm1intheamideIchiralspectrum,correspondingtotheantisymmetricandsymmetricamideImodes,respectively(Figure1),30,42butitdoesnotshowasignalinthechiralNHspectrum.
Incontrast,R-helicalrhodopsinandpHLIPshowchiralNHsignalsatabout3280cm1andLKR14showsachiralNHsignalatabout3300cm1.
However,allaresilentintheamideIchiralspectra(Figure1).
Conversely,theachiralSFGspectrumofeverypeptideorprotein,obtainedusingssppolarization(FigureS1),showsbothamideIandNHstretchsignalsregardlessoftheirsecondarystructures.
Similartoconven-tionalIRandRamanspectroscopy,achiralSFGspectroscopydoesnotallowdirectidenticationofsecondarystructures.
Moreover,therIAPPcontrolshowsanachiralsignalintheamideIspectrum(FigureS1),indicatingthatrIAPPadsorbsattheinterface.
How-ever,wecouldnotdetectachiralNHstretchoramideIsignal(Figure1),suggestingthatnoteveryproteinorpeptideatinterfacescangenerateachiralNHoramideIsignalunderourexperimentalconditions.
ItislikelythatrIAPPadoptsalargelydisorderedstruc-tureorapartiallyfoldedstructure43,whichdoesnotgeneratedetectablechiralsignals.
Ourresultsindicatethatparallelβ-sheetsexhibitchiralamideIsignalsbutaresilentinthechiralNHstretchspectrum,whereasR-helicesdisplaychiralNHstretchsignalsbutaresilentinthechiralamideIspectrum.
Becauserandomcoilsdonothaveachiralpeptidebackbone,theyshouldnotshowachiralNHoramideIsignalattheinterface.
Takentogether,theseresultsidentifyasetofchiralvibrationalSFGopticalmarkersthatcanbeusedtocharacterizeproteinsecondarystructuresatinterfaces.
Todemonstratetheuseoftheseopticalmarkers,westudiedhIAPPasamodelsystem,whichaggregatesintoβ-sheet-richstructures44,45depositedontopancreaticβ-cellsandcausestypeIIdiabetes.
46TheaggregationofhIAPPiscatalyzedbyinterac-tionswithnegativelychargedlipids2,3andisthoughttoundergoanR-helicalintermediatebeforeaggregatingintoβ-sheetstructures,Figure1.
ChiralSFGspectraofthemodelpeptidesandprotein.
(A)SchematicsofsecondarystructuresofthehIAPPaggregate,LKR14,rhodopsin,pHLIP,andrIAPP.
ThechiralSFGspectraattheairwaterinterfaceinthe(B)amideIregionand(C)NHstretchregion.
basedonextensivebiophysicalstudiesusingacombinationoftechniques,includingCD,uorescence,andIRreectionab-sorptionspectroscopy.
31,47FollowingadditionofDPPG,wemonitoredchiralSFGspectraofhIAPPintheNHstretchandamideIspectralregionsattheairwaterinterface(Figure2A).
ThesignaloftheNHstretchgraduallyincreasestoitsmaximumin3handdisappearsby10haftertheadditionofDPPG.
Incontrast,theamideIsignalappearsapproximately4hafteradditionofDPPGandreachesitsmaximumin10h.
Becausethekineticsofamyloidformationisdiculttoreproduceduetovariationsinfactorssuchasnucleationandagitation,48werepeatedeachmeasurementthreetimesandplottedtheNHstretchandamideIintensityasafunctionoftime(Figure2B).
OurndingsshowthattheNHstretchsignalconsistentlydisappearedpriortoaccumulationoftheamideIsignal.
Ascontrolexperiments,westudiedhIAPPintheabsenceofDPPG(FigureS2AB)andrIAPPinthepresenceofDPPG(FigureS2CD)becauseneitherpeptideaggregatesintoaβ-sheetundertheseconditionsinthetimescaleofhours.
1,30Inbothexperiments,weobservedtheachiralamideIsignals,suggestingbothpeptidesadsorbattheinterface.
30However,monitoringtheNHandamideIchiralspectraforapproximately10h,wecouldnotdetectanychiralsignal(FigureS2).
OurresultsindicatethatneitherhIAPPnorrIAPPformsaβ-sheetandthatneitherhIAPPnorrIAPPfoldsintoanR-helicalstructurethatcanbedetectedinourexperiments.
OnthebasisoftheNHsignalat3285cm1,whichcorres-pondstoanR-helix,andtheamideIsignalat1620cm1,whichcorrespondstoaparallelβ-sheet,theresultspresentedinFigure2showatransientR-helicalintermediateandanalparallel-β-sheetproductintheamyloidaggregationprocess.
TheinitialabsenceofanamideIsignalcouldmeanthathIAPPbeginsineitheranR-helicalorarandom-coilstructure.
However,theinitialabsenceoftheNHstretchsignalrevealsthathIAPPisarandomcoil.
DuetospectraloverlapintheamideIregion,itisdiculttodistinguishrandomcoilsandR-helicesusingIRandRamanspectroscopy.
WeconcludethathIAPPadsorbsatthelipidwaterinterfaceasarandomcoilandbeginsfoldingintoanR-helixwithin11.
5h.
ItconvertsfullytoanR-helixatapproxi-mately3handfoldsintoparallelβ-sheetsatapproximately9h.
