Structural and mutational characterisation of Shigella pathogenicity factors [Elektronische Ressource] / vorgelegt von Naomi Tidten

Naomi Tidten

Sujets

• Gesundheit

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 60
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Structuralandmutational
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ofShigellaPathogenicityFactors

Dissertation
zurErlangungdesDoktorgrades
derNaturwissenschaften
(Dr.rer.nat.)
demFachbereichPharmazie
derPhilipps!UniversitätMarburg
vorgelegtvon
Naomi&idten
ausBonn
Marburg/Lahn2007
DieUntersuchungenzurvorliegendenArbeitwurdenaufAnregungvonHerrn
PD Dr. Klaus Reuter und Herrn Prof. Dr. Gerhard Klebe am Institut für
Pharmazeutische Chemie des Fachbereichs Pharmazie der Philipps!
Universität Marburg in der Zeit von Mai 2004 bis Dezember 2007
durchgeführt.
VomFachbereichPharmaziederPhilipps!UniversitätMarburgalsDissertation
am15.02.2008angenommen.
Erstgutachter:Prof.Dr.GerhardKlebe
Zweitgutachter:PDDr.KlausReuter
TagdermündlichenPrüfung:15.02.2008InErinnerunganmeinenGroßvater
EsgibtmehrLeute,diekapitulieren,alssolche,diescheitern.
HenryFord(1863!1947)
Tableofcontents I
Tableofontents
Tableofcontents.......................................................................................... I
FiguresandTables ................................................................................... V
1 Introduction............................................................................................. 1
1.1 Bacillarydysentery–Shigellosis......................................................... 1
Shigellaeclassification........................................................................... 1
1.2 PathogenesisbyShigellae.................................................................. 2
1.3 MolecularbasisoftheShigellainvasionmechanism........................... 6
1.3.1 PathogenicityfactorIpaA,itschaperoneSpa15andthehostcell
proteinVinculin .......................................................................... 7
1.3.2 PathogenicityfactorIpgDanditschaperoneIpgE ........................ 9
1.3.3 PathogenicityfactorOspD1....................................................... 10
1.3.4 PathogenicityfactorIpgB2......................................................... 11
1.3.5 PathogenicityfactorVirF ........................................................... 11
1.4 tRNAguaninetransglycosylase,thegeneproductofvacCisatarget
forstructure!baseddrugdesign...................................................... 12
1.5 Aimsoftheproject ......................................................................... 14
1.5.1 PathogenicityfactorsIpaA,IpgE,IpgB2andOspD1.................... 14
1.5.2 SubstrateselectivityandspecificitystudyoftRNAguanine
transglycosylasesofthethreedomainsoflife,basedona
ZymomonasmobilisTGTmodelsystem..................................... 14
1.6 References ...................................................................................... 16
2 SelectedShigellaPathogenicityFactors................................................... 21
2.1 CloningandExpressionofipaA,spa15,vinc_hd,ipgB2,ospD1,
ipgE ................................................................................................ 22
2.2 PurificationofPathogenicityFactorsIpgB2,OspD1andIpgE ............ 26
2.2.1 IpgB2........................................................................................ 26
2.2.2 OspD1 ...................................................................................... 27
2.2.3 IpgE.......................................................................................... 27
2.3 CrystallisationofPathogenicityFactorsOspD1andIpgE .................. 28
2.4 HomologyModellingofPathogenicityFactorsIpaAandIpgE ............ 29
2.5 SummaryandOutlook..................................................................... 31
2.6 MaterialsandMethods .................................................................... 33
2.6.1 PrimersusedforPCRamplificationofpathogenicityfactorgenes
fromthevirulenceplasmidpCP301........................................... 33
II Tableofcontents
2.6.2 Buffers,Enzymes ...................................................................... 34
2.6.3 CleavageoftheGST!tag ........................................................... 34
2.6.4 Homolgymodelling................................................................... 35
2.7 References ...................................................................................... 35
2353 Glu iscriticalforsubstrateselectivityinTGT ...................................... 39
3.1 Results............................................................................................ 46
3.1.1 CrystalstructureofwildtypeZ.mobilisTGTincomplexwith
guanine .................................................................................... 46
3.1.2 EnzymaticcharacterisationofwildtypeZ.mobilisTGT.............. 47
3.1.3 ConstructionandenzymaticcharacterisationofZ.mobilis
235TGT(Glu Gln) .......................................................................... 48
2353.1.4 CrystalstructureanalysesofTGT(Glu Gln).............................. 49
3.2 Discussion ...................................................................................... 53
3.3 Summary ........................................................................................ 57
3.4 MaterialsandMethods .................................................................... 58
3.4.1 CloningandTGTpreparation .................................................... 58
Tyr3.4.2 PreparationoftRNA ............................................................... 58
3.4.3 Kineticparameters .................................................................... 58
3.4.4 Crystalstructureanalyses ......................................................... 60
3.4.5 ProteinDataBankAccessionCodes ........................................... 62
3.4.6 CrystallographicTables............................................................. 62
3.4.7 Acknowledgements................................................................... 65
3.5 References ...................................................................................... 65
4 CharacterisationofamodelsystemforthehumanTGTbindingpocket .. 69
4.1 Introduction.................................................................................... 69
4.2 Results............................................................................................ 73
4.2.1 Sequencecomparisonsandhomologymodelling....................... 73
4.2.2 ConstructionandenzymaticcharacterisationofZ.mobilis TGT
variants .................................................................................... 75
4.2.3 Crystalstructures...................................................................... 79
4.2.4 Moleculardynamicssimulation.................................................. 84
4.2.5 Co!crystallisationoftheTGTvariantswithpreQ derivatives ..... 87 1
4.3 Discussion ...................................................................................... 90
4.3.1 TheturnovernumbersoftheTGTvariantsdecrease
dramatically.............................................................................. 90
4.3.2 TheturnovernumbersofTGTvariantsincreaseforthesubstrate
basepreQ ................................................................................ 90 1Tableofcontents III
4.3.3 IncreasedK forpreQ .............................................................. 91 M 1
4.3.4 Z.mobiliswild!typeTGTiseventuallyabletobindqueuine....... 93
4.3.5 Outlook .................................................................................... 95
4.4 Summary ........................................................................................ 96
4.5 Supplement..................................................................................... 97
4.5.1 MALDI!TOF/MSanalysisoftheRNAproduct.............................. 97
4.5.2 MALDI!TOF/MSassaydrawbacks ............................................ 100
4.6 MaterialsandMethods .................................................................. 102
4.6.1 Homolgymodelling................................................................. 102
4.6.2 ConstructionoftheTGTmutantsC158V,V233G,C158V!V233G,
A232S!V233G,C158V!A232S!V233G..................................... 102
4.6.3 Expression,Purification........................................................... 103
4.6.4 Determinationofkineticparameters ....................................... 103
4.6.5 MALDI!TOF/MSanalysis.......................................................... 104
4.6.6 Crystallisation,datacollection,andprocessing........................ 105
4.6.7 MolecularDynamicsSimulation ............................................... 110
4.6.8 AlignmentandFigures ............................................................ 111
4.6.9 ProteinDataBankAccessionCodes ......................................... 111
4.7 Acknowledgements ....................................................................... 111
4.8 References .................................................................................... 111
5 Appendix............................................................................................. 119
5.1 X!rayrefinementToolkit!Manual ................................................ 119
5.1.1 Arrangementoffoldersandsubfolders ................................... 119
5.1.2 default.txt............................................................................... 120