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Histidine phosphorylation in proteins - A characterization of phosphohistidines in the bacterial transcription factor GlcT of Bacillus subtilis [Elektronische Ressource] / Sebastian Himmel

129 pages
Ernst-Moritz-Arndt-UnivHistidinedophosphorylationoinderproteinsSebastian-akA(Dr.chenharacterizationvofamphosphohistidinesAprilinhenthererumbacterialnat.)transcriptionh-Naturwissenscfactorakult?tGlcTGreifswofvBacilgeblusinsubtilisGreifswIdesnademiscaGradesuctorgnaturaliumurer.ranaMathematisclhaftlicdFidersersit?tsaldeorgelegtrontHimmelaorent30.03.1982iBeeskownald,zur2011Erlangung2.Dekhkinat...............................................an:......Prof.......Prof.Promotion:Dr.hK.H.Fagesser.....................................Juli1.GutacGutacter:hDr.ter:Osc......Prof.TDr.derK.......13.W2011.......................................................eisz......................................onResultsNo.ofinmyGriesingerwGriesingeroandterkol,wvere122,publishedticationoCorrelationrEd.,contributedS.toer,publications:eisD.inD.andteRo(2010)dr?guez,andC.Protein-PhosphorylationGrosse,throughS.atterns",Himmel,InC.ol.Gonz?lez,8971-8974I.S.M.BecdeLee,Ilarduyha,IdenS.Protein-Becmithilfekorrelationen",er,VG.47,M.C.Sheldric"Detectionk,IdenI.ofUs?nSites"CrystallographicHistidinesabHNPinitioPproteinAngewstructureChemiesolutiontern.bVelo49,w47,atomic(2010)resolution",Himmel,NatureWMethoS.ds,kVD.ol.C.6,"NacNo.w9,und651-653tizierung(2009)onS.PhosphorylierungenHimmel,HistidinenS.vWHNP-Kol,AngewS.Chemie,Becol.kNo.er,9155-9158D.Lee,&&&&&.Intro.duction(Ni-NT7.1.1.Histidine.phosphorylation.in.proteins..........2.2.5...........y............7.1.2.The27bacterial.sugar.Phosphotransferase.System.
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Ernst-Moritz-Arndt-UnivHistidinedophosphorylationoinderproteinsSebastian-akA(Dr.chenharacterizationvofamphosphohistidinesAprilinhenthererumbacterialnat.)transcriptionh-Naturwissenscfactorakult?tGlcTGreifswofvBacilgeblusinsubtilisGreifswIdesnademiscaGradesuctorgnaturaliumurer.ranaMathematisclhaftlicdFidersersit?tsaldeorgelegtrontHimmelaorent30.03.1982iBeeskownald,zur2011Erlangung2.Dekhkinat...............................................an:......Prof.......Prof.Promotion:Dr.hK.H.Fagesser.....................................Juli1.GutacGutacter:hDr.ter:Osc......Prof.TDr.derK.......13.W2011.......................................................eisz......................................onResultsNo.ofinmyGriesingerwGriesingeroandterkol,wvere122,publishedticationoCorrelationrEd.,contributedS.toer,publications:eisD.inD.andteRo(2010)dr?guez,andC.Protein-PhosphorylationGrosse,throughS.atterns",Himmel,InC.ol.Gonz?lez,8971-8974I.S.M.BecdeLee,Ilarduyha,IdenS.Protein-Becmithilfekorrelationen",er,VG.47,M.C.Sheldric"Detectionk,IdenI.ofUs?nSites"CrystallographicHistidinesabHNPinitioPproteinAngewstructureChemiesolutiontern.bVelo49,w47,atomic(2010)resolution",Himmel,NatureWMethoS.ds,kVD.ol.C.6,"NacNo.w9,und651-653tizierung(2009)onS.PhosphorylierungenHimmel,HistidinenS.vWHNP-Kol,AngewS.Chemie,Becol.kNo.er,9155-9158D.Lee,&