Giventhesendings,wecanusetheSFGopticalmarkerstoexplicitlyidentifytheR-helicalintermediateandfollowthekineticsofchangesinthesecondarystructureofhIAPPatthelipidwaterinterface.
Interestingly,theamideIandNHstretchbehavedierentlyinthechiralSFGspectra.
Atpresent,aquantitativedescriptionofthechiralNHandamideIsignalsfromR-helicesandβ-sheetsisbeingdeveloped.
OurobservedchiralamideIresponseisinqualitativeagreementwiththetheorydevelopedbyPerryetal.
,49whichpredictsalargercontributionofthechiralamideIresponsefromβ-sheetsandasmallercontributionfromR-helices.
TheobserveddierencesinthechiralSFGsignalsmaybeoriginatedfromthesymmetryofthevibrationalmodesandthecouplingofthevibrationalmodesinthepeptidebackbones.
ItisknownthatindividualNHstretchesarehighlylocalized,50whileindividualamideImodesarestronglycoupledinthepeptidebackbones.
9WespeculatethatthevibrationalcouplingcanplayaroleintheSFGchiral-opticalresponse.
Moreover,wearguethatthechiralSFGsignalsthatweobservedoriginatefromtheinterfacesduetothechiralmacro-moleculararrangementofthepeptidebackbones.
26AlthoughtheSFGchiral-opticalresponsecouldbeobservedfromthebulkofchiralliquid,51thisbulkchiralsignalisduetotheasymmetryoftheRamantensor,whichisveryweakandneedsthevisiblebeamtobeinresonancewiththeelectronictransitionofthemolecules.
Inaddition,thisbulkchiralsignalwasdetectedbythetransmissionopticalgeometryfromthebulkofpurechiralliquidasreportedpreviously.
51Incontrast,thechiralSFGsignalobservedinourexperimentiscomparabletotheachiralsignals,andtheserelativelystrongchiralsignalsweredetectedatlowconcentration(15μM)usingreectivegeometrywithoutelectronicresonance.
Hence,itisnotlikelythatourobservedchiralSFGsignalsarefromthebulk.
Overall,ourresultsshowtheadvantagesofusingchiralSFGtoprobeinterfacialproteinstructures.
First,thechiralSFGvibra-tionalsignaturesareopticallyclean.
TheamideIsignalfromaβ-sheetandtheNHsignalfromanR-helixdonotinterferewitheachother.
ThechiralSFGsignalisalsoinsensitivetothepresenceofachiralsolventsandachiralsolutesatinterfaces.
Hence,theSFGmarkersareopticallycleanandrelativelybackground-free.
Second,kineticinformationcanbereadilyobtainedbymonitoringtheSFGopticalsignals,whichenableskineticstudiesofproteinfoldingandmisfoldingatinterfacesandFigure2.
AggregationofhIAPP.
(A)Thetime-dependentchiralSFGspectrainthevibrationalregionsofNHstretch(left)andamideI(right)afteradditionofDPPG.
(B)TheintensityoftheNHstretchandamideIsignalsasafunctionoftime.
Resultsoftriplicateexperimentsareshown.
(C)TheaggregationmodelofhIAPPonamembranesurfaceasobservedintheSFGexperiments:adsorptionasarandomcoilleadstoformationofR-helicalintermediates,whichareconvertedtoβ-sheetaggregates.
surfacecharacterizationsofbiomaterialsandbiosensors,withouttheuseofspectroscopiclabels.
BecauseSFGissurface-selective,onlyamonolayerofproteinintheamountofmicrogramsisneededtoobtainSFGspectra.
Thissmallsamplesizeallowsthestudyofmostproteinsthatcanbepuriedfromnaturalsourcesorrecombinantexpressionsystems,includingmembranepro-teins,whicharediculttoobtain.
Moreover,SFGcanbeusedtomeasuretheorientationofsecondarystructuresatinterfaces,52andthuscanbeusedtostudybiologicalprocesses,whichofteninvolvesubtleconformationalchangesinproteins.
Furthermore,SFGcanbeusedtoprobedistinctvibrationalstructuresofindividualsidechainsandotherbiomolecules,allowingstudiesofhighlyspecicproteininteractionsatinterfaces.
Finally,asSFGusesultrafastlasers,whichprovidenanosecondtofemto-secondtimeresolution,itenablesstudiesofultrafastvibrationaldynamicsofproteins.
Forallthesereasons,chiralSFGspectros-copyisexpectedtobeusefulforsolvingavarietyofproblemsrelatedtostructures,functions,anddynamicsofproteinsatinterfacesthatconventionaltechniquescannotadequatelyaddress.
'ASSOCIATEDCONTENTbSSupportingInformation.
Materials,SFGSetup,Experi-mentalProcedureandDataAcquisition,FiguresS1andS2.
ThismaterialisavailablefreeofchargeviatheInternetathttp://pubs.
acs.
org.
'AUTHORINFORMATIONCorrespondingAuthorelsa.
yan@yale.
edu'ACKNOWLEDGMENTTheauthorsthankDr.
R.
Tykcoforprovidingthestructureoftheparallelβ-sheetaggregatesofhIAPP.
E.
Y.
istherecipientoftheStarterGrantAward,SpectroscopySocietyofPittsburgh.
J.
L.
isanAndersonPostdoctoralfellow.
TheauthorsthankDr.
S.
G.
Rayforhelpfuldiscussions.
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