Molecular analysis of adenylyl cyclase [Elektronische Ressource] : Bacillus anthracis edema factor exotoxin / presented by Hesham Hamada Taha Mohammed
179 Pages
English
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Molecular analysis of adenylyl cyclase [Elektronische Ressource] : Bacillus anthracis edema factor exotoxin / presented by Hesham Hamada Taha Mohammed

Downloading requires you to have access to the YouScribe library
Learn all about the services we offer
179 Pages
English

Description

The experimental part of this work was carried out between September 2006 andOctober2009attheDepartmentofPharmacologyandToxicologyattheUniversityofRegensburgunderthesupervisionofProf.Dr.R.Seifert. ThePhD–thesiswassubmittedon:October2009Thecolloquiumtookplaceon:27.November2009BoardofExaminers: Prof.Dr.J.Schlossmann(Chairman)stProf.Dr.R.Seifert (1 Expert)ndProf.Dr.S.Elz(2 Expert)Prof.Dr.A.Göpferich (Examiner)Acknowledgement First and foremost, I would like to thank God for blessing me with thisopportunitytopursuemyPhDintheGermany.IthankHimforsupportingmethroughthebestandtoughestyearsofmylife.Next,IamextremelythankfulandgratefultomysupervisorProf.Dr.RolandSeifert for his invaluable guidance, support and for sharing his expertise andresearchinsight.Prof.Seiferttaughtmetheaccuracyintheworkandseekingfortheexcellence.Thanksforbelievinginmeduringthetoughesttimesandboostingmymorale to do better every day.

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Published 01 January 2009
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Exrait



The experimental part of this work was carried out between September 2006 and
October2009attheDepartmentofPharmacologyandToxicologyattheUniversityof
RegensburgunderthesupervisionofProf.Dr.R.Seifert.






















ThePhD–thesiswassubmittedon:October2009


Thecolloquiumtookplaceon:27.November2009





BoardofExaminers: Prof.Dr.J.Schlossmann(Chairman)
stProf.Dr.R.Seifert (1 Expert)
ndProf.Dr.S.Elz(2 Expert)
Prof.Dr.A.Göpferich (Examiner)

Acknowledgement


First and foremost, I would like to thank God for blessing me with this
opportunitytopursuemyPhDintheGermany.IthankHimforsupportingmethrough
thebestandtoughestyearsofmylife.

Next,IamextremelythankfulandgratefultomysupervisorProf.Dr.Roland
Seifert for his invaluable guidance, support and for sharing his expertise and
researchinsight.Prof.Seiferttaughtmetheaccuracyintheworkandseekingforthe
excellence.Thanksforbelievinginmeduringthetoughesttimesandboostingmy
morale to do better every day. Thanks for helping me develop my presentation,
writingandresearchskills.IamdeeplygratefultohimandIwouldn’thavebeenable
to fulfil this dream without his constant encouragement and support. I hope that I
havethechancetocontinueworkwithhimforever.

I am deeply grateful to Prof. Dr. Frieder Kees for helping me initiating the
project,forhelpingmeintheproteinpurificationandhelpingmerealizewhatIwas
capableofandencouragedme.

IwouldliketothankProf.Dr.J.Schlossmann,Prof.Dr.S.ElzandProf.Dr.A.
Göpferich(InstituteofPharmacy,UniversityofRegensburg,Germany)forbeingpart
oftheexaminationboard.

IwouldliketothankalsodueallthecollaboratorsatvariousstagesofmyPhD
studies. I also wish to thank Dr. Wei!Jen Tang (Ben!May Institute of Cancer
Research,UniversityofChicago,Chicago,IL,USA)forkindlyprovidinguswithEF,
EF3,EF3F586AbacteriaandvariousmutantsEF3proteins.

IwouldalsoliketothankDr.StefanDove(DepartmentofPharmaceuticaland
MedicinalChemistryII,UniversityofRegensburg,Germany)forprovidingmewithall
thehelponmolecularmodeling.IwouldlikealsotoacknowledgeDr.Burkhardkönig
and my colleague Dr. Jens Geduhn (Institute of Organic Chemistry, University of
Regensburg, Germany) for providing us with various purine and pyrimidine
substitutednucleotides.


