生物化學(xué)英文版課件：Chapter 5-4 Protein Function,modulation and evolution-myosin and actin

Chapter5-4

Proteinfunction,modulationandevolution:MyosinandactinMyosinbeingtheprototypeofamolecularmotor,convertingchemicalenergyofATPintomechanicalenergybycyclicalinteractionwiththeactinfilament,generatingforceandmovement.Keyinmusclecontraction;celldivision;organellestreaming,etc.BiochemistryLectureforOct16,2012EMstudiesBiochemicalstudiesProtein-basedmolecularmotorsalloworganismsto“move”atvariouslevelsManythingsaremovinginlivingorganisms,e.g.:Musclecontractioninvertebrates(myosinandactin);Migrationoforganellesalongmicrotubulesandchromosomeseparationindividingcells(kinesinsanddyneins);Rotationofbacterialflagella(acomplexrotationalmotorproteins);DNAmetabolism(helicasesandpolymerases).Viacyclicconformationalchangesofproteins,consumingchemicalenergysuppliedbyATP.Achievingexceptionallyhighlevelsofspatialandtemporalorganization.SarcomeremakethestructuralandfunctionalunitofmyofibrilsZlineStriatedappearance:Alternatinglightanddarkbands;I-band:Zoneofthinfilaments(ofactin)alone;Z-line:themiddleofthinfilaments;A-band:Entirelengthofasinglethickfilament(ofmyosin);M-line:themiddlecross-linkedregionofthickfilaments;Hzone:Zoneofthickfilamentsalone;(TheAbanddoesnotchange,butIbandandHzoneshortensduringmusclecontraction!)

AnisotropicisotropicLeeuwenhoekfirstdiscoveredsuchmusclecross-striationsin1682!Thestriatedmusclesarcomereismadeupofthick(myosin)andthin(actin)filaments,andotherproteinsproteinsfromskeletalmuscle;&smoothmuscle(c)Electronmicrographofalongitudinalsectionofskeletalmusclefiber.CrosssectionsMyosinandactinaretwomajorproteincomponentsofstriatedmuscleG-actinF-actinMyosin:520kDa(twoheavychain,eachof220kDa;fourLightchains,each20kDa)Actin:42kDaCoiledcoilsATPasebindsactinCyclestopshereinrelaxedlivingmuscle(duetoremovalofmyoplasmiccalcium)Ca2+*HighactinaffinityADP+PiADP+PiPiADP*Lowactinaffinity*ATPATPRestingmusclePowerstroke(ATPboundformofmyosin)(ADPboundformofmyosin)Musclecontractionmodel:Powerstrokecausedbycross-bridgeformation,tiltingandsliding.MyosinActinCross-bridgeTiltingandslidingMyosinwasinitially

describedbyWilhelmF.Kuhne(1864)Aviscousproteinwasextractedfromthepressjuiceofthefrogskeletalmusclewithconcentratedsaltsolution.Itwasconsideredtoberesponsiblefortherigorofmuscle.Hecoinedtheterms“myosin”(also“enzyme”).Kuhne(1837-1900)Myosinwasfoundtobeextremelyasymmetric(1930)Muralt&Edsall(1930)Studiesinthephysicalchemistryofmuscleglobulin,IV.Theanisotropyofmyosinanddoublerefractionofflow,J.Biol.Chem.89:351–386.Asindicatedbytheobservationsofdoublerefractionofflowandviscosity.Bothacidandalkali(denaturization)destroyedthedoublerefraction.MyosinwasfoundtobeanATPaseEngelhardtWA,LiubimovaMN.(1939)Myosineandadenosinetriphosphatase,Nature,144:688-189

AcidificationtopHbelow4rapidlydestroysthisactivity;completelylostafter10minat37oC,butthepresentofATPstabilizesit.Engelhardt(1941):EngelhardtATPproposedtobethemoleculethatpromotemusclecontraction(1942)Engelhardt,W.A.(1942)EnzymaticandMechanicalPropertiesofMuscleProteinsYaleJBiolMed.15:21–38.(translated)OnlyATPwasfoundtoproduceamarkedeffectonthemyosinthread.

