LisaLi

BIO/CHM376
Dr.FredNaider
Outline

AllostericPropertiesofChaperoninGroEL

Introduction

Chaperoninsareaclassofmolecularmachinesthatassistinproteinfolding.Their

structureconsistsoftwobacktobackstackedringswithacavityateachendlinedwith

hydrophobicbindingsitesfornonnativeproteins.Theringsundergocoordinatedrearrangements

suchastiltingandrotatinginresponsetoATPbindingandhydrolysis.Theinteractionsbetween

thetwosevenmemberedringsofthechaperoninGroEL(fromEscherichiacoli)regulatethe

conformationalchangesresultinginthebindingandreleaseofsubstrates,thebinding

cochaperoninGroES,andthealternationofringactivity.Amongtheseinteractionsofthe

GroELGroESsystemarethenestedcooperativityoftheATPasemachineryandnegative

cooperativitybetweentherings.ATPhydrolysisisnotnecessaryfortheencapsulationof

substratebutisneededforthebindingofATPtotheoppositering.Furthermore,ATPbindingis

responsiblefortheejectionofthesubstratefromtheactivering.Here,wedescribetheallosteric

mechanismsoftheGroELGroESsystemwithrespecttoATPandthestructuralbasisforsuch

processestooccur.
Methods

CooperativityinATPhydrolysisbyGroELisincreasedbyGroES

ATPaseassays
GroELandGroESmixturewasincubatedin50mMTrisHCIpH7.78,10mMKCI,10mM
MgCI,and10mMglucoseat25"C.Thereactionwasinitiatedbytheadditionof[814C]ATP
incubatedat25C.Thereactionwasquenchedbyadding4ulaliquotsofthereactionmixtureto
5ulof0.7MLiCI,IMHCOOHonice.Samplesofthequenchedreactionmixturewerespotted
onpolyethyleneiminesheets.Chromatographywasperformedin0.7MLiCI,1MHCOOHto
separatereactionproducts.Afterdrying,ATPandADPspotswerelocated,cutandcounted.
RadioactivitywasexpressedasthefractionofATPhydrolysed.

PositiveCooperativityintheFunctioningofMolecularChaperoneGroEL

GroELandGroESwerepurifiedfromEscherichiacoli.40molofGroELparticlesand600
molofGroESparticleswereincubatedfor15minat25"Cin40lofbufferA(20mM
triethanolamineacetate,100mMpotassiumacetate,10mMmagnesiumacetate,0.1mMEDTA,
2mMDTT,pH7.5)withorwithout2mMadeninenucleotides.Highlypurifiedrhodanesefrom
bovineliverwasmethylatedinthepresenceofNaB[3H]4purifiedbyaffinitychromatography,
andstoredat20Cin0.01Msodiumphosphate,pH7.1,and1mMDTT.Rhodanesewas
denaturedin6MguanidineHCl,10mMDmfor15minatroomtemperature.1l(15mol)of
thedenaturedrhodanesewasaddedto100lofbuffercontaining50mMTrisHC1,35mM
KCl,25mMNH4Cl,10mMMgC12,pH7.5,44molofGroEL,52molofGroES,or1mM
ATP.Afterincubationfor1hat25C,renaturationwasmeasuredbyarhodaneseactivityassay
performedfor30minat25C.

DissociationoftheGroELGroESAsymmetricComplexIsAcceleratedbyIncreased
CooperativityinATPBindingtotheGroELRingDistaltoGroES

SurfacePlasmonResonanceMeasurements
ATPdependentformationanddissociationoftheGroELGroEScomplexwasmonitoredby
surfaceplasmonresonancewithaBIAcore2000apparatus.GroESwasimmobilizedonaCM5
chipbyaminecouplingchemistryusingstandardprotocolswithHBSasabuffer.After
activationwithapreparedmixtureofNhydroxysuccinimide(50mMinwater)and1ethyl3(3
dimethylaminopropyl)carbodiimide(195mMinwater)for4min(flowrate10L/min),GroES
(5g/mLin150mMsodiumacetatebuffer,pH3.8)wasinjectedfor510min(flowrate10
L/min).Injectionof1Methanolaminehydrochloride,pH8.6,for5min(flowrate10L/min).
wasadministeredtodeactivateremainingcarboxylicgroups.Aftercoupling,thebufferwas
changedto50mMTrisHCl(pH7.5)containing1mMDTT,10mMMgCl2,10mMKCl
(bufferA)andATPatdifferentfixedconcentrations.TheassociationofATPboundGroELwith
GroESwasmonitoredbyaddingdifferentfinalconcentrationsofATPto10nMGroELinbuffer
Aandinjectingthissolutionfor5minataflowrateof20L/min.ThedissociationofGroEL
fromGroESwasinvestigatedbyinjectingdifferentconcentrationsofATPinbufferAfor1530
minataflowrateof20L/min.
Significance
AllostericbehaviorsnotonlywithintheGroELringsbutalsobetweenthemarevitalto

theproperactionofthechaperoninmachine.ATPbindingplaysamajorroleinallostery,

directingthechaperoninsactionthroughbindingandhydrolysis.Perhapsmostsignificantly,

ATPisresponsibleforthealternatingbehaviorofGroEL.ATPbindingdrivestheactivityofthe

cisringbyactingthroughtheequatorialregionandthecontactsbetweentheringstoinhibit

foldingactivityinthetransring.Furthermore,ATPbindinginthetransringisresponsiblefor

thereleaseofthefoldedproteinencapsulatedinthecisring.Bystudyingthekineticproperties,

suchaspositiveandnestedcooperativity,andthestructuralbasisforthesemechanismsofthe

GroELGroESsystem,wecanbetterunderstandtherangeofactionsthatthesystemusesto

regulateproteinfolding.

1.Introduction
1.1OverviewofAllostericModels
1.2StructureandFunctionofGroELChaperonins
1.3IntraRingAllostery:PositiveCooperativity
1.4InterRingAllostery:NestedCooperativtity
2.Methods
2.1CooperativityinATPHydrolysisofGroELbyGroES
2.2PositiveCooperativityintheFunctioningofMolecularChaperoneGroEL
2.3DissociationoftheGroELGroESAsymmetricComplexIsAcceleratedbyIncreased
CooperativityinATPBindingtotheGroELRingDistaltoGroE
3.Results
4.Discussion
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ArticlesUsed:
AllostericMechanismsinChaperoninMachines(Primaryreviewarticle)

PuttinghandcuffsonthechaperoninGroEL(Reviewarticleforbackgroundinfo)
TheChaperoninATPaseCycle:MechanismofAllostericSwitchingandMovementsof
SubstrateBindingDomainsinGroEL(Backgroundinfo)

ATPInducesNonidentityofTwoRingsinChaperoninGroEL(Backgroundinfo)

PositiveCooperativityintheFunctioningofMolecularChaperoneGroEL(Article#1)

NestedcooperativityintheATPaseactivityoftheoligomericchaperoninGroEL(Article#2)

CooperativityinATPhydrolysisbyGroELisincreasedbyGroES(Article#3)