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Structural and functional studies of the reovirus attachment protein _s631 [sigma-1] and its interaction with the receptor JAM-A [Elektronische Ressource] = Strukturelle und funktionelle Studien am Zelladsorptionsprotein-_s631 [Zelladsorptionsprotein-sigma-1] des Reovirus und seiner Interaktion mit dem Rezeptor JAM-A / vorgelegt von Eva Kirchner

111 pages
2009functionalChemiestudiesDISSERofzurtheKircreodervirusEbattacDoktorshmenvtRezeptorproteinAandakult?t1Pharmazieandersit?tieintsNaturwissenscinorteractionEvwithdemtheJAM-AreceptorTJAM-ATIONStrukturelleFundf?rfunktionelleundStudienderamerhard-Karls-UnivZelladsorptionsproteinT?bingenStructuralErlangungdesGrades1esdesderReohaftenvirusvundgelegtseineronInateraktionhnermitRapapTStehleagterstatter:derterstatter:mBeric?ndlicDr.henBericPr?fung:Dr.26.1.Maih2009Prof.DekT.an:2.Prof.hDr.Prof.L.D.Wortesemann csubsequenisttheviraltheenformedtryatareAbstractandkineyvstepsreceptor,inwvi-umraloinfection.tReceptorer-CARrecognivirusesthiniion1alsotservofesreoanthreeessenThetialhargedfunctionciationintrifugationtargetccuringcelldimerselepublishedc-btion.theMammalianMoreorthoreoD345Nvirusesconforma(reo1viruses)tryareorhighlye.tractableeenexpanderimenexamined.talbmoofdelseforThestudiestheofinvirus-receptorcompriseipntheteractionsonandtheviraldeterminedpathogenesis.theFthermourthermore,ttheyhoshostructurewaspromiseheasbvstateectorsacidsforholdingoncolyticstrimerandvvofaccines.givSimilarlyabtoimaninyccuringotherviralviruses,probablyreoavirusesdetrimerizeduseucell-surfaceurthermore,carbetohattacydrhmenareotwecrystalscomplexesandwa1cellwadhesionprototmoleculandedetermined.astrimersreceptors.culesHoD1whomotheofusagetactoflargemerultipleandrecesuggestingptnoendenrThestconAtrwianalyticalbucomparedteofscomplex.toforcesviraltattacohmenractionsttheisterface.
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2009functionalChemiestudiesDISSERofzurtheKircreodervirusEbattacDoktorshmenvtRezeptorproteinAandakult?t1Pharmazieandersit?tieintsNaturwissenscinorteractionEvwithdemtheJAM-AreceptorTJAM-ATIONStrukturelleFundf?rfunktionelleundStudienderamerhard-Karls-UnivZelladsorptionsproteinT?bingenStructuralErlangungdesGrades1esdesderReohaftenvirusvundgelegtseineronInateraktionhnermitRapapTStehleagterstatter:derterstatter:mBeric?ndlicDr.henBericPr?fung:Dr.26.1.Maih2009Prof.DekT.an:2.Prof.hDr.Prof.L.D.Wortesemann









csubsequenisttheviraltheenformedtryatareAbstractandkineyvstepsreceptor,inwvi-umraloinfection.tReceptorer-CARrecognivirusesthiniion1alsotservofesreoanthreeessenThetialhargedfunctionciationintrifugationtargetccuringcelldimerselepublishedc-btion.theMammalianMoreorthoreoD345Nvirusesconforma(reo1viruses)tryareorhighlye.tractableeenexpanderimenexamined.talbmoofdelseforThestudiestheofinvirus-receptorcompriseipntheteractionsonandtheviraldeterminedpathogenesis.theFthermourthermore,ttheyhoshostructurewaspromiseheasbvstateectorsacidsforholdingoncolyticstrimerandvvofaccines.givSimilarlyabtoimaninyccuringotherviralviruses,probablyreoavirusesdetrimerizeduseucell-surfaceurthermore,carbetohattacydrhmenareotwecrystalscomplexesandwa1cellwadhesionprototmoleculandedetermined.astrimersreceptors.culesHoD1whomotheofusagetactoflargemerultipleandrecesuggestingptnoendenrThestconAtrwianalyticalbucomparedteofscomplex.toforcesviraltattacohmenractionsttheisterface.stilltunclear,complexandwithgeneralofrulesvirusforThereceptorwrecogniticomplexesonofatasparticanandatomicforceslevtheel1hatogether.voeer,notterpretationytheetstructurebeseentsestablished.outIntthisonalthesis,hangesstructuralofpropoertiedursngandenreceptor,bindinginmecolvinghanismspartlyoffullythetreomolecviruslattacFhmencomplexestbproteinwlsthe1hmenwproteinere1analyzed.thecelvirus1JAM-A,coneretaiThenstructuresstheanformedunetueensalattactrimerizationproteinsmotif,ttheoasparticvirusacidypcluster,strainsatJAM-AitseretrimerThein1terfacengagee.moleAofhostJAM-A1domainproteithen,dimericinterfacewhicJAM-A.hcononeareasofathenasparticbacidsofofolarthecclusterresidues,wthatasimteractionutateddeptotasparaginepH.(D345N),dissowconstanasofanalyzedJAM-regardinghombdimeriasndusingiultracenngandtowiththeonereothevirus1-JAM-AreceptorTheJAM-A,dynamicandoitsinchetow1indisruptedimersdemonstratenwtorcanystalJAM-AstructureiworderasengagesolvJAM-AedintoThehighofresolution.heThe1-JAM-AanalysiswofcomparedthetheD345Nstructuremtutanadenotbprocomplex.videssimilaritiesinformationetabeenoutoththeIprotonationcell-surfacesuppcellortunderstandingthestrategieststudieshehanismsorandyvirusesofogether,antoevtheolutionaryreolinkhmenbealetbwengageeenIttheseheaddcelltheattacofhmenmectofmecvirushanismsattacusedtbrevycommonreousedvirusyantodtheiradenoreceptors.virus.IT

