膜蛋白的结构与功能三

膜蛋白的结构与功能三第1页，共47页，2023年，2月20日，星期四BacteriorhodopsinIntroductiontobacteriorhodopsinArchaebacteriaHalobacteriaSalinariumarethesourceofbacteriorhodopsinTheyarehalophilicbacteria(foundinverysaltywatere.g.GreatSaltLake)第2页，共47页，2023年，2月20日，星期四BacteriorhodopsinThepurplemembranepatchesareareasonthemembranewhereBRisconcentratedBRabsorbslightat570nm(visiblegreenlight)RedandBluelightisreflected,givingmembraneitspurplecolour第3页，共47页，2023年，2月20日，星期四BacteriorhodopsinThepurplemembranewasbuiltbyreplicatinginspaceahexagonalunitcellusingperiodicboundaryconditions.Theunitcellcontains3monomericbRmolecules,28lipidmolecules,and8410watermolecules,givingatotalnumberof23783atoms.Thisstructureisreplicatedinalldirectionsofspace,followingahexagonalsymmetry,toconstructamultilammelarPMmodel.第4页，共47页，2023年，2月20日，星期四BacteriorhodopsinThefunctionofbacteriorhodopsin—bacteriorhodopsinfunctionsasaprotonpump—BacteriorhodopsinuseslightenergytopumpH+totheexteriorofthecell,thusgeneratingaH+electrochemicalgradient,orproton-motiveforce—Photonsreactwithaboundretinalgroupcausingconformationalchangeinbacteriorhodopsin—Thecellsusethisproton-motiveforcetosynthesiseATPandtoperformothervitalfunctionsrequiringenergyexpenditure第5页，共47页，2023年，2月20日，星期四BacteriorhodopsinBacteriorhodopsintakesenergyfromphotonsThisenergyisconvertedandcreatesaprotongradientbypumpingprotonsoutsidethecellProtonsareallowedbackintothecellbyanATPsynthaseInanutshell:Photonsareusedtopowerthecell第6页，共47页，2023年，2月20日，星期四BacteriorhodopsinExtracellularmatrixCytosolOKHH-D85deprotonated-PRSreprotonated-backtosquare1untilanotherprotonisomerizestheAll-transretinalPRSHH+

EarlyElectronMicroscopyStudiesofBacteriorhodopsin

Bacteriorhodopsinnaturallyforms2-dimensionalcrystals.Lipidis25%ofpurplemembrane&bRistheother75%.Firstmembraneproteinstructure:Henderson&Unwin,Nature257,28-32(1975).Electronmicrographsfrom1mm2

×45Åsamples(120,000proteinmolecules).Diffractionpatternisnotathreedimensionallatticeofpoints,butatwodimensionallatticeofcontinuouslines.Electronmicroscopyimagesgivephasesdirectly,butatlowerresolutionthanadiffractionpattern.Needtotiltthesampletogetinformationperpendiculartotheplaneofthemembrane→technicalcomplications.Structureshowedsevenahelices,andthattheproteinpackedasatrimerwithinthemembrane.第7页，共47页，2023年，2月20日，星期四Bacteriorhodopsin7TMhelicesFormsahomotrimerHomotrimersaggregatetoformthepurplemembraneStabilityoftrimerby:G113,I117,L48Moststabilitycomesfromsurroundinglipids第8页，共47页，2023年，2月20日，星期四BacteriorhodopsinProtonpumpingchannel&watermoleculesGroundstaterevealsaprotonpumpingchannel:-Chargedresidueslineuplikearelayteam.-Schiffbaselinkageatthecentreofthischannel.Watershaveafunctionalroleinproton-transport.-Anextensivehydrogen-bondnetworklinkstheSchiffbasetotheextracellularmedium.-Nosuchwatersarevisibletothecytoplasmicside.第9页，共47页，2023年，2月20日，星期四BacteriorhodopsinThecomplex-counterion

