Association of bacterial respiratory complexes [Elektronische Ressource] / von Mohd Khalid Siddiqui

Khalid Siddiqui

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2007
• Nombre de lectures: 19
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Association of bacterial respiratory complexes






Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften











vorgelegt im Fachbereich
Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main




von
Mohd Khalid Siddiqui
aus Lucknow, Indien



Frankfurt am Main 2006
(DF1)























vom Fachbereich Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität als Dissertation angenommen.

Dekan: Prof. Dr. H. Schwalbe
Gutachter: Prof. Dr. B. Ludwig
Prof. Dr. T. Prisner


Datum der Disputation



Diese Doktorarbeit wurde vom 17. Februar 2002 bis zum 28 August 2006 unter Leitung von
Prof. Dr. Bernd Ludwig in der Abteilung für Molekulare Genetik, Institut für Biochemie
der JW Goethe Universtät Frankfurt am Main durchgeführt.


















Eidesstattliche Erklärung

Hiermit versichere ich, dass ich die vorliegende Arbeit selbständig angefertigt habe
und keine weiteren Hilfsmittel und Quellen als die hier aufgeführten verwendet habe.




Mohd Khalid Siddiqui
Frankfurt am Main, den 31. August 2006

Table of contents
Summary ................................................................................................................................. I
Zusammenfassung .................................................................................................................V
1. Introduction........1
1.1 Oxidative phosphorylation ..............................................................................................................................1
1.2 Electron transport chain of Paracoccus denitrificans......................................................................................2
1. 3 Structures of the respiratory chain complexes................................................................................................3
1.3.1 NADH:ubiquinone oxidoreductase.....................................................................................................5
1.3.2 Succinate:ubiquinone oxidoreductase...........5
1.3.3 Ubiquinone:cytochrome c oxidoreductase..........................................................................................6
1.3.4 Cytochrome c oxidase.........................................................................................................................6
1.4 Arrangements of the complexes ......................................................................................................................7
1.4.1 Solid state or supercomplex model .....................................................................................................8
1.4.2 Random diffusion model.....................................................................................................................9
Objectives of this dissertation..............................................................................................................................12
2. Materials and Methods ....................................................................................................15
2.1 Suppliers........................................................................................................................................................15
2.2 Chemicals....................................15
2.3 Column chromatography ...............................................................................................................................17
2.4 Proteins..........................................................................................................................................................18
2.5 Strains............................................................................................................................................................18
2.6 Instruments ....................................................................................................................................................18
2.7. Experimental setup .......................................................................................................................................19
2.7.1 FRAP and FCS..................................................................................................................................19
2.7.2 Pulsed EPR .......................................................................................................................................19
2.7.3 Flash photolysis ................................................................................................................................19
2.8 Software......................................20
2.9 Solutions..................................................................................................................20
2.9.1 Growth Medium................................................................................................................................20
2.9.2 Antibiotics.........................................................................................................................................22
2.9.3 Molecular biology.............................................................................................................................22
2.9.4 Protein estimation .............................................................................................................................24
2.9.5 Electrophoresis and Western-blot .....................................................................................................24
2.10 Molecular biology methods.........................................................................................................................32
2.10.1 Plasmid isolation......................32
2.10.2 Competent E. coli cells ...................................................................................................................32
2.10.3 Transformation..........................................................................................................33
2.11 Growth of P. denitrificans...........................................................................................................................33
2.12 Membrane preparation.................................................................................................................................33
2.12.1 Osmotic pressure method...............33
2.12.2 Sonication method ..........................................................................................................................34
2.12.3 Manton-Gaulin Press (MG) method................................................................................................34
2.12.4 Purification of membrane................................................................................................................35
2.13 Membrane vesicle size measurements.........................................................................................................35
2.14 Expression and purification of respiratory protein complexes ....................................................................35
2.14.1 Cu fragment...................................................................................................................................35 A
2.14.2 Cytochrome c fragment...............................................................................................................36 552
2.14.3 Cytochrome cent .................................................................................................................37 1
2.14.4 Cytochrome c from Thermus thermophilus.................................................................................37 552
2.14.5 Cytochrome c (full length) ..........................................................................................................38 552
2.14.6 Purification of cytochrome c oxidase..............................................................................................38
2.14.7 Isolation of P. denitrificans supercomplex .....................................................................................39
2.15 Horse heart cytochrome c............................................................................................................................39
2.16. Oxidation of cytochrome c and c ............................................................................................................39 552
2.17 Fv fragment expression ...............................................................................................................................40
2.18 Purification and labelling of Fv fragment....................................................................................................40
2.18.1 Labelling of Fv antibody fragment .................................................................................................40
2.18.2 Fluorescein isothiocyanate (FITC)...........41
2.18.3 Separation of labelled Fv fragment ..........................................................