IAcknowledgement


I would like to acknowledge and extend my appreciation to the following
personswhohavemadethecompletionofthisthesisispossible:

Dr. R. Schupfner (Center for Chemical Analysis, University of Regensburg,
Germany)forthesupportinhandlingofradiochemicals.

Dr.E.SchneiderandDr.K.Wenzel!Seifert(InstituteofPharmacy,University
ofRegensburg,Germany)forinspiringscientificdiscussions.

Mrs. R. Prenzyna, Mrs. A. Seefeld, Mrs. S. Brüggemann, and Mrs. K.
Wohlfahrt(InstituteofPharmacy,UniversityofRegensburg,Germany)forsupport,
understandingandexperttechnicalassistance.

MycolleaguesMrs.H.Appl,Ms.I.Brunskole,Mr.M.Desch,Ms.M.Erdorf,
Ms. S. Geiger, Mr. B. Hieke, Ms. M. Hübner, Ms. N. Pop, Ms. K. Salb, Ms. E.
Schinner,Mr.D.Schnell,Ms.A.SchrammandDr.M.Göttleforcontributingtothe
friendlyatmosphereinourgroup.

I don’t have enough words to describe how grateful I am to my parents.
Withoutthem,mydreamtobecomearesearcherwouldalwaysbeunfulfilled.They
havebeentheguidingangelsinmylife,helpedmerealizewhatIwascapableof.
Theyencouragedme,helpedmeachievemygoalsandtostayfocused,inshort,I
owethemallmysuccess.

Ithankallmyfamily,mysisters(Shereen,Ashgan),mybrother(Mohammed)
for being very supportive and proud of my achievements. In particular, I would
specially like to acknowledge my father!in!law Mr. Abdelwahab for his constant
encouragementthatwillalwayshelpmeremaingroundedandworkevenharderto
achievemygoals.

I would like to thank my colleagues at the Department of Biochemistry,
Facultyofpharmacy,Al!AzherUniversity,Egyptforthecontinuousencouragements.
Also,IwouldliketothanktheEgyptiangovernment, MinistryofHigherEducation,
andCulturalaffairsmissionforthefinancialsupportofthescholarship.
IIAcknowledgement


Lastly,andmostimportantly,IthankHager,mywife,andmykidsforbeing
mysourceofstrengthwithoutthemthisjourneywouldhavebeenincomplete.Iam
extremelygratefultomywifeforalwaysbeingthereforme,forbelievinginmeand
mycapabilities,andforherlove.




































IIIAbstracts and publication


Abstracts and Publications

Parts of this thesis were published or presented as posters or short lectures:

Original Publications:

2008

Taha, H.M.,Schmidt,J.,Göttle,M.,Suryanarayana,S.,Shen,Y.,Tang,W.J.,Gille,
A.,Geduhn,J.,König,B.,Dove,S.,Seifert,R.(2009)Molecularanalysisofthe
interactionofanthraxadenylylcyclasetoxin,edemafactor,with2´(3´)!O!(N!
(methyl)anthraniloyl)!substitutedpurineandpyrimidinenucleotides.MolPharmacol
75:693!703.

2009

In preparation:

Taha, H.M.,Shen,Y.,Tang,W.J.,Gille,A.,Geduhn,J.,König,B.,Dove,S.,Seifert,
R.(2009)Molecularanalysisoftheinteractionofanthraxadenylylcyclasetoxin,
edemafactor,withbis!2´,3´!O!(N!(propyl)/(methyl)anthraniloyl)!substitutedpurine
andpyrimidinenucleotides.