ASchiffbaseisformedbyacovalentdoublebondtoanitrogen.-ArchaealrhodopsinshaveawellconservedlysineinhelixG.AprotonatedSchiffbaseispositivelycharged.-Buriedchargesusuallyenergeticallyunfavoured.-A`counter-ion'mustcompensatetheSchiffbasecharge.BacteriorhodopsinhasseveralchargedgroupsneartheSchiffbase.-WatermoleculesstabilisethepositivelychargedSchiffbase&Arg82,andthenegativelychargedAsp85&Asp212.-Wholearrangementiscalledthecomplexcounter-ion.第10页，共47页，2023年，2月20日，星期四BacteriorhodopsinPi-bulgeBelow2ÅresolutionpossibletoseedetailsintheH-bondingnetworkofa-helices.AlonghelixGnearLys216,thenormalH-bondnetworkofana-helixisdisrupted:-CarbonyloxygensofAla215&Lys216formH-bondstowatermolecules.-Inthisregionthen→n+4a-helixpatternislocallydisruptedinfavorofan→n+5p-helixpattern.Inducesanon-prolinekinkinhelixG.第11页，共47页，2023年，2月20日，星期四Lipids

30lipidmoleculesareassociatedwitheachtrimer.-Sixwithinthetrimer.-24surroundingthetrimer.-Inter-trimerinteractionsaredominatedbylipidcontacts.Welldefinedprotein-lipidcontactsexplainthestrongstabilityofthepurplemembrane.CytoplasmicExtracellular第12页，共47页，2023年，2月20日，星期四BacteriorhodopsinProtein:lipidcontactsPrimarilyhydrophobicinteractionsofalkylchainswithhydrophobicresidues.Tyrosinehydroxylgroupscanformhydrogenbondswiththeoxygenatomsofthelipidethers.第13页，共47页，2023年，2月20日，星期四BacteriorhodopsinPrerequisitesforprotonpumpingWorksoneedsamotor.Canbelightdriven(eg.photosyntheticreactioncentres&bacteriorhodopsin).Classicalprotonpumpneedsapathwayforprotontransport(orelectronsinoppositedirection).Protontransportchannelformedbychargedgroups.Sequencespecificexchangebetweenmembersofthe``proton-pumping-relay-team''.Mustpreventback-leakageof200mV/30Åprotongradient.Hydrophobicplugmustexistinground-state.The``switch''transientlyopensthisplug.Vectoriality.Protonexchangebetweenspecificgroupsmusthappeninaspecificsequence.Structuralrearrangementsdictatetheorderingofevents.第14页，共47页，2023年，2月20日，星期四BacteriorhodopsinThemotorBacteriorhodopsincontainsanall-transretinalchromophoreburiedwithintheprotein'score.-RetinalcovalentlyboundtoLys216viaaprotonatedSchiffbase.-Lightabsorptiontriggersanisomerisationto13-cisconfigurationwith64%quantumefficiency.-Initiatesaphoto-cycle.-Stericclashesdrivestructuralrearrangements.第15页，共47页，2023年，2月20日，星期四BacteriorhodopsinTransportchannelMutagenesis&spectroscopyhaveidentifiedkey-groups.Event1:(L→Mtransition,40msec).-ProtontransferfromSchiffbasetoAsp85.-Alwaysfirst,irrespectiveoftemperature,pHormutation.Event2:(aroundL→Mtransition,~100msec).-Protonreleasetoextracellularsurface(fromGlu204&Glu194).-Timingdependsdramaticallyontemperature,pH&mutation.第16页，共47页，2023年，2月20日，星期四Bacteriorhodopsin

Event3:(M→Ntransition,5msec).-RetinalreprotonationfromAsp96.-Sloweddowndramaticallybymutation&temperature.Event4:Asp96reprotonatedfromcytoplasm.Event5:(aroundN→Otransition,10msec).-ProtontransferfromAsp85toGlu194/204.OnlymutationsofAsp85orretinaldestroypumping.第17页，共47页，2023年，2月20日，星期四fspsμsms4.ms3.Asp96μs1.Lys216RetinalAsp85ms5.Arg822.Glu194Glu204FExtracellularsideCytoplasmicside第18页，共47页，2023年，2月20日，星期四Hydrophobicplug&the"switch"Inthegroundstateaseriesofwatermoleculesdefineapathfromtheretinaltotheextracellularsurface.-Structuralchangescutretinalofffromextracellularside.Onthecytoplasmicsideno-suchwaterpathwayexists.-Structuralchangesmustfacilitatelocalorderingofwaters.Nearcytoplasmicsurfaces,bulkyhydrophilicgroupsblockentrancechannel.-Entrancechannelmust``open-up''duringphotocycle.第19页，共47页，2023年，2月20日，星期四BacteriorhodopsinVectorialityPrimaryprotontransfereventiskey(SB=Schiffbase)Asp85-+SB+

