GAF-Tandem Signal-Transduktion in Chimeren mit der CyaB1-Adenylat-Cyclase als Reporter [Elektronische Ressource] = Interdomain signal transduction in GAF tandem chimeras using the CyaB1 Adenylyl Cyclase as a reporter [[Elektronische Ressource]] / vorgelegt von Iman Mansi

Iman Farag

Sujets

• Biologie

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2008
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

GAF-Tandem Signal Transduktion in Chimeren mit der CyaB1
Adenylat Cyclase als Reporter




Interdomain Signal Transduction in GAF Tandem Chimeras
Using the CyaB1 Adenylyl Cyclase as a Reporter



DISSERTATION


der Fakultät für Chemie und Pharmazie
der Eberhard-Karls-Universität Tübingen



zur Erlangung des Grades eines Doktors
der Naturwissenschaften




2008


vorgelegt von

Iman Mansi





































Tag der mündlichen Prüfung: 18. Juli 2008
Dekan: Prof. Dr.L.Wesemann
Erster Berichterstatter: Prof. Dr. J.E. Schultz
Zweiter Berichterstatter: Prof.Dr. P. Ruth Allah sagte:
’’ihr Menschen! Wir erschufen euch aus einem Mann und einer Frau und machten
euch zu Völkern und Stämmen, damit ihr einander kennenlernt. Doch der vor Allah
am meisten Geehrte von euch ist der Gottesfürchtigste unter euch. Allah ist fürwahr
wissend, kundig’’
(Quran, 49:13)



۞۞۞۞

All thanks goes to Allah who created me, send me my living, guided me to the right
path and gave me power and good health to finish this work.

۞۞۞۞

Acknowledgment

The experimental part of this work was performed within the period from August 2004 to
September 2007 under the supervision of Prof. Dr. J.E.Schultz.
I would like to thank prof. Schultz for accepting me in his lab., for the beneficial scientific
discussions and the different subjects he gave.
I am so grateful to prof. Ruth for taking part in the evaluation of my thesis.
Prof. Werz and prof. Laufer, I do appreciate your participation in the thesis examination.
I express my gratitude to Jost Weber and Jürgen Linder for the great help and interest they
showed at the beginning of my work in the lab and for training me on the handling of the
different kits and methods. Special thanks to J.Linder for the beneficial discussions, support
and interest and for providing some of the constructs and templates. Anita Schultz, thanks a
lot for providing some of the templates and your help in the cloning of three of my constructs.
And not to forget to thank Gottlieb Daimler und Karl Benz Foundation represented by the
company managers Prof. Putlitz and Dr. Schade, the manager of the foundation, Dr. Klein and
his secretary Mrs. Jung and all other employees in the foundation for providing my personal
finance for two years and for the annual nice meetings with beneficial lectures and interesting
journeys.
I am so thankful to Dr. I.Tews and his four-week diploma student, F.Voigts-Hoffmann from
Heidelberg University, for the efforts they spent to solve the crystal structure of CyaB1 PAS
domain and from the department of Biochemistry (Tübingen University), Dr. C.Schal for
measuring the crystals of CyaB2 GAF ( ∆253-A255) and J.Romer for his help in operating the
crystallization robot.
‘Meine Deutsch Lehrerin, Frau. Voegt, Ich danke ihnen sehr für ihre Bemühung und Geduld
während Deutsch Unterrichte’
My Lab colleagues, my friends in Germany and in Jordan, my family in Germany and in
Jordan, thank you all, without your love, encouragement and prayers, I will never be able to
finish this work.


Content
Content

Content…………………………………………………………………………… I

List of Constructs………………………………………………………………... IX

Abbreviations…………………………………………………………………….. XII

Amino acid abbreviations……………………………………………………….. XIV

1. Introduction………………………………………………………………….. 1
Signal transduction………...………………………………………………….. 1

1.1. Small molecule binding domains……………………………….………... 2
1.1.1. GAF domains…...……….……………………………………….. 3
1.1.2. PAS domains...………………………………….………………... 5

1.2. Phosphodiesterases…………………………............................................. 6
1.2.1. PDE regulatory domain..………………………………………….. 8
1.2.2. PDE catalytic domain...………………………….………………... 10

1.3. Class III adenylyl cyclase……………………………………………....... 12

1.4. Cyanobacterium anabaena sp. PCC 7120 Adenylyl cyclases, CyaB1 and
CyaB2……………………………………………………………………. 12

1.5. Aim of work…………………………………………………….………... 14

2. Materials and equipments…..………………………………………………. 17

2.1. Chemicals and Materials…………...…………………………………….. 17

2.2. Equipments………………………………………………………………. 19

2.3. Bacterial strains.....……………………………………………………….. 20

2.4. Plasmids………………………………………………………………….. 21
IContent
2.5. Oligonucleotides…………………………………………………………. 21
2.5.1. Sequencing primers……………………………………………….. 22
2.5.2. Cloning primers………………………………….………………... 23
2.5.2.1.CyaB1 holoenzyme…………………………………………… 23
2.5.2.2.CyaB2 holoenzyme and mutants..…………………………….. 24
2.5.2.3.hPDE5 GAF domain………………………………………….. 26
2.5.2.4.rPDE2 GAF doma27
2.5.2.5.hPDE2 GAF doma28

