Spectroscopic studies on flavoproteins [Elektronische Ressource] / Monika Joshi

Monika Graetzer-Tortorella

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Lehrstuhl für Organische Chemie und Biochemie der
Technischen Universität München



Spectroscopic Studies on Flavoproteins


Monika Joshi


Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität
München zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. Johannes Buchner

Prüfer der Dissertation:
1. Univ.-Prof. Dr. Dr. Adelbert Bacher
2. Sevil Weinkauf



Die Dissertation wurde am 26.04.2007 bei der Technischen Universität München eingereicht
und durch die Fakultät für Chemie am 22.05.2007 angenommen. Acknowledgement i
___________________________________________________________________________
Acknowledgement
I would like to express my deep and sincere gratitude to Professor Dr. Dr. Adelbert Bacher,
for providing me an opportunity to perform my Ph.D. study in Germany. His wide knowledge
and logical way of thinking have been of great value for me. His understanding, encouraging
and personal guidance have provided a good basis for the present thesis.
I am deeply grateful to my supervisor, PD Dr. Wolfgang Eisenreich, for introduction to
the experimental NMR spectroscopy and its constant discussion, his detailed and constructive
comments, helpfulness and for his important support throughout this work.
I wish to express my warm and sincere thanks to Professor Dr. Markus Fischer, University
of Hamburg, who introduced me to the field of molecular biology. His ideas and concepts
provided a remarkable influence on my entire research. I owe my sincere gratitude to
Professor Dr. Gerald Richter of Cardiff University, my former supervisor for his support and
the constant helpfulness.
I warmly thank PD Dr. Stefan Weber and Dr. Erik Schleicher of Free University of Berlin
for outstanding co-operation within the ranges of EPR and ENDOR spectroscopy. Their
valuable advice and extensive discussions around my work have been very helpful for this
study. My sincere thanks are due to Dr. Boris Illarionov and PD Dr. Felix Rohdich for their
kind support and guidance and special thanks to PD Dr. Nediljko Budisa of Max Planck
Institute of biochemistry, Martinsried for tryptophan auxotrophic E. coli strain.
I am grateful to Mr. Fritz Wendling for his professional assistance with computer and
HPLC problems and Mr. Richard Feicht for his help and advice in protein purification.
I have great regard and I wish to extend my warmest thanks to all my colleague; Miss
Susan Lauw, Mrs. Heidi Hofner, Miss Ryu-Ryun Kim, Dr. Victoria Illarionova, Dr. Werner
Römisch, Mrs. Astrid König, Mrs. Elena Ostrojenkova, Dr. Tanja Radykewicz, Mr. Christoph
Grassberger, Miss Martina Winkler, Mr. Matthias Lee, Mrs. Christine Swartz, Dr. Johannes
Kaiser, Dr. Ferdinand Zepeck, Dr. Tobias Gräwert, Dr. Stefan Hecht, Dr. Ralf Laupitz, Miss
Brigit Keil, Miss Katrin Gärtner, Miss Silke Marsch, Miss Eva Sicklinger, Miss Eva Eylert,
Miss Sabine Saller, Mr. Stefan Kraut, Mr. Thomas Wojtulewicz, Dr. Lilla Margl, Dr. Ilka
Haase, Miss Young-Eun Woo, Miss So-Young Kim, Mr. Oliver Ladebeck, Dr. Chan Yong
Lee.
My special gratitude is due to my entire families for their loving support, without their
encouragement and understanding it would have been impossible for me to finish this work.
Table of Contents ii
___________________________________________________________________________
Table of Contents
Acknowledgement i
Table of Contents ii
Abbreviations v