sitets17Abstract.I.Abbreviations.VI.1dIn.tro.duction.1.1.12.1.2Viruses7.............15...lysis.............................ds.........General.....y............1.1.1.1.Dandenition18andunctionstructure......JAM.......1.3.3.................2...........dw....1.1.1.2.Itcosahedral.symmetry..y...........c.......and.......and.....2.1.3.2.....2.1.3.3.......1.3.12JAM-A1.1.3.V.iral.life.cycle....11.structure...............cted.1...........1.4.....................and.2.13.1.2.Reo.viruses............H.................15.ose.........2.1.1.2.c.........2.1.1.3.y...........grap.w..4.1.2.1.Reo.virus.structure..Ki...............Concen.l...........a.culture.........puricatio.........I4.1.2.211IFnfectionofof.cells..........................1.3.2.-A.............................116Predi1.2.2.1bindingAfortenttac.hmen.t.and.en.try............13.Aims..........................6.1.2.2.2.Disassem.bly.and.transfer.to14theMaterialscytoplasmMetho.15.Materials..............6.1.2.3.The.attac.hmen.t.protein.of.reo.virus....2.1.1.ar.are............................8.1.2.3.1.The2.1.1.1structurepurpofequipmenCon..1..............15.Cell.and.hromatograph.................16.X-ra.crystallograph..8.1.2.3.2.The.aspartic.acid.cluster........16.Crystallo.hi.soft.are......................9.1.2.42.1.3Reotsvirusesconsumablesas.therap.eutic.agen.ts................1.2.1.3.1.trators..ters................10.1.317JunctionalCloningadhesionnmolbacterialecule-A................17.Protein.n.analysis...............I.I.














the.42...3.2.....Structural.screens..18.2.1.4alPl.asmids.and.primers........Data.........tation...T3D...1.....49.p.......ys..........18.2.1.4.1.Plasmids41...v.....D345N.......D345N.D345N.........the.....com.stabilit...........Crystallization....18.2.1.4.2.Primers........2.2.3.4.....2.2.3.5.............e.....41.y.....ts...3.1.........Purication18.2.2.Methoofds..of.....Binding.....com.......49.compl...3.2.1.1.D1...the...51.T3D...Structure...crystallograph.............29.crystal.......2.2.3.2.data19.2.2.1.M.olecular.biology31.cessing.............determinatio...........renemen...........2.2.4.trifugation...........Sample..19.2.2.1.1.Comp.eten.t.cSedimenellsci.erimen.......2.2.4.3.exp.......Results...................45.crystallization.1....19452.2.1.2functionalGlycerol.sto.c.ks3.1.2.1...........47.T3D.to.........l...............Purication......19.2.2.1.3.Puricationofofandplasmid.DNA....Generation.Crystallization.l.....Structure.of.1-JAM-A.ex...51.n.comple...IV19y2.2.1.4.PCR,.agarose.gel.electrophoresis,.and.PCR.purication.....20.2.2.1.5.Restriction,.ligation2.2.3.1andandtransformationfreezing.............29.X-ra.and.collection21.2.2.1.6.Expression.of.recom.binan.t.proteins....2.2.3.3.pro....................21.2.2.2.Protei33nStructurepuricationnand.analysi.s..............35.Structure.t.....................3822Analytic2.2.2.1ultracenGeneral......................2.2.4.1.pr.paration.......................2.2.4.2.tation.elo.t22exp2.2.2.2tsAssessmen.t.of.protein.concen.tration..41.Sedimen.equilibrium.erimen.............3.4522T3D2.2.2.31SDS-P.A.GE..............................3.1.1.and.of...D345N...........3.1.2.and2analysis2T3D2.2.2.41Purication.of.T3D...147.Structure.T3D.1...................3.1.2.2.of...D345N.JAM-A....23.2.2.2.5.Purication.of48JAM-AT3D.1-JAM-A.p.ex.............................3.2.1.of.T3D251-JAM-A2.2.2.6exPurication.of.the.T3D...1-JAM-A.D149complexPurication.T3D.1.JAM-A......27.2.2.2.7.Purication.of3.2.1.2theofT1LT3D.1-JAM-A1-JAM-ApD1excomplex........3.2.2.and.y27the2.2.2.82.1.3.4Analyticalcomscalelsize-exclusion...1-JAM-A.x.513.2.2.1OvdeterminatiostructureoftheT3D.ccomplehromatograph.y3.2.2.2.erall.of.T3D.1-JAM-A.x..28.2.2.352X-ray














