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Firefly Luciferase Mutants As Sensors of Proteome Stress

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Firefly Luciferase Mutants As Sensors of Proteome Stress Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Firefly Luciferase Mutants as Sensors of Proteome Stress Rajat Gupta aus New Delhi India 2012 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. F. Ulrich Hartl betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, am 18.09.2012 Rajat Gupta Dissertation eingereicht am: 18.09.2012 1. Gutachter: Prof. Dr. F. Ulrich Hartl 2. Gutachter: PD Dr. Konstanze Winklhofer Mündliche Prüfung am: 06.11.2012 Acknowledgements I would like to express my deepest gratitude to Prof. Dr. F. Ulrich Hartl for giving me an opportunity to work in his international and interdisciplinary team. His constant support, faith and encouragement have been extremely helpful for the development of the work presented in this thesis. The stimulating discussions and exchange of ideas with Prof. Hartl helped me to nurture the scientific and analytical skills. I also like to thank Dr. Manajit Hayer-Hartl for her constant advice and suggestions during the course of the work. I would like to thank PD Dr. Konstanze Winklhofer for being a co-referee of my thesis committee. I am grateful to Dr. Mark Hipp for his constant support and suggestions for the development of this project and for his help in my thesis corrections. I would also like to thank Dr. Niti Kumar, Dr. Frédéric Frottin and Dr. Prasad Kasturi for their immense help during scientific discussions and correction of this thesis and for their constant encouragement. I sincerely acknowledge Dr. Prasad Kasturi for performing the crucial experiments in C. elegans. Many thanks to Dr. Andreas Bracher and Dr. Swasti Raychaudhuri for their contributions in the work presented in this thesis. I am also grateful to Evelyn, Silke and Andrea for their kind help in all my administrative-related queries and to Nadine, Emmanuel, Elisabeth, Verena, Romy and Albert for their expert technical and logistical help. Without their support the journey would have been difficult. I would like to take this opportunity to express my sincere thanks to Dr. Niti kumar, Dr. Sathish Lakshmipathy, Dr. Rashmi Gupta, Dr. Jyoti Sinha, Christian Löw, Andreas Wörner, Florian Rüßmann, Matthias Antonin, Dr. Frédéric Frottin, Dr. Julian Micoud, Ayano Konagai, Yury Kukushkin and rest of the Hartl department for all the endless fun in the lab. All these people have significantly contributed in smoothing my stay in Germany and without their support and fun my Ph.D. journey would have been incomplete. Lastly, I am indebted to my family members whose constant faith and motivation has allowed me to reach this stage where I can express my gratitude to all the people associated with me in these last four years. The work presented in this thesis is accredited to my family members for their unconditional love. Contents Contents I Summary .................................................................................. 1 II Introduction ............................................................................ 4 II.1. Proteins ................................................................................................................................ 4 II.2. Protein Folding .................................................................................................................... 4 II.3. Energy Landscape Perspective on Protein Folding ............................................................. 6 II.4. Proteostasis .......................................................................................................................... 7 II.4.1. Molecular Chaperones in Proteostasis .......................................................................... 9 II.4.1.1. Different Classes of Chaperones .......................................................................... 11 II.4.1.1.1. Ribosome Associated Chaperones................................................................. 12 II.4.1.1.2. Non-ribosome Associated Chaperones .......................................................... 13 II.4.1.1.2.1. The Hsp70 Chaperone System ................................................................ 14 II.4.1.1.2.2. The Hsp90 Chaperone System ................................................................ 19 II.4.1.1.3. The Chaperonins ............................................................................................ 23 II.4.2. HSF1 and Stress Response .......................................................................................... 26 II.4.3. Protein Misfolding and Aggregation .......................................................................... 28 II.4.3.1. Huntington’s Disease ........................................................................................... 32 II.4.4. Cellular Defense Mechanisms against Protein Misfolding ......................................... 34 II.4.4.1. Chaperone Mediated Refolding and Disaggregation ........................................... 34 II.4.4.2. The Degradation Arm of Proteostasis .................................................................. 36 II.4.4.2.1. The Ubiquitin-Proteasome System (UPS) ..................................................... 37 II.4.4.2.2. Autophagy Mediated Degradation................................................................. 40 II.4.5. Small Molecule Regulators of Proteostasis ................................................................ 42 II.4.6. Proteostasis in Aging .................................................................................................. 43 II.4.7. Sensors of Proteostasis ................................................................................................ 46 III Aim of the Study .................................................................. 48 IV Materials and Methods ......................................................... 49 IV.1. Materials........................................................................................................................... 49 i Contents IV.1.1. Chemicals .................................................................................................................. 49 IV.1.2. Materials and Instruments ......................................................................................... 53 IV.1.3. Enzymes .................................................................................................................... 55 IV.1.4. Bacterial Strains ........................................................................................................ 55 IV.1.5. Softwares ................................................................................................................... 55 IV.1.6. Kits Used ................................................................................................................... 56 IV.1.7. Antibodies ................................................................................................................. 56 IV.2. Media and Buffers ............................................................................................................ 57 IV.2.1. Media ......................................................................................................................... 57 IV.2.2. Buffers ....................................................................................................................... 57 IV.3. Molecular Cloning Methods ............................................................................................ 58 IV.3.1. Preparation and Transformation of Chemically Competent E. coli Cells ................. 59 IV.3.2. PCR Amplification .................................................................................................... 59 IV.3.3. Site-Directed Mutagenesis PCR ................................................................................ 60 IV.3.4. DNA Restriction and Ligation .................................................................................. 60 IV.3.5. Generation of Expression Constructs ........................................................................ 61 IV.3.5.1. Constructs ........................................................................................................... 64 IV.4. Protein Analytical Methods ............................................................................................. 66 IV.4.1. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) .......... 66 IV.4.2. Western Blotting........................................................................................................ 66 IV.4.3. Protein Quantification by Bradford’s Assay ............................................................. 67 IV.5. In Vitro Characterization of Fluc Mutants ....................................................................... 67 IV.5.1. Thermal Denaturation of Fluc Variants in RRL ........................................................ 67 IV.5.2. Measurement of Fluc Specific Activity in RRL ........................................................ 67 IV.5.3. Assessment of Co-translational Folding Efficiency of Fluc Variants in RRL .......... 68 IV.5.4. Limited Proteolysis by Proteinase K ......................................................................... 68 IV.5.5. Guanidinium Chloride Denaturation ......................................................................... 68 IV.5.6. Cold Denaturation Method .......................................................................................
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