↔Asp85+SB-OnlymutationofAsp85(toaneutralresidue)orreplacementofretinal,destroyspumping.Asp85andtheSBhaveH-bondstothesamewater.-Yetprotontransfertakes40msec!Structuralrearrangementsmustexplain:-Whytheprimaryprotontransfereventtakessolong.-HowthepKa'softheSchiffbase&Asp85changetofacilitateprotontransfer(doesnotoccurinthegroundstate).-WhatpreventsSchiffbasereprotonationfromAsp85.第20页，共47页，2023年，2月20日，星期四IntermediatetrappingRetinalisomerisationisdrivenbylight.Thermalenergyrequiredtocrossdown-streamenergybarriers.-Loweringthetemperatureenablesintermediatestobecaptured.-Millisecondintermediatesmaybecapturedbyflashingandfreezing.第21页，共47页，2023年，2月20日，星期四BacteriorhodopsinDifferenceFourierAnalysisMeasurediffractionamplitudeswhenanintermediateistrappedwithincrystals.Measurediffractionamplitudesforthegroundstate.Subtractthetwo&usephasesfromtheground-statemodeltocalculateadifferenceFouriermap.-Least-biasedmethodforobservingelectrondensitychanges.-Immediatelyseethenoise-levelofthemap.Quantifyusingrefinement.第22页，共47页，2023年，2月20日，星期四BacteriorhodopsinEarlyrearrangementsKLTwastrappedusinggreenlightat110K.-Differencespectrafromsinglecrystalsconfirmedthis.IsomerisationrotatesSchiffbasewhichpointupwards.-LosesstabilisingH-bondtoWat402.-Schiffbasepointsintoahydrophobicenvironment.SchiffbasepKadecreases(makesitwanttogiveupaproton).第23页，共47页，2023年，2月20日，星期四Bacteriorhodopsin

Keywatermolecule(Wat402)becomesdisordered.-KickedoutlikeasoccerballbyLys216.-H-bondlosttoAsp85,increasingitspKa.-Asp85approachesAsp212,enhancingtheirinteraction.第24页，共47页，2023年，2月20日，星期四RearrangementsfacilitatingprotontransferLLTtrappedat170K(lateralsoredlight150K).-MoreH-bondstoAsp85lost,increasingitspKa.-LossofstabilisingH-bondscauseshelixCtobend.-Asp85furtherapproachesAsp212(enhancesinteraction).-AwatermoleculereordersbetweenAsp85andAsp212(enhancesinteraction).

pKaofAsp85dramaticallyincreasesuntilitsabletoacceptaprotonfromtheSchiffbase.第25页，共47页，2023年，2月20日，星期四RearrangementsfollowingprotontransferSchiffbaseandAsp85becomeneutralisedbyprotontransfer.-Mutualelectrostaticattractioncanceled.-HelixCstraightensout,drawingAsp85&Schiffbaseapart.-Retinalflexesupwards,drawingAsp85&Schiffbaseapart.L-statewaterspreserved.L-stateM-state第26页，共47页，2023年，2月20日，星期四

AnticipatingSchiffbasereprotonationLocalunwindingofhelixGnearLys216.-Hydrophobicregionbecomeshydrophilic.-Watersorderinthisregion.-DefinespathwayforreprotonationfromAsp96.StericclashofretinalwithTrp182.-TiltshelixFoutwards&opensupcytoplasmic``plug''.-FacilitatesreprotonationofAsp96fromthecytoplasm.第27页，共47页，2023年，2月20日，星期四StructuralModelNowaterlinkedpathwayfromAsp96toRetinalinbRstate.WaterlinkedpathwayalmostfromAsp96toRetinalinM.bR-stateM-state第28页，共47页，2023年，2月20日，星期四SummaryofstructuralchangesRetinalisomerisationdisordersakeywatermolecule.SubsequentdisorderingofwatersenableshelixCtoflex&mediatestheprimaryprotontransferevent.Stericclashesdrivetheopeningofthecytoplasmicside.Relaxationrecoverstherestingstate.