1. Introduction 1
1.1 Phototropin 5
1.1.1 General background 5
1.1.2 LOV domain architecture and chromophore environment 7
1.1.3 Photoexcited–state structural dynamics of LOV domains 7
1.1.4 Photochemistry of LOV domain/Reaction mechanism 10
1.2 DNA photolyase 14
1.2.1 General background 14
1.2.2 Escherichia coli DNA photolyase 15
1.2.3 Reaction mechanism 17
1.2.4 Escherichia coli DNA photolyase E109A mutant 17
1.3. Flavodoxin 18
1.3.1 General background
1.3.2 Overall structure of Escherichia coli flavodoxin 19
2 Materials and Methodology 21
2.1 Materials 21
2.1.1 Instruments
2.1.2 Chromatographic materials 22
2.1.3 Chemicals and enzymes
2.1.4 Culture medium 23
2.1.5 Buffers and solution 25
2.1.6 Bacterial strain and plasmid 29
2.2 Molecular-biological method 31
2.2.1 Isolation of plasmid with PeQlab Plasmid Isolation Miniprep Kit 31
2.2.2 Agarose gel electrophoresis 32
2.2.3 Competent cells and transformation 33
2.2.4 Construction of an expression plasmid for LOV domain 34
2.2.4.1 Construction of expression plasmid for Avena sativa LOV1 NPH1-1 domain 34 Table of Contents iii
___________________________________________________________________________
2.2.4.2 Construction of an expression plasmid for Avena sativa LOV2 NPH1-1 domain 34
2.2.4.3on of expression plasmid for Adiantum capillus-veneris
phy3 LOV2 domain 34
2.2.5 Construction of recombinant Bacillus subtilis strain expressing DNA photolyase
E109A mutant 5
2.2.6 Construction of recombinant Escherichia coli strain expressing flavodoxin 35
2.3 Protein-chemical methods 35
2.3.1 Culture preservation
2.3.2 Microbial culture
2.3.3 Expression test 36
2.3.4 SDS-polyacrylamide gel electrophoresis 36
2.3.5 Protein expression 37
2.3.5.1 Expression of LOV domain
2.3.5.2 Expression of DNA photolyase E109A mutant 38
2.3.5.3 Expression of Escherichia coli flavodoxin 38
2.3.6 Protein extraction 38
2.3.7 Protein purification 39
2.3.7.1 Purification of LOV domain
2.3.7.2 Purification of DNA photolyase E109A mutant 40
2.3.7.3 Purification of Escherichia coli flavodoxin 42
2.3.8 Protein concentration determination 42
2.3.9 Concentrating protein solution through ultrafiltration 43
2.3.10 Preparation of cofactor 44
2.3.10.1 Preparation of random isotpologue libraries of 6,7-dimethyl-8-ribityllumazine
by in vivo biotransformation 44
2.3.10.2 Preparation of random isotopologue libraries of riboflavin by enzymatic
synthesis 45
2.3.10.3 Preparation of ordered isotopologue libraries of riboflavin 45
2.3.10.4 Preparation of flavin mononucleotide (FMN) 45
2.3.10.5 Preparation of 5-deaza-FMN 45
2.3.10.6 Preparation of tetraacetylriboflavin (TARF) 46
2.3.11 Isolation of tryptophan 46
2.4 Spectroscopic method 47 Table of Contents iv
___________________________________________________________________________
2.4.1 Optical spectroscopy 47
2.4.2 NMR spectroscopy
2.4.3 Circular dichroism 48
3 Results and Discussion 49
3.1 Phototropin LOV domain 49
3.1.1 Carbon isotopologue editing of FMN bound to LOV domain 49
3.1.1.1 Isolation of LOV domain
3.1.1.2 Optical spectroscopy 51
3.1.1.3 NMR spectroscopy
3.1.1.4 Discussion 59
3.1.2 CIDNP study on Avena sativa LOV2 domain C450A mutant 64
3.1.2.1 Discussion 78
3.1.3 CIDNP study on LOV2 domain C450A mutant reconstituted with 5-deaza-FMN 80
3.1.3.1 Discussion 86
3.1.4 ENDOR spectroscopy of LOV2 domain C450A mutant 88
3.2 Escherichia coli DNA photolyase E109A mutant 92
3.2.1 Isolation of Escherichia coli DNA photolyase E109A mutant 92
3.2.2 Optical spectroscopy 92
3.2.3 ENDOR spectroscopy of Escherichia coli DNA photolyase E109A mutant 93
3.3 Escherichia coli flavodoxin 94
3.3.1 Isolation of Escherichia coli flavodoxin 94
3.3.2 Optical spectroscopy 94
3.3.3 NMR spectroscopy 95
3.3.4 Discussion 103
3.3.5 ENDOR spectroscopy of Escherichia coli flavodoxin 107
4 Summary 109
5 References 112
Abbreviations v
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Abbreviations
8-HDF 8-Hydroxy-5-Deazaflavin
Å Angstrom
ADP Adenosine-5’-diphosphate
AM ’-monophosphate
APS Ammonium peroxide sulphate
ATP Adenosine-5’-triphosphate
bp Base pair
CD Circular Dichroism
CIDNP Chemically Induced Dynamic Nuclear Polarisation
CPD Cyclobutane pyrimidine dimers
Da Dalton
DNA Deoxyribonucleic acid
dNTP Deoxynucleotide triphosphate
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
ENDOR Electron Nuclear Double Resonance
EPR Electron Paramagnetic Resonance
FAD Flavin adenine dinucleotide
fldA Flavodoxin
FMN Flavin mononucleotide
FPLC Fast Protein Liqiud Chromatography
FTIR Fourier Transform Infrared Spectroscopy
GTP Guanosine triphosphate
h hour
HEPES