......71..54.3.2.2.4.StabilitMutationsy4.2.4.2of.the.T3D.terface.1-JAM-A.com.p.l75ex..ok...D345N.T3D.....ts...Disruption..56reo3.3.T1L.in741-JAM-A.compleer-CARxfor.e...sequences...d.............85...References.101.......dimers.....hmen.1.....1-JAM-A....58complexes3.3.1.Purication.ofthethe.T1Las1-JAM-A.1supcomplexviral.....endix.......Crystallographic.....6.2.1...............com58.3.3.2.Structure.determinationtrifugationof.the.T1L.T3DA1-JAM-AcademiccompleVx..........71.JAM-A59.3.3.3.Structure.of.the.T1LCelltheb1-JAM-AT3Dcomple.x......73.the.terface.........Related..........61Comparison3.3.3.1virusStructure.of.T1Landinp1viruses...4.2.5.mem.s.eptors.......5.6.6.1.structures...........81.a.qualit..61.3.3.3.2.The.T1L.tacts.1-JAM-A.in.terface........82.............6.2.3..l........63.3.4.StabilitAnalyticaly.of.the.JAM-A.homo.dimer..86.Publications.kno.0.her.vitae.........................4.2.1.of.homo..65.3.4.1.Mixi.n.g.of.JAM-A.D1.and.D1D24.2.2.attac.t.y.virus.....................4.2.3.in...in....65.3.4.2.Analytic.al.ultracen.trifugation....4.2.4.virus-receptor.....................4.2.4.1.to.adeno.b.complex.....7566JAM-A4CARDiscussionrece67tors4.1otherThe.aspartic.aci.d77clusterIg.erfamily.b.r.as.rec...............77.Outlo.79.App.81.Protein.and..................67.4.1.1.Mutations.in6.2thestatisticsasparticnacidstructuresandwicyh..............82.T3D.1............67.4.1.2.Comparison.of.T1L.and.T3D.Con.1..6.2.21-JAM-Ax.comple.........................83.T1L.1-JAM-A.p.ex....68.4.1.3.The.aspartic.acid.c.lust.e.r.as.a6.3molultracenecular.switc.h......................7.8768994.2cThewledgmenreo10virusA3.2.2.3teac1-JAM-AscomCurriculump102lex
immTheicommonlyBertaniusedS-transferaseabbreviationsvirionare2-methusedliquidforethcisopropheKmicalmanddiaphfelineysicalGSHunits,HEPEStheacidcIghemicalISVPelemenJAM-Ats,constantheelihoessen2-(N-morpholino)tiNcoallinaaminoacidacidsfastashromatographwGSTellhasydrotheerazbaseshodeciencyccurringunoglobulinin-D-1-thiogalactopDNA.termediateAtidadhesionadenodissovirusLBAPSlogammoniumdpurineersulfateacidASUenasymmetriccunitorCARtcocxsacfkievirusFPLCandproteinadenocvirusyreceptorglutathioneCCDglutathionechharge-coupledumand4-(2-hexyviceyl)-1-pipCHESineethanesulfonicN-cyclohexyl-2-aminoHIVethanesulfonicumanacidunoCNSviruscenimmtralIPTGnervylAbbreviationsousyranosidesysteminfectious/inDNAsubdesoparxyribcleonjunctionaluclmolecule-AeicDacidciationdNTPtdesoLuriaxynLLGucleotideliktriphosphatesodsgaindouble-strandedmE.MEScethanesulfonicoliMPDEscyl-2,4-pherictanediolhiaNocoliarEDTcAalethVIylenediaminetetraaceT3DNCSSourcenon-crystallographicrissymmetryeloODT600ypopticalSSMdensit1yngatTEMED600violetnmSLSPCRoptimalpSVolT1Lymeraseypc3haintempreactionTPEGTfBpyl)amiolystomatitisethholcicyleneLighglycolsupPfussPyrmatcotationcitoypcT2Jcus2furiosusypPMSFarphenAylmethTylsulphonEDTylThermusuorideylethr.buerm.ydros.UVd.vrowtotXhomeanVIsquareSwissdeviationtRNASOBriberonbrothucleicsingle-strandedacidsecondary-structurerpmhingrsedimenevvcolutionsyptereminLangutetReTJonesrotometempDeeratureiSATsialicannealingacideratureSAPAEshrimpris-acetatealkAalineaqphosphataseaquaticusSDS-PtetramethAylenediamineGEtransformingsoTdiumtris(hdoxymethdecylnomethanesulfateultrapVSVolyesicularacrylamidevirusgelwild-telectrophoresiseSEXanthomonassedimenolatationIequilibrium

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