中山大學(xué)生物化學(xué)課件中山大學(xué)生物化學(xué)課件中山大學(xué)生物化學(xué)課件§4ProteinFunction§4.1Generalfeatures§4.2Oxygen-bindingproteins§4.3Immunoglobulins§4.4Musclecontractionliguofu§4ProteinFunction§4.1Generalfeatures§4.2

Oxygen-bindingproteins§4.3Immunoglobulins§4.4Musclecontractionliguofu§4.1GeneralfeaturesVersatileinfunctionBeinghardtostudyFunctionviainteractionTheinteractionwithothermolecule(ligand)isreversible.Theinterfacebetweenthebindingsiteiscomplementaryinstructure,makingsuchinteractionhighlyspecific.Structuredynamicnessofaproteinisusuallyessentialforsuchinteractions.liguofu§4.1Generalfeatures§4.2

Oxygen-bindingproteins§4.3Immunoglobulins§4.4Musclecontraction§4ProteinFunctionliguofuQuantitativedescriptionofinteraction(1)Protein+nLigand

PLnliguofuQuantitativedescriptionofinteraction(2)IfkdisconstantliguofuQuantitativedescriptionofinteraction(3)IfkdisnotconstantKdliguofuQuantitativedescriptionofinteraction(4)liguofuQuantitativedescriptionofinteraction(5)liguofuThe

iron-porphyrin

inhemoglobin

accountsforthe

red

colorofblood,

thecopper-porphyrin

inhemocyanin

forblue

colorofblood,andthemagnesium-porphyrin

inchlorophyllisresponsibleforthe

green

ofplants.

liguofuStructureofPorphyrinPyrroleringMethenebridgeliguofuOxygencanbeboundtoaheme(1)Heme=ProtoporphyrinIX+Fe2+

NoneoftheaasidechainsinproteinsissuitedforreversiblebindingO2Heme

prostheticgroupTransitionmetals,Fe&Cu,haveastrongtendencytobindO2needmorecommonlyliguofuOxygencanbeboundtoaheme(2)Heme=ProtoporphyrinIX+Fe2+

FreeIronpromotestheformationofhighlyreactiveoxygenspeciessuchashydroxylradicals.CoordinatedNatomshelppreventtheconversionofFe2+toFe3+liguofuMbhasasinglebindingsiteforoxygenMyoglobin:153residues,16700DaHis93/F8His64/E7liguofuOxygencanbeboundtoaheme(3)Infreehememolecules,reactionofoxygenatoneofthetwo“open”coordinationbondsofironcanresultinirreversibleconversionofFe2+toFe3+Inheme-containingproteins,thisreactionispreventedbysequesteringthehemedeepwithinaproteinstructurewhereacesstothetwo“open”coordinationbondsisrestricted.Oneofthesetwocoordinationbondsisoccupiedbyaside-chainNofaHisresidue.NOCOAlsoliguofuQuantitativedescriptionofMbbindingO2liguofuProteinstructureaffectsligandsbind(1)InfreehemeorInmyoglobinInfreehemeInmyoglobinhemeorliguofu“Breathing”:MolecularmotionsThebindingofO2tohemeinmyoglobindependsonits“breathing”InmyoglobinProteinstructureaffectsligandsbind(2)liguofuHbisthemost-studiedandbest-understoodproteinHemoglobinwas

thefirstproteintobecrystallized(in1849);thefirsttobeassociatedwithaspecificphysiologicalfunction(around1875);oneofthefirstproteinstohaveitsmolecularweightdeterminedcorrectly(64,500);thefirsteukaryoticmessenger(mRNA)tobeisolatedandsubsequentlysequenced.thefirsteukaryoticproteintobesynthesizedinacell-freesysteminvitro;thesecondproteinhavingits3-Dstructuredetermined(1969).liguofuThe“redcoloringmatter”(hemoglobin)ofanimalbloodcouldbebroughttocrystallization,withcrystalformscharacteristicoftheirbiologicalorigins(1840s-1860s).Reversibleinterconversionofoxyhemoglobinanddeoxyhemoglobinwasrevealedbyusingspectroscope(1860s).Treatmentofhemoglobinwithacidgaveacolorlessalbuminoidconstituent(globin)andarediron-containingmaterial(by1870).Thestructureofhemewaselucidatedtobeatetrapyrrol(porphyrin)derivativebychemicalsynthesis(HansFischer,1929).Similartetrapyrrolderivativesarealsousedasprostheticgroupsofotherproteins(e.g.,thecytochromesthatfunctioninbiologicaloxidationandphotosynthesis!liguofuHbhasfoursubunitsMr=64,500141residues146residuesliguofuHbsubunitsarestructurallysimilartomyoglobin

subunitlackstheshortDhelixDhelixliguofuconservedinallknownglobinsconserved27positionsareidentical,~18%82143liguofu30residuesInteractionsinthefoursubunits19residues19residuesHydrophobicinteractionsHbondsSaltbondsliguofuThisiscalledTensestate(deoxyHb)SomeionpairsarenotshownhereIonpairsatthe1/2and2/1interface(1)liguofuIonpairsatthe1/2and2/1interface(2)IonpairsstabilizetheTstateofdeoxyHbliguofuQuantitativedescriptionofHbbindingO2（1）liguofuHillplotnH:hillcoefficientQuantitativedescriptionofHbbindingO2（3）liguofuHbundergoesastructuralchangeonbindingO2(1)TensestateismorestableandthusthepredominantconformationofdeoxyHbRelaxedstateisthepredominantconformationofhigheraffinitytoO2BindingO2ValE11liguofuIntheTstate,theporphyrinisslightlypuckered,causingthehemeirontoprotrudesomewhatontheproximalHis(HisF8)side.ThebindingofO2causesthehemetoassumeamoreplanarconformation,shiftingthepositionofHisF8andFhelix.HbundergoesastructuralchangeonbindingO2(2)liguofuAllostericproteinisoneinwhichthebindingofaligandtoonesiteaffectsthebindingpropertiesofanothersiteonthesameprotein.Modulatorisamoleculethatbindstoanallostericproteinandaffectsitsbindingproperties.

