The conformational dynamics of BsoBI, analyzed by fluorescence spectroscopy down to the single molecule level [Elektronische Ressource] / vorgelegt von Jasmina Dikić

Dikicjasmina

Sujets

• Biologie

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2009
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

The conformational dynamics of BsoBI, analyzed by
fluorescence spectroscopy down to the single molecule level





Inauguraldissertation



zur Erlangung des Grades
Doktor der Naturwissenschaften
Dr. rer. nat.
Im Fachbereich Biologie und Chemie
der Justus-Liebig-Universität Gießen



vorgelegt von

Dipl. Molekularbiologin und Physiologin
Jasmina Dikić





Gießen, 2009
The present study has been carried out within the Marie Curie Research Training Network
“DNA enzymes”, at the Institute of Biochemistry, Justus-Liebig-University Giessen, between
April 2006 and October 2009, under the supervision of Prof. Dr. Alfred Pingoud.













Advisor Prof. Dr. Alfred Pingoud
Institut für Biochemie
Justus-Liebig-Universität
Heinrich-Buff-Ring 58
35392 Gießen

Co-Advisor Prof. Dr. Michael U. Martin
Institut für Immunologie
Justus-Liebig-Universität
Winchesterstraße 2
35394 Gießen
Erklärung

Hiermit versichere ich, die vorliegende Arbeit selbständig verfasst und keine anderen als die
angegebenen Hilfsmittel benutzt zu haben. Stellen, die ich anderen Arbeiten und
Veröffentlichungen dem Wortlaut oder Sinn entsprechend entnommen habe, sind durch
Quellenangaben gekennzeichnet.




Gießen, den 9.11.2009
Acknowledgements

I would like to thank:
Prof. Dr. Alfred Pingoud, for invaluable discussions, ideas, motivation, and great knowledge
which inspired me to learn more and work better every day. Thank You for the opportunity to
be the part of the Institute of Biochemistry.
Prof. Dr. Michael U. Martin, for taking over the reading of this manuscript.
Dr. Wolfgang Wende, for constant help, great patience, productive discussions, helpful
criticisms, and for supporting and encouraging me all the time. Above all, thank You for
teaching me to enjoy my work, even when it didn’t go as planned.
My friends all over Europe, whose diverse life experiences and cultures made me a better
person:
Michele, my single-molecule fellow, for helpful discussions and a great time together, in
Giessen, Düsseldorf and all other places we had fun at.
George, for your experience, knowledge and passion for science, and Silke, for your great
spirit and fun times together.
Jadranka, Laura, Daniel, Lena, Ines W, thank you for all the fun we had, in different periods
of my life in Giessen; you were all an important part of it.
My labmates, Ines F. for nice working atmosphere, and Dr. Vera Pingoud for all helpful
advices.
All the people at the Institute of Biochemistry, for always creating an exciting working
atmosphere.
My friends from the Marie Curie Network, for great scientific discussions and amazing fun
we had in the past three years.
Our collaboration partners, Prof. Dr. Claus Seidel, for introducing me to single-molecule
world, Dr. Paul Rothwell and Evangelos Sisamakis, for their help and patience, and all the
people at the Institute of Molecular Physical Chemistry in Düsseldorf, who made my long
days in their lab much easier.
I would especially like to thank my parents and my sister, for their constant support for
everything I did, for their love, encouragement, sacrifice, and for understanding my decision
to continue my scientific life in Germany.