ATPpromotesmusclerelaxation(notcontraction).Myosin:chemicaltransformerwhichconvertschemicalenergyintomechanicalaction.TwoformsofMyosinswererevealed(1942)MyosinAandmyosinBfrommincedrabbitskeletalmuscle.MyosinBhadamuchhigherrelativeviscositywhichonadditionofATPwasreducedtoavaluesimilartothatofMyosinATheviscosityofmyosinAchangedlittlebytheadditionofATP.(inHungary)MyosinB(renamedactomyosin)threadswasobservedtocontractonadditionofATP!(1942)+ATP(boiledmusclejuice)-ATPThreadofMyosinBThecontributionofonmusclebiologyfromthelabofAlbertSzent-Gyorgyi"Discoveryisseeingwhateverybodyelsehasseen,andthinkingwhatnobodyelsehasthought."AlbertSzent-Gyorgyi

Szent-Gyorgyi(1893-1986)

ByAndrewSzent-GyorgyiNobelPrizeinPhysiologyorMedicine,1937:Forhisdiscoveriesinconnectionwiththebiologicalcombustionprocesses,withspecialreferencetovitaminCandtothecatalysisoffumaricacid.(bothrevelationwasNOTcorrect!)ActinwasdiscoveredasasecondcomponentinMyosinB(1942)ActinwasidentifiedasasecondproteincomponentinmyosinB(Straub,1942),thelatterrenamedas“actomyosin”.IttransformsMyosinAintothecontractileone,thusnamedas“actin”.Itexistsasglobularorfibrousforms(G-actinandF-actin).ThestructureofmyosinwasexaminedviaproteolyticcleavageMihalyiE.&Szent-GyorgyiAG.(1953)Trypsindigestionofmuscleproteins.III.Adenosinetriphosphataseactivityandactinbindingcapacityofthedigestedmyosin.JBiolChem.201:211-9.Loweyetal.(1969)Substructureofthemyosinmolecule.I.Subfragmentsofmyosinbyenzymicdegradation.JMolBiol.42:1-29.Shorttrypsincleavageledtodecreaseofviscosityandgeneratetwofragments:“fast”(heavy)fractioncontainingtheATPaseandactinbindingactivities.Sedimentation:thefastfractionbindsactin!Digestedmyosinalone+actin3minutesafterreachingfullspeed.95minutesafterreachingfullspeed.IsolatedslowcomponentIsolatedfastcomponentFormingfiber-likestructureATPase&actinbindingslowfastslowslowWithATPaseNoATPaseMyosinwasrevealedtocontaintwo(notthree)longpolypeptidechainsSlayterHS,LoweyS.(1967)Substructureofthemyosinmoleculeasvisualizedbyelectronmicroscopy.PNAS.58:1611-8.

Bipartiteheadsofhighflexibility.(singlelobeaftercompletepapaindigestion)TheHeadstructureofmyosinwasdetermined(1993)Raymentetal.(1993)Structureoftheactin-myosincomplexanditsimplicationsformusclecontraction.Science.261:58-65.Raymentetal.(1993).Three-DimensionalStructureofMyosinSubfragment-1:AMolecularMotor."Science

261:50-65.

