膜蛋白的结构与功能(三)

第十二章：膜蛋白的结构与功能(三)Bacteriorhodopsin•Introductiontobacteriorhodopsin•ArchaebacteriaHalobacteriaSalinariumarethesourceofbacteriorhodopsin•Theyarehalophilicbacteria(foundinverysaltywatere.g.GreatSaltLake)Bacteriorhodopsin•ThepurplemembranepatchesareareasonthemembranewhereBRisconcentrated•BRabsorbslightat570nm(visiblegreenlight)•RedandBluelightisreflected,givingmembraneitspurplecolourBacteriorhodopsinThepurplemembranewasbuiltbyreplicatinginspaceahexagonalunitcellusingperiodicboundaryconditions.Theunitcellcontains3monomericbRmolecules,28lipidmolecules,and8410watermolecules,givingatotalnumberof23783atoms.Thisstructureisreplicatedinalldirectionsofspace,followingahexagonalsymmetry,toconstructamultilammelarPMmodel.Bacteriorhodopsin•Thefunctionofbacteriorhodopsin—bacteriorhodopsinfunctionsasaprotonpump—BacteriorhodopsinuseslightenergytopumpH+totheexteriorofthecell,thusgeneratingaH+electrochemicalgradient,orproton-motiveforce—Photonsreactwithaboundretinalgroupcausingconformationalchangeinbacteriorhodopsin—Thecellsusethisproton-motiveforcetosynthesiseATPandtoperformothervitalfunctionsrequiringenergyexpenditureBacteriorhodopsin•Bacteriorhodopsintakesenergyfromphotons•Thisenergyisconvertedandcreatesaprotongradientbypumpingprotonsoutsidethecell•ProtonsareallowedbackintothecellbyanATPsynthase•Inanutshell:PhotonsareusedtopowerthecellBacteriorhodopsinExtracellularmatrixCytosolOKHH-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.Bacteriorhodopsin•7TMhelices•Formsahomotrimer•Homotrimersaggregatetoformthepurplemembrane•Stabilityoftrimerby:–G113,I117,L48–MoststabilitycomesfromsurroundinglipidsBacteriorhodopsinProtonpumpingchannel&watermolecules•Groundstaterevealsaprotonpumpingchannel:-Chargedresidueslineuplikearelayteam.-Schiffbaselinkageatthecentreofthischannel.•Watershaveafunctionalroleinproton-transport.-Anextensivehydrogen-bondnetworklinkstheSchiffbasetotheextracellularmedium.-Nosuchwatersarevisibletothecytoplasmicside.BacteriorhodopsinThecomplex-counterion•ASchiffbaseisformedbyacovalentdoublebondtoanitrogen.-ArchaealrhodopsinshaveawellconservedlysineinhelixG.•AprotonatedSchiffbaseispositivelycharged.-Buriedchargesusuallyenergeticallyunfavoured.-A`counter-ion'mustcompensatetheSchiffbasecharge.•BacteriorhodopsinhasseveralchargedgroupsneartheSchiffbase.-WatermoleculesstabilisethepositivelychargedSchiffbase&Arg82,andthenegativelychargedAsp85&Asp212.-Wholearrangementiscalledthecomplexcounter-ion.BacteriorhodopsinPi-bulge•Below2ÅresolutionpossibletoseedetailsintheH-bondingnetworkofa-helices.•AlonghelixGnearLys216,thenormalH-bondnetworkofana-helixisdisrupted:-CarbonyloxygensofAla215&Lys216formH-bondstowatermolecules.-Inthisregionthen→n+4a-helixpatternislocallydisruptedinfavorofan→n+5p-helixpattern.•Inducesanon-prolinekinkinhelixG.Lipids•30lipidmoleculesareassociatedwitheachtrimer.-Sixwithinthetrimer.-24surroundingthetrimer.-Inter-trimerinteractionsaredominatedbylipidcontacts.•Welldefinedprotein-lipidcontactsexplainthestrongstabilityofthepurplemembrane.CytoplasmicExtracellularBacteriorhodopsinProtein:lipidcontacts•Primarilyhydrophobicinteractionsofalkylchainswithhydrophobicresidues.•Tyrosinehydroxylgroupscanformhydrogenbondswiththeoxygenatomsofthelipidethers.BacteriorhodopsinPrerequisitesforprotonpumping•Worksoneedsamotor.–Canbelightdriven(eg.photosyntheticreactioncentres&bacteriorhodopsin).•Classicalprotonpumpneedsapathwayforprotontransport(orelectronsinoppositedirection).–Protontransportchannelformedbychargedgroups.–Sequencespecificexchangebetweenmembersofthe``proton-pumping-relay-team''.•Mustpreventback-leakageof200mV/30Åprotongradient.–Hydrophobicplugmustexistinground-state.–The``switch''transientlyopensthisplug.•Vectoriality.–Protonexchangebetweenspecificgroupsmusthappeninaspecificsequence.–Structuralrearrangementsdictatetheorderingofevents.BacteriorhodopsinThemotor•Bacteriorhodopsincontainsanall-transretinalchromophoreburiedwithintheprotein'score.-RetinalcovalentlyboundtoLys216viaaprotonatedSchiffbase.-Lightabsorptiontriggersanisomerisationto13-cisconfigurationwith64%quantumefficiency.-Initiatesaphoto-cycle.-Stericclashesdrivestructuralrearrangements.BacteriorhodopsinTransportchannel•Mutagenesis&spectroscopyhaveidentifiedkey-groups.•Event1:(L→Mtransition,40msec).-ProtontransferfromSchiffbasetoAsp85.-Alwaysfirst,irrespectiveoftemperature,pHormut