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.Intro.duction(Ni-NT7.1.1.Histidine.phosphorylation.in.proteins..........2.2.5...........y............7.1.2.The27bacterial.sugar.Phosphotransferase.System.(PTS).and.the.role.of.a.transcriptional.anytiterminator:.GlcTGST-fusion.33.........23.....agarose...........A8.1.3.Characteristicsofof.phosphohistidines29.......29.......291.....lysis.......Nucleic.ylenimine.Nic.y11.1.4.Detection.of32histidine.phosphorylation..v...trating...............PCR:Contents.........2.2.412electrophoresis1.5.Goal.of.this.dissertationtransformation...........Denaturating...........2.2.7.concen.......Protein..............14Protein2.Materials.&.Metho.ds.16.2.1.GeneralProteinmaterials..........2.5.1...............31.precipitation.oly...31.el-nitrilotriacetic.agarose.hro-...............Purication16.2.1.1.Imp.ortan.tProteolyticcwithhemicals....2.5.6.proteins.........34...................2.2.3.Cloning.Mutagenesis....16.2.1.2.Imp.ortan.t.materials.of25EnzymesDNA.gel.................26.Plasmid..........18.2.1.3.Soft.w.are....2.2.6.SDS-P.GE...................27.Determination.protein.tration.............2.3.sequence........20.2.1.4.Gro.wth.media.able.An.tibiotics......2.4.constructs............................202.52.1.5expressionVPuricationectors..................31.Cell...............................2.5.2.acid.b.p.eth22(PEI)2.2.General.metho2.5.3dsk.acid.A).anit.c.matograph.............................2.5.4.of.proteins............23.2.2.12.5.5DNAcleaplasmidagepuricationTEV-proteaseT....Sequencing......33.Concen.of............23.2.2.2.PCR:.DNA.cloningprimers&
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.of.Con.ten.ts55.2.5.7.Size.exclusionSummaryc5.2hromatograph.yConclusion(gelTheltration)hemistry...Histidine.....I...ecicit..34922.5.8.PRDIwledgement.4.1.....sp.63.....y.......GlcT.....Histidine.....5.5.....w.....112...antiterminato.the...59..34.2.5.9SamplePRDI.I..of.....PRDI...66...............of.c.....PRDI.....PRDI.....I............361,3-diphosphohistidines2.5.10compaRBD-PRDIPRDI.........102.127...57.of.GlcT.osition.phosphorylation.......Regio.PRDI.......62.for.........Quan39phosphorylation2.5.11.Enzyme.I.(EI)64.sites.........Sp.PRDI.........71.................PRDI.antiterminato.5.1.of....41.2.5.12.EnzymeticationIphosphorylationI-AB.(EI.I-AB)..81.sites.........5.4.of........................42.2.5.1391HPrp.Discussion.b.PRDI.94.................8.App.Curriculum.A.......4.PRDI.the.r.58.Comp.of.PRDI.mix......43.2.6.Sp.ecialized4.2methocdsof.phosphohistidines...............4.3.optimization.mass.ectrometry...............4.4.tication.PRDI........45.2.6.1.Phosphoramidate.(P.A)4.5synphosphorylationthesisin.................4.6.ecicit.of.phosphorylation......45.2.6.2.Analytical.gel.ltration.able.Sample4.7preparation.................45.2.6.3.Mass.sp.ectrometry.T...Sample76preparationThe.I.the.r.77.Regio.hemistry.PRDI.phosphohistidines....45.2.6.4.RNA.syn.thesis78(RAQuanTofRNA)I.................5.3.phosphorylation.in.I............47.2.6.584ElectrophoreticSpmobilityyPRDIshiftphosphorylationassa.y.(EMSA)............85.Summary....48.2.6.6.Nuclear.magnetic.resonance.sp.ectroscop.y.(NMR)............649in3roteinsThe7newandHNP-exprisonerimentet51een3.1andSummaryI.7.1.................................99.References.9.endix.10.Vitae.11.ckno.128.δ
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