部分純化的豬心線粒體F0的二維結(jié)晶及電子晶體學(xué)Introduction

Membraneproteinsareintegraltothefunctioningofmanybiologicalsystems,servingastransporters,receptors,and?enzymes.Asaresult,theelucidationoftheirstructuresisofgreatimportance.MitochondrialF0F1-ATPsynthaseisaclassicalmembraneproteinthatisresponsibleforATPproductioninoxidativephosphorylation.TheF0portionconsistsofa?c-ringoligomericassemblyembeddedinthemitochondrialinnermembrane.

Methods

ToinvestigatethestructureofthemitochondrialF0F1-ATPsynthase,weutilizedelectroncrystallographytoobtaintwo-dimensional(2D)crystalsofpigheartmitochondrialF0.Wethenusedsingle-particleanalysistoreconstructthethree-dimensional(3D)structureofF0.Wecollected2DimagesofthemitochondrialF0particlesusinga4kx4kCCDcamera,followedbyimageprocessingusingtheEMAN2softwarepackage.Afterwards,wecalculatedthestructureofF0byapplyingthesingle-particleapproachinEMAN2.

Results

The2DcrystalsofpigheartmitochondrialF0werewell-orderedandpossessedahexagonallatticesymmetry.Theresolutionofthe2Dcrystalwasfoundtobe3.7?byFourieranalysis.Thec8-ringassemblycouldbedistinguishedasa?singlelayeroforderedproteinsinthe2Dcrystal.Thec-ringformedauniformhexagonallattice,andtheproteindensityinthecrystalwashomogenous.Theimageprocessingtechniqueallowedustoimprovethesignal-to-noiseratioanddistinguishtheproteindensityfromthenoise.

The3DstructureofF0wasreconstructedfromthe2Dcrystalusingsingle-particleanalysis.Theresolutionofthefinalmapwasdeterminedtobe5.5?basedontheFouriershellcorrelation(FSC)curve.Thec-ringwasshowntoformacylinder-likestructurecomprisingeightsubunits.The3Dstructureofthec-ringshowedanunexpectedtiltofapproximately13°relativetothenormalaxis.Furthermore,therewereindicationsofstructuralvariationsintheproteindensitythatwereobservedinsomeregionsofthemap.

Conclusion

Insummary,ourstudypresentsanew2DcrystalstructureofthepigheartmitochondrialF0,alongwitha3Dstructureoftheproteinobtainedfromthecrystal.Wehaveimprovedtheexistingunderstandingofthec-ringsubunitstructureinF0anddeterminedthatithasanunexpectedtiltofapproximately13°relativetothenormalaxis.ThefindingsofthisstudymayprovidenewinsightsintotheworkingmechanismofthemitochondrialF0F1-ATPsynthaseandcouldcontributetothedevelopmentofnewdrugsfordiseasesassociatedwithoxygenenergymetabolism.Discussion

ThestructureoftheF0F1-ATPsynthasehasbeenextensivelystudied,butthestructureoftheF0subunithasbeenlesswellcharacterized,especiallyintermsofthedetailedconformationofthec-ringassembly.Thec-ringislocatedwithintheinnermitochondrialmembraneandfunctionsasaprotontranslocator.TheF0subunitoftheF0F1-ATPsynthaseisconsideredapromisingtargetfordrugdevelopmentagainstdiseasesrelatedtooxidativephosphorylation,suchasmitochondrialdisorders,neurodegenerativediseases,andcancer.

Thepresentstudyobtaineda2DcrystalofpigheartmitochondrialF0andreconstructedits3Dstructurebysingle-particleanalysis.The2Dcrystalpossessedahexagonallattice,andthec-ringwasclearlydistinguishedasasinglelayeroforderedproteinsinthe2Dcrystal.Theproteindensityinthecrystalwashomogenous,andtheresolutionofthe2Dcrystalwasdeterminedtobe3.7?byFourieranalysis.Thereconstructed3DstructureofF0showedthatthec-ringsubunitformedacylinder-likestructurecomprisingeightsubunits.Theresolutionofthefinal3Dmapwasdeterminedtobe5.5?basedontheFSCcurve.

Thetiltofthec-ringsubunitrelativetothenormalaxiswasobservedtobeapproximately13°,whichwasanunexpectedfinding.ThistiltmayhavestructuralimplicationsforthemechanismofprotonconductionandATPsynthesisintheF0subunit.Previousstudieshavesuggestedthatprotontransportacrossthec-ringiscoupledtorotationalmovement,whichdrivesATPsynthesisintheF1subunit.Theunexpectedtiltobservedinthec-ringsubunitmayindicateamorecomplicatedmechanismforthiscouplingthanpreviouslyassumed,andfurtherinvestigationiswarranted.

Thepresenceofstructuralheterogeneityinsomeregionsofthe3Dmapisofinterestforfutureresearch.StructuralvariationsinregionsoftheproteindensitymayindicatelocalconformationalchangesordomainmovementsthatarerelevanttothefunctioningoftheF0F1-ATPsynthase.Itwouldbevaluabletoinvestigatethecauseofthisheterogeneityanditspotentialimpactontheproteinfunction.

Onelimitationofthepresentstudyistheuseofpigheartmitochondriaasthesamplesource.Whilepigheartmitochondriaareacommonlyusedmodelsystem,itispossiblethatthestructureoftheF0subunitcoulddifferinotherspeciesortissues.Futurestudiesusingothermodelsystemscouldhelpaddressthisissue.

Conclusion

Inconclusion,thisstudypresentsa2DcrystalstructureofthepigheartmitochondrialF0subunitanda3Dreconstructionofthec-ringassemblyobtainedbysingle-particleanalysis.Theunexpectedtiltobservedinthec-ringstructuremayhaveimplicationsforthemechanismofprotontranslocationandATPsynthesisintheF0F1-ATPsynthase.Thestructuralheterogeneityobservedinsomeregionsofthe3Dmaphighlightstheneedforfurt