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Dnak Functions As a Central Hub in the E. Coli Chaperone Network

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Dnak Functions As a Central Hub in the E. Coli Chaperone Network Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München DnaK Functions as a Central Hub in the E. coli Chaperone Network Taotao Chen aus Henan, China 2011 Erklärung Diese Dissertation wurde im Sinne von §13 Absatz 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 (in der Fassung der sechsten Änderungssatzung vom 16. August 2010) von Herrn Prof. F. Ulrich Hartl betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfsmittel erarbeitet. München, am 25. 10. 2011 Taotao Chen Dissertation eingereicht am 25. 10. 2011 1. Gutacher Prof. Dr. F. Ulrich Hartl 2. Gutachter Prof. Dr. Walter Neupert Mündliche Prüfung am 12. 12. 2011 Acknowledgements First of all, I would like to express my deepest gratitude to Prof. Dr. F. Ulrich Hartl for giving me the opportunity to study and learn the extremely interesting subject in his laboratory. I would like to thank him for the encouragement and the continual support throughout the entire period of my PhD study. Uncountable thanks go to my direct supervisor Dr. Manajit Hayer-Hartl, Dr. Giulia Calloni, Dr. Sonya Scherrman and Dr. Roman Körner for their invaluable advice and constant support, Especially Dr. Manajit Hayer-Hartl .She is not only a good advisor of my work but also a good mentor in my personal life. I would like to thank Prof. Dr. Walter Neupert for his help in correcting my dissertation and being the co-referee of my thesis committee. I thank colleagues in the department of cellular biochemistry for providing accommodative environment to a foreigner like me and many helps. In particularly, I would like to thank Andrea, Silke, Evelyn, Elisabeth, Emmanuel, Nadine, Albert, Romy, Andreas and Bernd Grampp for keeping the laboratory at good running. Special thanks to Nadine and Dirk for their excellent technical assistance. Many great thanks to Andreas.Bracher, Kausik, and Martin for generously sharing their specialty opinions and many insightful discussions. Their friendships and the good working atmosphere became the main basis for the success of this work. The deepest thanks go to my wife, Jiawen Qin, for her enormous support and patience and valuable discussions. The same deep thanks belong to my parents and my family in China for their understanding and support. CONTENTS 1 Summary ............................................................................................................................. 1 2 Introduction ......................................................................................................................... 3 2.1 protein stucture ................................................................................................................ 3 2.1.1 Protein folding and aggregation .............................................................................. 5 2.2 The challenge of protein folding in the cell .................................................................... 8 2.3 Molecular chaperone ..................................................................................................... 10 2.3.1 Ribosome-associated chaperone -Trigger factor .................................................. 13 2.3.2 E. coli Hsp70 - DnaK ............................................................................................ 20 2.3.3 E. coli Chaperonin GroEL/ES ............................................................................... 25 2.3.3.1 Structure and function of GroEL and GroES ................................................... .25 2.3.3.2 Mechanisms of GroEL-mediated protein folding ............................................. 27 2.3.3.3 Substrates of GroEL………...………………………………………………...31 2.4 Aim of the study............................................................................................................ 32 3 Materials and methods ...................................................................................................... 33 3.1 Materials ....................................................................................................................... 33 3.1.1 Chemicals ................................................................................................................ 33 3.1.2 Buffers and medium……………………………………………………………....34 3.1.2.1 Buffers……………………………………………………………..………….34 3.1.2.1 Medium……………………………………………………………………….37 3.1.3 Materials and instruments ..................................................................................... 37 3.1.4 E. coli strains and plasmid .................................................................................... 39 3.2 Methods......................................................................................................................... 40 3.2.1 DNA analytical methods ....................................................................................... 40 3.2.1.1 General molecular biology methods ............................................................. 40 3.2.1.2 Expressing a chromosomal C-terminally His6-tagged DnaK ....................... 40 3.2.1.3 Electrocompetent E. coli cells and electroporation ...................................... .41 3.2.2 Protein analytical methods ................................................................................... 41 3.2.2.1 Apyrase purification ......................................................................................... 41 3.2.3 Quantification of proteins .................................................................................... .42 3.2.4 SDS-PAGE .......................................................................................................... .42 3.2.5 Western blotting .................................................................................................... 44 3.3 Biochemical and biophysical methods ......................................................................... .44 3.3.1 Functional analysis of His-tagged DnaK .............................................................. 44 3.3.2 ATP depletion from cell lysate ............................................................................. 44 3.3.3 In vivo isolationof DnaK/GrpE/interactor complexes…………………………...45 3.3.4 Fractionation of the whole cell lysate ................................................................... 46 3.3.5 Expression of Luc-SecM-GFP stalling sequence ................................................. 46 3.3.6 GroEL/EL depletion .............................................................................................. 47 3.3.7 LC-MS/MS ........................................................................................................... 47 3.4 Analysis of MS data ..................................................................................................... .48 3.4.1 Determination of SILAC Ratios ........................................................................... 48 3.4.2 Determination of the DnaK interactome ............................................................... 49 3.4.3 Determination of the DnaK interactome changes ................................................. 50 3.4.4 Determination of substrate enrichement of DnaK (PD/Lysate SILAC) ............... 50 3.4.5 Determination of proteome differences between E. coli chaperone mutants ....... 51 3.4.6 Pulse and pulse-chase SILAC ............................................................................... 52 3.4.7 Bioinformatic anaylsis .......................................................................................... 54 4 Results ............................................................................................................................... 55 4.1 Analysis of DnaK interactome in WT .......................................................................... 55 4.1.1 Depletion of ATP from cell lysate ........................................................................ 55 4.1.2 Functional examination of cells with his-tagged DanK........................................57 4.1.3 Isolation and identification of DanK-bound proteins ........................................... 59 4.1.4 Properties of DnaK interactome ............................................................................ 60 4.1.5 Classification of DnaK-interactors by enrichement on DnaK .............................. 64 4.1.6 specific changes in proteome composition upon DnaK deletion .......................... 70 4.2 The Kinectics of DnaK and substrates interaction ........................................................ 74 4.2.1 DnaK functions in de novo colding and conformational maintenance ................. 74 4.2.2 Protein flux through DnaK .................................................................................... 78 4.3 Fucntional redundancy of DanK and Trigger factor is only partial .............................. 79 4.4 Interplay of DnaK and GroEL/GroES .......................................................................... 84 4.5 Proteostasis collapse upon combined deletion of DanK and TF .................................. 90 5 Discussion ....................................................................................................................... 101 5.1 Contribution of DnaK chaperone system to protein folding in E. coli ……....……...101 5.2 DnaK closely cooperate with Trigger factor …………………………...…………....105 5.3 The interplay of the DanK and chaperonion systems………………………………. .107 5.4 DnaKdnaJ and TF are involved in the biogenesis of ribosome ……………………...108 6 References
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