I would like to thank the Marie Curie Research Training Network “DNA Enzymes” for
funding my work.
Abbreviations

2-ME 2-Mercaptoethanol
A Ampere
a.u. Arbitrary unit
APS Ammoniumpersulfate
ATP Adenosine triphosphate
bp Base pair(s)
BSA Bovine serum albumin
dATP Deoxyadenosine triphosphate
DNA Deoxyribonucleic acid
DTT 1,4-dithiothreitol
EDTA Ethylene diamine tetraacetate
e.g. Exempli gratia; for example
EMSA Electrophoretic mobility shift assay
FPLC Fast protein liquid chromatography
FRET Fluorescence resonance energy transfer
g Gram
i.e. Id est; that is
IPTG Isopropyl-β-D-1-thiogalactopyranoside
l Liter
LB Luria-Bertani
m Milli
M Molar
MFD Multiparameter fluorescence detection
min Minute
MW Molecular weight
n Nano
NaOAc Sodium acetate
o/n Overnight
OD Optical density
p pico
PAGE Polyacrylamide gel electrophoresis
PCR Polymerase chain reaction
rpm Rotations per minute
SDS Sodium dodecyl sulfate
sec Second
TCEP Tris(2-carboxyethyl)phosphine
TEMED Tetramethylethylenediamine
UV Ultraviolet
vs. versus
v/v Volume/volume
w/v Weight/volume
μ Micro

| 5
Table of Contents
Abbreviations ............................................................................................................................. 5
1. Introduction ......................... 9
1.1. Restriction-modification systems in bacteria .................................. 9
1.2. Type II restriction enzymes ........................................................................................... 11
1.3. BsoBI restriction enzyme .............................. 14
1.4. Fluorescence spectroscopy of single molecules in solution .......................................... 16
1.5. Structural dynamics of proteins probed by single-molecule techniques ....................... 18
1.6. Aim ................................................................................................ 20
2. Materials and methods ...................................... 21
2.1. Materials ........................................................................................................................ 21
2.1.1. Chemicals and biochemicals ................... 21
2.1.2. Plasmids .................. 22
2.1.3. Bacterial strains ....................................................................................................... 22
2.1.4. Buffers ..................... 22
2.1.5. Enzymes .................. 23
2.1.6. Markers ................................................................................................................... 24
2.1.7. Fluorophores ........... 24
2.2. Methods ......................... 25
2.2.1. Microbiological methods ........................................................................................ 25
2.2.1.1. Culture media ................................... 25
2.2.1.2. Preparation of electrocompetent cells .............................. 25
2.2.1.3. Electroporation ................................................................. 25
2.2.2. Molecular biology methods .................... 25
2.2.2.1. Electrophoresis ................................. 25
2.2.2.1.1. SDS-PAGE ................................................................ 26
2.2.2.1.2. Polyacrylamide gel electrophoresis ........................... 26
2.2.2.1.3. Agarose gel electrophoresis ....................................... 26
2.2.2.1.4. EMSA ........................................................................ 26
2.2.2.1.5. Denaturing polyacrylamide gel electrophoresis ........ 27
2.2.2.2. Isolation of DNA .............................. 27
2.2.2.2.1. Minipreparation ......................................................................................... 27
2.2.2.2.2. Midipreparation 27
2.2.2.2.3. PCR purification ........................ 27
2.2.3. Creating single-cysteine BsoBI variants ................................................................. 27
2.2.3.1. Site-directed mutagenesis ................................................................................. 27
2.2.3.2. Screening .......................................... 28
2.2.3.3. Overexpression of BsoBI ................. 28
2.2.3.4. Purification of BsoBI ....................................................................................... 29
2.2.3.5. Determination of protein concentration ........................................................... 29
2.2.3.6. Determination of protein binding and cleavage activity .................................. 30
2.2.3.6.1. Determination of binding activity .............................. 30
2.2.3.6.2. Determination of cleavage activity ............................................................ 30
2.2.4. Michaelis-Menten kinetics ...................................................... 30
2.2.5. Site-specific fluorescent labeling of single-cysteine variants ................................. 31
2.2.5.1. Labeling and purification of proteins used in steady-state and stopped-flow
experiments ................................................................................................................... 32
2.2.5.2. Labeling and purification of proteins used in single-molecule experiments.... 33
2.2.5.3. Determination of labeling efficiency ................................................................ 35
2.2.5.4. Determination of binding and cleavage activity............... 35
2.2.6. Fluorescence methods ............................................................. 35
2.2.6.1. Introduction to fluorescence .............................................................