IVAbstracts and publication


Poster presentations:

2008

Molecularanalysisoftheinteractionofanthraxadenylylcyclasetoxin,edemafactor,
with2´(3´)!O!(N!(methyl)anthraniloyl)!substitutedpurineandpyrimidinenucleotides
Taha, H.M.,Geduhn,J.,König,B.,Dove,S.,Seifert,R.
SymposiumausAnlassderVerabschiedungvonProf.Dr.med.K.Reschunddes
DienstantrittsvonProf.Dr.med.R.SeifertalsDirektordesInstitutsfür
PharmakologieanderMedizinischenHochschuleHannover,Hannover(Germany),
November2008

2009

Synthesisofahydrolyticallystable,fluorescent!labeledATPanalogasatoolfor
probingadenylylcyclases
1 2 3 3 2 1 Emmrich T. , El!Tayeb A. ,Taha H.M. ,Seifert R. ,Müller C.E. ,Link A .
DPhG!DoktorandentagungFürStudentenundDoktoranden,Irdning(Austeria),
November2009.

Short Talk:

2009

Molecularanalysisoftheinteractionofanthraxadenylylcyclasetoxin,edemafactor,
withBis!2´,3´!O!(N!(propyl)/(methyl)anthraniloyl)!substitutedpurineandpyrimidine
nucleotides.
50.JahrestagungderDeutschenGesellschaftfürExperimentelleundKlinische
PharmakologieundToxikologie(DGPT),Mainz(Germany),March2009







VContents


Contents

General Introduction 2I
21 Adenylyl Cyclases
1.1 Mammalian Adenylyl Cyclases 2
1.2 Bacterial Adenylyl Cyclase Exotoxins 3
1.2.1 Anthraxadenylylcyclasetoxin,edemafactor 3
Anthrax!Biologicalwarfareandincidenceindeveloping
1.2.1.2 8
countries
Anthraxtripartitetoxinandmechanismoftoxintranslocation
1.2.1.3 10
intohostcells
1.2.1.4 Anthraxtoxincomponentsandtheirfunction 13
1.2.1.5 EdemaFactor!structureandintracellularactivation 14
1.2.1.6 CatalyticmechanismofEF 19
Challenges,limitationandfuturedirectionsofanthraxresearch
1.2.1.7 22
withspecialfocusonEF
Nucleotide-analogues as inhibitors for mammalian and
1.3 23
bacterial adenylyl cyclases
1.3.1 Anthraniloyl!substitutedpurineandpyrimidinenucleotides 24
1.4 Fluorescence spectroscopic studies of adenylyl cyclases 26
VIContents


1.5 The aim of this thesis 27
282 References
Purification of recombinant anthrax edema factor
42II from Escherichia coli and calmodulin from bovine
brain
421 Abstract
2 Introduction
463 Materials and Methods
3.1 Materials 46
EF/EF3(F586A) plasmid amplification and protein
3.2 46
expression
3.3 Lysis of the E. coli 47
3.4 Chromatographic purification of EF 48

IMACchromatographicpurificationusingHisTrapfast!flow!rate
3.4.1 482+Ni column

3.4.2 AnionexchangechromatographicpurificationofEF 49
3.5 Chromatographic purification of EF3(F586A) mutant 51
IMACchromatographicpurificationusingHisTrapfast!flow!rate
3.5.1 512+Ni column
3.5.2 CationexchangechromatographicpurificationofEF3(F586A) 51
VIIContents


3.6 Calmodulin purification from bovine brain 52
3.6.1 Extractionofcalmodulinfrombovinebraintissues 52
3.6.2 Hydrophobicchromatographypurificationofcalmodulin 53
3.7 Concentration and characterization of the purified proteins 54
4 57Results and discussion
4.1 Purification and characterization of the Full length EF 57
4.2 Purification and characterization of EF3(F586A) 63
4.3 Purification of calmodulin from bovine brain 66
Analysis of the interaction of MANT-nucleotides with EF3
4.4 69
in FRET experiments
705 Summary and conclusion
716 References
Molecular Analysis of the Interaction of Anthrax
Adenylyl Cyclase Toxin, Edema Factor, with 2’(3’)-
74III
O-(N- (methyl)anthraniloyl)-Substituted Purine and
Pyrimidine Nucleotides
741 Abstract
752 Introduction
763 Materials and Methods
VIII