2.6. Buffers and solutions………..…………………………………………… 29
2.6.1. Molecular biology...………………………………………………. 29
2.6.1.1.solutions for DNA treatment…...……………………………... 29
2.6.1.2.Solutions for bacterial culture media.………………………… 29
2.6.1.3.Solutions for blue/white screening....…………………………. 29
2.6.2. Protein chemistry…...…………………………….………………. 30
2.6.2.1.Protein purification with Ni+2 –NTA- Agarose…….………... 30
2.6.2.2.SDS-Polyacrylamide gel electrophoresis...…………………… 30
2.6.2.3.Western blot……………………......…………………………. 31
2.6.2.4.Cyclase enzyme Assay……………………………………….. 31
2.6.2.5.Dimerization buffers………………………………………….. 32
2.6.2.6.Other solutions……………………………………………….. 33

3. Methods………………………………………………………………………. 33

3.1. Bioinformatics and databases……………………………………………. 33

3.2. Molecular biology methods…………………............................................ 33
3.2.1. Polymerase chain reaction………………………………………... 33
3.2.2. Isolation of DNA by agarose gel electrophoresis………………… 35
3.2.3. Purification of DNA fragment from agarose gel…………………. 36
3.2.3.1.Use of DNA purification kit………………………………….. 36
3.2.3.2.Use of squeeze-freeze method………………………………... 36
3.2.4. Generation of blunt ends…………………………………………. 36
3.2.5. 5’-phosphorylation of the PCR product………………………….. 36
3.2.6. Dephosphorylation of the plasmid vector………………………… 37
3.2.7. Photometric determination of the DNA concentration…………… 37
3.2.8. Ligation of the DNA fragments…………………………………... 37
3.2.9. Transformation of the recombinant DNA………………………… 37
3.2.9.1.Preparation of competent cells………………………………... 37
II Content
3.2.9.2.Transformation of DNA plasmid into competent cells...……... 38
3.2.10. White-blue colony screen…………………………………………. 38
3.2.11. Isolation of DNA………………………………………………….. 38
3.2.11.1. Isolation and purification of DNA from bacterial cultures
(minipreps)…………………………………………………….. 38
3.2.11.2. Phenol/chloroform extraction……………………………... 38
3.2.11.3. Ethanol precipitation……………………………....……..... 39
3.2.12. Enzymatic digestion………………………………………………. 39
3.2.13. Sequencing………………………………………………………... 39
3.2.13.1. Chain terminating sequencing…………………………….. 39
3.2.13.2. Capillary DNA sequencing….…………………………….. 40
3.2.14. Glycerol stock…………………………………………………….. 41

3.3. Protein chemistry………………………………………………………… 41
3.3.1. Preculture…………………………………………………………. 41
3.3.2. Expression………………………………………………………… 41
3.3.3. Cell harvesting and washing……………………………………… 42
3.3.4. Purification of soluble protein from E.coli……………………….. 42
3.3.5. Protein dialysis……………...…………………………………….. 42
3.3.6. Bio-Rad protein determination……………………………………. 42
3.3.7. Sample concentration……………………………………………... 42
3.3.8. SDS-PAGE………………………………………………………... 43
3.3.9. Western blot………………………………………………………. 44
3.3.10. Adenylyl cyclase enzyme assay…………………………………... 45
3.3.11. Crystallization…………………………………………………….. 46
3.3.11.1. Hanging-drop vapour diffusion method…………………... 46
3.3.11.2. Sitting-drop vapour diffusion method...…………………... 46
3.3.12. Size exclusion chromatography (Gel filtration)…………………... 47
3.3.13. Dimerization studies…………………………………………….... 49

3.4. Cloning…………………………………………………………………… 50
3.4.1. Cloning of hPDE2 constructs……………………………………... 53
3.4.1.1. Cloning of hPDE2NGAF/CyaB1 chimera…………………… 53
3.4.1.2. Cloning of ΔN-hPDE2GAF/CyaB1 chimera………………… 54
3.4.1.3. Cloning of hPDE5N-hPDE2GAF/CyaB1 chimera…………... 55
3.4.1.4. Cloning of rPDE2GAF/CyaB1………………………………. 57
3.4.2. Cloning of CyaB2 constructs and mutants………………………... 58
3.4.2.1. Cloning of ( Δaa or + aa)/CyaB1……………………………... 58
3.4.2.2.Cloning of CyaB2 ( ΔR253-A254)/CyaB1……………………. 60
IIIContent
3.4.2.3.Cloning of CyaB2 ( ΔR253-A255) GAFcr for crystallization… 61
3.4.2.4.Cloning of CyaB2 (256 + NAA)cr, CyaB2
(M258L/M259L)GAF and CyaB2 (M258S/M259T)GAF…….. 62
3.4.2.5.Cloning of CyaB2 (M74G/L75G)/CyaB1 and CyaB2
(M74S/L75T)/CyaB1………………………………………….. 63
3.4.2.6.Cloning of CyaB2 α2-mutants (CyaB2 (M87S)/CyaB1,
CyaB2 (L88S)/CyaB1 and CyaB2(M8