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E. Coli Cell Preparation and Transformation Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Structure and Function of Hip, an Attenuator of the Hsp70 Chaperone Cycle Zhuo Li aus Dalian, Liaoning, China München, 2013 Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Structure and Function of Hip, an Attenuator of the Hsp70 Chaperone Cycle Zhuo Li aus Dalian, Liaoning, China München, 2013 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung von 28. November 2011 von Herrn Prof. Dr. F. Ulrich Hartl betreut. Eidesstattliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfsmittel erarbeitet. München, den 03.12.2013 ………………………………… Zhuo Li Dissertation eingereicht am: 03.12.2013 1. Gutachter Prof. Dr. F. Ulrich Hartl 2. Gutachter: Prof. Dr. Roland Beckmann Mündliche Prüfung am: 13.02.2014 Acknowledgement I am particularly thankful to Prof. Dr. F. Ulrich Hartl for giving me the opportunity to work in his inter- national and interdisciplinary department at Max Planck Institute of Biochemistry. I am grateful to Dr. Manajit Hayer-Hartl for her support and advice. I especially express my deepest gratitude to my supervisor Dr. Andreas Bracher. It has been my pleasure and my luck to work with him, a knowledgeable, efficient, conscientious and distinguished scientist, who gave me many valuable and critical suggestions and managed many details throughout my entire PhD study. He patiently and meticulously guided me and encouraged me to undertake and keep our project going on schedule. I am especially thankful for his constant help and suggestions in my thesis corrections. I would like to extend my gratitude to Dr. Swasti Raychaudhuri for his crucial contributions to this pro- ject. I am also deeply grateful to Dr. Mathias Stotz for his help and discussion during the various phases of my project. Furthermore, many great thanks to Dr. Qiaoyun Shi, Dr. Guoxin Jiang, Ayano Konagai, Dr. Yujin Kim, Dr. Joshi Jidnyasa, Dr. Young-Jun Choe, Dr. Niti Kumar, Sneha Kumar, Dr. Frédéric Frottin, Dr. Rajat Gupta and Dr. Taotao Chen. I extend my sincerest appreciation and thanks to my of- fice members Dr. Sigrun Polier, Leonie Mönkemeyer, Tobias Neudegger, Dr. Cuimin Liu, Dr. Oliver Müller-Cajar, Dr. Candace Tsai, Dr. Amanda Windhof, Dr. Paulo Durao and Thomas Hauser. I am thankful to Evelyn Frey-Royston, Silke Leuze-Bütün, Andrea Obermayr-Rauter and Emmanuel Burghardt, Albert Ries as well as Dr. Stefan Pinkert for their invaluable supports. To the rest of the de- partment, I really appreciated the open-minded scientific atmosphere and enjoyable working environ- ment. I want to thank all the support from Karina Valer and Sabine Pleyer for initial screening of crystalliza- tion conditions in MPIB Crystallization Facility. I am especially grateful to Dr. Debora Makino for col- lecting diffraction data and to Dr. Claire Basquin for her help with SAXS data analysis. I am thankful for my thesis advisory board members Prof. Dr. Elena Conti and Dr. Roman Körner. I also want to thank for the members of my Ph.D committee Prof. Dr. Roland Beckmann, Prof. Dr. Elena Conti, Prof. Dr. Jörg Nickelsen, Prof. Dr. Don Lamb and Dr. Petra Wendler for critical evaluation of this thesis. I am grateful for my good friends Zijing Gong, Dr. Weihua Qin and Xixi Jia. I am really appreciated all the fun times we have shared and all the support they gave me. Finally, I give a very special note of gratitude to my parents Xiuling Shi and Shijie Li, who have sup- ported and encouraged me over the years. Contents Contents 1 Summary ............................................................................................................................. 10 2 Introduction ......................................................................................................................... 12 2.1 Protein folding and protein aggregation ....................................................................... 12 2.2 Molecular chaperones ................................................................................................... 14 2.2.1 Chaperonins .......................................................................................................... 16 2.2.2 The Hsp90 system ................................................................................................. 19 2.2.3 The Hsp70 system ................................................................................................. 22 2.2.3.1 Diversity of Hsp70 isoforms .......................................................................... 22 2.2.3.2 Cellular functions of Hsp70 ........................................................................... 22 2.2.3.3 Structure and working cycle of Hsp70 .......................................................... 23 2.2.4 Hsp40 co-chaperones ............................................................................................ 26 2.2.5 Nucleotide exchange factors (NEFs) .................................................................... 28 2.2.5.1 Family I .......................................................................................................... 29 2.2.5.2 Family II ........................................................................................................ 29 2.2.5.3 Family III ....................................................................................................... 30 2.2.5.4 Family IV ....................................................................................................... 30 2.2.5.5 Cellular functions of NEFs ............................................................................ 31 2.3 Hsp70 protein-protein network..................................................................................... 32 2.3.1 Eukaryotic Hsp70 protein-protein network ........................................................... 32 2.3.2 Hsp70 interacting protein (Hip) ............................................................................ 33 2.3.2.1 Predicted domain structure of Hip ................................................................. 34 2.3.2.2 The N-terminal dimerization domain of Hip ................................................. 35 2.3.2.3 The TPR domain of Hip................................................................................. 37 2.3.2.4 The GGMP repeat region ............................................................................... 37 2.3.2.5 The DP domain of Hip ................................................................................... 38 2.3.3 C-terminus of Hsc70 interacting protein (Chip) ................................................... 38 2.3.4 The Hsp70-Hsp90 organizing protein (Hop) ........................................................ 39 6 Contents 2.3.4.1 Similarities between Hip and Hop ................................................................. 41 2.4 Role of Hip in cellular protein quality control ............................................................. 42 2.4.1 Regulation of progesterone receptor maturation ................................................... 42 2.4.2 Aggregation prevention in models of neurodegenerative disease ......................... 43 2.4.3 Hip promotes degradation of misfolded protein ................................................... 45 2.5 Aim of study ................................................................................................................. 46 3 Materials and Methods ........................................................................................................ 48 3.1 Chemicals ..................................................................................................................... 48 3.2 Enzyme and antibodies ................................................................................................. 50 3.3 Strains ........................................................................................................................... 50 3.4 Instruments ................................................................................................................... 51 3.5 Media and buffers ......................................................................................................... 53 3.6 Molecular biology methods .......................................................................................... 58 3.6.1 DNA analytical methods ....................................................................................... 58 3.6.2 Polymerase chain reaction (PCR) ......................................................................... 59 3.6.3 Site-directed mutagenesis...................................................................................... 60 3.6.4 DNA restriction and ligation ................................................................................. 61 3.6.5 Plasmid DNA and DNA fragment purification ..................................................... 62 3.6.6 Cloning strategies .................................................................................................. 62 3.6.7 Competent E. coli cell preparation and transformation ........................................ 63 3.6.7.1 Chemocompetent E. coli cells and chemical transformation ......................... 63 3.6.7.2 Electrocompetent E. coli cells and electroporation ....................................... 64 3.6.8 Lithium acetate transformation of S. cerevisiae cells ..........................................
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