第29页，共47页，2023年，2月20日，星期四NM2M1OKLbRFC第30页，共47页，2023年，2月20日，星期四pKa&protontransferInitialH-bondsstabilisehighSchiffbasepKa&lowAsp85pKa.Retinalisomerisation&waterdisorderingdisruptsH-bonds.-SchiffbasepKadecreases.-HelixbendexaggeratesmovementofAsp85towardsAsp212.-Asp85linkedonlytoAsp212viaawater.-Asp85becomeshighlypolarised&pKaincreases.StructuralchangesreversetherelativepKaofSchiffbase&Asp85.-Directorwatermediatedprotontransferpossible.第31页，共47页，2023年，2月20日，星期四BacteriorhodopsinProtonpathwaySimulationsshowSchiffbaseprotonrotates180ouponK-to-Ltransition.-HelixCbendchangesitselectrostaticenvironment.L-stateshowstwoplausibleprotontransferpathways.Direct.ViaThr89.Multiplepathwaysplausible.第32页，共47页，2023年，2月20日，星期四BacteriorhodopsinMechanisticConclusionsBacteriorhodopsinbestcharacterisedmembraneprotein.Whydoestheprimaryprotontransfertake~40msec?Facilitatedbyatransientbendofana-helix.HowarethepKa'sofkeygroupsperturbed?RetinalisomerisationbreaksaH-bondfromtheSBtoawatermolecule.SBreorientatesintoahydrophobicpocket.WaterdisorderingdisruptsH-bondstoAsp85.HelixCflexexaggeratesinteractionofAsp85&Asp212.WhatpreventsSchiffbasereprotonationfromAsp85?StraighteningofhelixCdrawsAsp85&Schiffbaseapart.StraighteningofretinaldrawsAsp85&Schiffbaseapart.Techniquesused&underlyingprinciplesshouldfindapplicationwithotherproton&ionpumps.第33页，共47页，2023年，2月20日，星期四PorinIntroduction第34页，共47页，2023年，2月20日，星期四PorinPorinsareaclassofporeformingproteinsintheoutermembranesofgram-negativebacteria,mitochondriaandchloroplasts.Theyshowmostlyß-barrelstructure,havelargerporediametersandarelessspecificforsubstratesthanionchannels.Theirmaintaskistomediatetheregulateddiffusionalsubstratetransportacrosstheoutermembrane.Becauseofthecommonspreadofimmunityagainstantibiotics,porinsbecomemoreandmoreimportantinpharmacology.第35页，共47页，2023年，2月20日，星期四PorinPorinsaretubeswithadiameterofabout1nmwhicharefilledwithwater.Non-specificporinsallowthediffusionofionsandmoleculesuptoamolecularweightof600.Thediffusionspeeddependsonboththedifferenceofconcentrationintheperiplasmandoutsideandthemolecularweightofthesolute.第36页，共47页，2023年，2月20日，星期四ompFOmpFporinisanon-specifictransportchannelthatallowsforthepassivediffusionofsmall,polarmolecules(600-700Dainsize)throughthecell'soutermembrane.Suchmoleculesincludewater,ions,glucose,andothernutrientsaswellaswasteproductsOmpFalsoservesasacell-surface-exposedreceptorformanyphagesandcolicins.Severalantibiotics,includingß-lactams,usetheporinpathwaytocrosstheoutermembraneandtofindtheirtargets第37页，共47页，2023年，2月20日，星期四ompFOmpFwasthefirstmembraneproteintoyieldcrystalsofasizeandorderthatwereamenabletohighresolutionstructuralanalysisbyX-raycrystallography(1980).Ittookmorethan10yearsbeforetheatomicstructurewaselucidated(1992).第38页，共47页，2023年，2月20日，星期四ompFTheoverallstructureofompF—Ineachmonomer,16b-strandsspantheoutermembrane—Thetiltofthestrandsrelativetotheß-barrelaxisvariesbetween35to50degree.—Theporeliesoff-centerandatanangleof16degreetothebarrelaxis.—TheL3loopconstraintheporesize—Themajorcontributiontotrimerformationishydrophobicinteraction.—Thechannelareseparatedoveradistanceofabout35Å第39页，共47页，2023年，2月20日，星期四ompFTheexternalsurface—Ahydrophobicbandwithawidthof25Åencirclesthetrimer.—TheboundariesofhydrophobicbandaremostlycomposedofYandF,theremainderofthebandarecomposedofsmallandmediumaliphaticresidues.YandFmayinteractwithLPS.—HydrophilicresiduesofthesmoothendofompFcontactwiththeheadgroupoflipid.TheconservedresiduesinhydrophilicportionofroughendcouldformhydrogenbondwithYinthearomaticboundaryandwithLPSthroughdivalentcations.roughendsmoothend第40页，共47页，2023年，2月20日，星期四ompFStabilityoftheß-barrel—ThemonomerofompFlackofhydrop