Homotropic:thenormalligandandmodulatorareidentical.

Heterotropic:thenormalligandandmodulatorarenotidentical.Someproteinshavetwoormoremodulatorsandthereforecanhavebothhomotropicandheterotropicinteractions.HemoglobinbindsoxygencooperativelyliguofuTwomodelforcooperativebindingConcertedModelMWCModelSequentialModelliguofuN-terminalresidueCarbamino-terminalresidueH+HemoglobintransportsCO2carbonicanhydrase15%to20%80%to85%liguofuHemoglobinalsotransportsH+BohreffectH+bindsto:NHofHis146inb4N-terminalNH2Others:Arg,Lys…liguofuO2bindingtoHbisregulatedbyBPG(1)liguofuO2bindingtoHbisregulatedbyBPG(2)liguofuBindingofBPGtodeoxy-HbstabilizestheT-state,thuslowerstheO2affinityofHb.

OnlyoneBPGbindstoeachdeoxy-HbtetramerliguofuTheBPGbindingpocketexistsintheT-state,butdisappearsintheR-stateofHb.T-state(deoxy-Hb)R-state(oxy-Hb)PositivelychargedgroupsthatBPGinteractswithInfetushemoglobin(a2g2),theconversionofb-His143tog-ser143resultsinthedecreaseoftheaffinityofBPGMutantHbsprovidedauniqueopportunitytostudystructure-functionrelationshipsinproteinsMany(~500)Hbgeneticvariants,mostly(~95%)withasingleaminoaciddifference,havebeenidentifiedinthehumanpopulation.Mostofsuchvariationhaveaveryminoreffectontheproteinstructureandfunction.Somedocausedebilitatingdiseases(e.g.,thosefromsicklecellanemia.)Deleteriousmutationsaremostlyclusteredaboutthehemepocketsandinthevicinityofthea-bcontactregionthatisimportantintheallosterictransition.liguofuDistributionofmutationsinhumanhemoglobin

Glu→ValSicklecellanemialiguofuliguofuSicklecellanemiaiscausedbyasingleaminoacidreplacementonthebsubunit:Glu6→ValliguofuGlu→ValliguofuliguofuElectronmicrographofdeoxy-HbSfibersspillingoutofarupturederythrocyte.Thesickledcellsarefragile.Theirbreakdownleadstoananemiathatleavesthevictimsusceptibletoinfectionsanddiseases.liguofuTheelongatedcellstendtoblockcapillaries,causinginflammationandconsiderablepain

liguofuByLinusPaulingin1949.19031987NormalTraitPatient(Normal)(defective)liguofuTheHbSalleleconfersasmallbutsignificantresistancetomalariainheterozygousindividualsTheHbSallele(i.e.,sicklecelltrait)wasfoundcommonincertainpartsofAfrica.Plasmodium(aprotozoancausingmalaria)increasestheacidityoferythrocyte,favoringtheformationofdeoxy-HbS(theBohreffect),thusprobablyallowingthespleentopreferentiallyremovetheinfectedredcells.liguofu§4.1Generalfeatures§4.2

Oxygen-bindingproteins§4.3Immunoglobulins§4.4Musclecontraction§4ProteinFunctionliguofuliguofuStructureofimmunoglobulin(1)liguofuStructureofimmunoglobulin(2)H=HeavychainL=LightchainC=ConstantV=VariableCHO=carbohydrate

liguofuStructureofimmunoglobulin(3)liguofuStructureofVariableRegion(1)LightChainHeavyChainCDRs:complementarity-determiningregionsHypervariableloopsliguofuStructureofVariableRegion(2)HeavyChainliguofuAntibodybindtightly&specificallytoantigen(1)SmallantigenliguofuSmallantigenAntibodybindtightly&specificallytoantigen(2)Largeantigen:lysozymeAntibodyIAntibodyIIVC1liguofuliguofuFiveclassesofimmunoglobulins(1)liguofuliguofu抗體的作用：例liguofuPolyclonal/Monoclonalantibodyliguofuliguofu§4.1Generalfeatures§4.2

Oxygen-bindingproteins§4.3Immuneproteins§4.4Musclecontraction§4ProteinFunctionliguofuMusclestructureliguofuMusclestructureliguofuMusclecontractionliguofuMusclecontractionliguofuActinmoleculesTropomyosinmoleculesTroponinmoleculesCrossbridges(headgroupsofmyosinmolecules)ActinTropomyosinTroponinMyosinmoleculeheadgroupsSplitbypapainThinfilamentThinfilamentThickfilamentliguofuMyosin(1)(Twoheavychain)(four)Mr=540,000MrH=220,000MrL=20,000~325nmliguofuMyosin(2)liguofuMyosin(3)：S1structureliguofuMyosin(3)：S1structureliguofuGactin

Factin(1)liguofuGactin

Factin(2)=Ca2+orMg2+liguofuGactin

Factin(3)liguofuInteractionbetweenmyosinandF-actin

liguofu

Step1Step2The“powerstroke”modelofmusclecontraction(ATP-consumingMotor)Twoconformationsofthecross-bridgeweredetectedininsectmuscle(1965)liguofuStep3Step4Step1liguofuTropomyosin:lyingalongthegrooveintheF-actinhelix.Troponin:TnC,TnI,TnTTnC:theCa2+bindingsubunitTnI:th