Analysis of TF interactions with ribosome-nascent chain complexes -photocrosslinking and fluorescence spectroscopic approaches [Elektronische Ressource] / Sathish Kumar Lakshmipathy

Sathish Kumar Lakshmipathy

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München


Analysis of TF interactions with ribosome-nascent chain complexes
-photocrosslinking and fluorescence spectroscopic approaches


Sathish Kumar Lakshmipathy

aus
Cuddalore, Tamilnadu
India

2009

Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 der Promotionsordnung vom
29. Januar 1998 von Herrn Professor Dr. F. Ulrich Hartl betreut.



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfen erarbeitet.

München, am ....26-03-2009...................................




......................................................................
Sathish Kumar Lakshmipathy


Dissertation eingereicht am 26-03-2009
1. Gutachter: Professor Dr. F. Ulrich Hartl
2. Gutachter: Professor Dr. Roland Beckmann
Mündliche Prüfung am 19-05-2009


Acknowledgements

I would like to thank Prof. F. Ulrich Hartl for providing me the opportunity
to work under his guidance and introducing me to the fascinating field of
Chaperone mediated protein folding. His constant support, encouragement, ideas
and often stimulating discussions were important for the fruitful completion of this
work. I also take this opportunity to thank Dr. Manajit Hayer-Hartl for her advice
and discussions during the course of the work.
I would like to thank Prof. Dr. Roland Beckmann for being a co-referee of
my thesis committee.
My special thanks to Dr. Stephanie Etchells for her supervision of my work
throughout the last five years. Her guidance, suggestions and support during this
whole period forms the basis of my work. I would like to thank her and Dr. Alex
Hastie for their help in my thesis corrections.
I would like to thank Dr. Kausik Chakraborty and Dr. Hung-Chun Chang for
their expert and valuable suggestions. I would also like to thank Dr. Sladjana
Tomic, Florian Ruessmann, Florian Brandt and Dr. Raluca Antonoae for engaging
in regular weekly discussions.
My thanks to Andrea for all her help during my entire stay. I would like to
thank all the members of the Department of Cellular Biochemistry for providing a
congenial atmosphere during my stay.
My thanks to Rashmi not only for her loving support and patience but also
for standing by me during the ups and downs.
Last but not the least; I would like to thank my parents and sisters in India
for their tireless support without which this work would not have been possible.
CONTENTS i

Contents 
I Summary ......................................................................... 1 
II Introduction ................................................................... 4 
II.1 Translation and protein folding ................................................................................ 4 
II.1.1 Levels and determinants of protein structure .................................................... 4 
II.1.2 Protein folding in vitro ...................................................................................... 6 
II.1.3 Protein folding in the cell .................................................................................. 8 
II.2 Molecular chaperones .............................................................................................. 9 
II.2.1 The DnaK chaperone system and it’s interaction with nascent chains ........... 11 
II.2.2 Chaperonins ..................................................................................................... 14 
II.2.2.1 Group I chaperonins ................................................................................. 14 
II.2.2.2 Group II chaperonins15 
II.2.3 Other chaperone systems16 
II.2.4 Ribosome associated chaperones .................................................................... 17 
II.2.4.1 Eukaryotic ribosome associated chaperones ............................................ 17 
II.2.4.2 Prokaryotic ribosom ........................................... 17 
II.2.4.2.1 The Signal Recognition Particle and Trigger Factor ......................... 18 
II.3 Trigger Factor ......................................................................................................... 19 
II.3.1 TF-ribosome interactions ................................................................................ 20 
II.3.2 Structure of TF and explanation for nascent chain interactions ...................... 21 
II.3.3 TF-substrate interactions ................................................................................. 26 
II.4 Aim of the study ......................................................... 29 
III Materials and Methods ............................................... 30 
III.1 Chemicals .............................................................................................................. 30 
III.2 Materials and Instrumentation .............................................................................. 31 
III.3 Media and buffers ................................................................................................. 32 
III.3.1 Media ............................................................................................................. 32 
III.3.2 Buffers ............................................................................................................ 32 
III.4 DNA manipulations .............................................................................................. 33 
III.4.1 General molecular biology methods .............................................................. 33 
III.4.2 Cloning of E. coli TF in pBAD vector ........................................................... 36 
III.4.3 Cloning of S7 in pET 22b vector ........................................................ 37 
III.4.4 Site directed mutagenesis ............................................................................... 37 
III.4.5 Other constructs used in this study ................................................................ 38 
III.4.6 Preparation of linear template DNA for in vitro translation in the PURE
system ....................................................................................................................... 38 
III.5 Protein preparative methods ................................................................................. 38 
III.5.1 Expression of TF proteins in the presence of the pBpa ................................. 38 
III.5.2 Purification of pBpa labeled TF ..................................................................... 39 
III.5.3 Purification of TF ........................................................................................... 40 
III.5.4 In vitro translation in the PURE system ........................................................ 40 
III.5.5 Photocrosslinking of pBpa-TF to RNCs40 CONTENTS ii

III.5.6 Separation of ribosome-nascent chain complexes (RNCs) ............................ 41 
III.5.7 RNase A Digestion ........................................................................................ 41 
III.5.8 Site specific labeling of single cysteine TF proteins ..................................... 41 
III.6 Protein analytical methods .................................................................................... 42 
III.6.1 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
................................................................................................................................... 42 
III.6.2 Autoradiography ............................................................................................ 43 
III.6.3 Western blotting ............................................................................................. 43 
III.6.4 Immunoprecipitation ...................................................................................... 44 
III.6.5 Quantification of proteins .............................................................................. 44 
III.6.6 In vivo functionality test for TF single site mutants ...................................... 44 
III.6.7 Luciferase activity measurements .................................................................. 45 
III.7 Fluorescence measurements .................................................................................. 45 
III.7.1 Overview ........................................................................................................ 45 
III.7.2 RCM-RNase T1 refolding45 
III.7.3 Equilibrium fluorescence measurements ....................................................... 46 
III.7.4 Kinetic fluorescence measurements ............................................................... 46 
III.7.5 Competition experiments ............................................................................... 47 
III.7.6 Evaluation of the kinetic data ........................................................................