RegulatoryDomain(leverarm)ThemyosinlightchainsareconsideredtostiffentheboundregionoftheheavychainOriginallythoughttoregulatetheactinbindingandATPaseactivities.Mayfunctiontostiffentheboundregionoftheheavychainandthustoaidinforcegenerationandtransmission.PurifiedmyosinandactinareabletoformfilamentsunderinvitroconditionsPurifiedmyosinformsbipolaraggregates:withthetailstackingoneonanother.(Eachismadeofseveralhundredmyosinmolecules)Purifiedactin(G-actin)associatestoformlongfilamentsorF-actin.G-actinDetailEMstructureanalysisrevealsregularthick-thinfilamentpatterns(1953)HANSONJ,HUXLEYHE.(1953)Structuralbasisofthecross-striationsinmuscle.Nature.172:530-2.Whenmyosinwasextracted,theA-bandsdisappeared.Duringstretch,thelengthofAbanddoesnotchange,butthatoftheIbandshortens.beforeafterbeforeafterHexogonalarrangementofthickandthinfilamentsHughE.HuxleyEMobservationsrevealtheregularpatternchangeduringmusclecontractionandstretch(1954)HuxleyH&Hanson,J.(1954)Changesinthecross-striationsofmuscleduringcontractionandstretchandtheirstructuralinterpretation.Nature.173:973-6.HuxleyA&Niedergerke,R.(1954)Structuralchangesinmuscleduringcontraction;interferencemicroscopyoflivingmusclefibres.Nature.173:971-3.ThelengthoftheI-band,butnottheA-bandchangesduringmusclecontractionandstretch(relaxation)!Actinfilamentsseemtoslideoutoforintothethinkfilaments.Thesamefibril(of4sarcomeres)undergoingATP-inducedcontractionunderinvitroconditions.A-bandI-bandHughE.HuxleySirAndrewFHuxley(NobelPrize1963)Afilamentslidingmodelwasproposedtoexplainmusclecontraction(1957)AnisotropicIsotropicHuxley,A.F.(1957)."Musclestructureandtheoriesofcontraction."Prog.Biophys.Biophys.Chem.7:255-318.Majorobservation:TheAbandmaintainedconstantwhiletheIbandshortensduringthecontractionprocess.Cross-bridgesfoundtoreorientduringcontraction-relaxation(1965)Reedyetal(1965)InducedChangesinOrientationoftheCross-BridgesofGlycerinatedInsectFlightMuscle.Nature,207:1276-1279.Rigorrelaxed(-ATP+ATP)Rigorrelaxed(-ATP+ATP)Tropomyosinwasisolatedfrommuscleasanasymmetricprotein(1946)Bailey,K.(1946)Tropomyosin:Anew

asymmetricproteincomponent

ofmuscle.

Nature.157:368–369.Bailey,K.(1948)Tropomyosin:anewasymmetricproteincomponentofthemusclefibril.BiochemJ.43:271-9.Fibrous:highviscosity(similartomyosin)homogeneousWithasizeofabout90kDa;X-raydiffractionpatternofthea-type;Proposedtobetheprototypeofmyosin(thusthename)CrystalsareformedTroponinwasrevealedasaonewhichallowsthenaturalactomyosintobecontrolledbyCa2+(1963)

Ebashi,S.1963.Thirdcomponentparticipatinginthesuperprecipitationof“naturalactomyosin”.Nature.200:1010.Ebashi,S.,andF.Ebashi.1964.AnewproteincomponentparticipatinginthesuperprecipitationofmyosinB.J.Biochem.55:604–613.Superprecipitationoftrypsin-treated“naturalactomyosin”wasCa2+insensitive;BecameCasensitivewiththenewComponentadded.NewproteinComponentaddedThelargestpolypeptide,TitinwasdiscoveredinmuscleWangetal(1979)Titin:Majormyofibrillarcomponentsofstriatedmuscle,PNAS,76:3698.PresentinM&Zlines,thejunctionsofA&Ibands,andperhapsthroughouttheentireAbands.Withabout27,000aminoacids.(c)SmoothmuscleImmunofluorescentstainingoftitininchickenbreastmyofibrils(titinispresentwidelyinsarcomere)Titin:ExtendsfromtheZdisktotheMdisk.

Titinisthelargestpolypeptidesofarfoundinnature.IgdomainTroponin-C1)F-actin-apolymerofmanyG-actinmonomersF-Actin[TropomyosinTroponin-ITroponin-TG-Actinmonomer2)Tropomyosin（原肌球蛋白）-liesinthegroovebetweenF-actinstrandsandblocksmyosinbindingsitesonactin.3)Troponins(肌鈣蛋白；Tn):Tn-Ibindstoactin&inhibitsactomyosininteraction:Tn-Tbindstotropomyosin;Tn-CbindstoCa2+.AcomplexofmultipleinteractingproteinsonF-actinregulatemyosin-actininteraction.OtherproteinsfoundinmuscleTheyformtheZdisk(e.g.,a-actinin,desmin,andvimentin)andtheMdisk(e.g.,paramyosin,C-protein,andM-protein);organizethearraysofthickandthinfilaments(e.g.,titinandnebulin);

regulatemyosin-actininteraction(e.g.,tropomyosinandtroponin).Nebulin(伴肌動蛋白),having~7,000residues,isthoughttobestructuresasanahelixspendingthelengthofthethinfilament.ThecurrentmodelofmusclecontractionMyosinwithboundATPhasalowaffinityforactin(ATP