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Structural and Functional Characterization of TMEM16 Family Members Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2016 Structural and functional characterization of TMEM16 family members Lim, Novandy Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-129639 Dissertation Published Version Originally published at: Lim, Novandy. Structural and functional characterization of TMEM16 family members. 2016, University of Zurich, Faculty of Science. Structural and Functional Characterization of TMEM16 Family Members Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Novandy Karunia Lim aus Indonesien Promotionskomitee Prof. Dr. Raimund Dutzler (Vorsitz) Prof. Dr. Markus Seeger Prof. Dr. Martin Jinek Zürich, 2016 Acknowledgements I would like to take this opportunity to thank all the people who have supported and helped me throughout my stay and project in the lab. Firstly, I would like to thank Prof. Raimund Dutzler for the opportunity to work on this highly exciting project. I am sincerely grateful for his constant support and his faith in me throughout my time in his group. I would like to thank Prof. Martin Jinek and Prof. Markus Seeger for being part of my thesis committee. I would like to thank Janine Brunner and Stephan Schenck for the helpful discussion on TMEM16 project. A big thank you to Alessia Dürst too for helping us with the homologue screen during her Masters thesis. I am grateful to all the past and present members of the Dutzler for their friendship, support and scientific discussions. In particular to Yvonne, I can’t thank you enough for the support and for always looking out for me. Thank you for bearing the brunt of the lab to make our lives here as pleasant as it could be. I would also like to thank the infrastructure team in Department of Biochemistry. Special thanks to: Beat Blattmann and Celine Stutz-Ducommun from the protein crystallization facility for their help with crystallization trials. IT department and Workshop: for their fast troubleshooting with the frequent spontaneous problems in the lab. Sascha Weidner: for taking the initiative to invent the ultimate cell lyser, HPL6, and taking care of its maintenance. I want to thank my friends outside the lab environment. The Thursday Frisbee ASVZ group always ensured that I had a little weekly workout to clear the mind. Last but not least, thank you to my family for their constant support despite being half the world away. I Contents Acknowledgements .......................................................................................................................................... I List of Figures ................................................................................................................................................. IV List of Tables .................................................................................................................................................... V List of Abbreviations ....................................................................................................................................... VI Abstract ......................................................................................................................................................... VII Zusammenfassung .......................................................................................................................................... IX 1. Introduction ................................................................................................................................................. 1 1.1 Ion transport in cell homeostasis .......................................................................................................... 1 1.2 Chloride channels: Overview................................................................................................................. 2 1.3. Ca2+-activated chloride channel (CaCC): Role in Physiology ................................................................ 5 1.4. Members of the TMEM16 protein family mediate are CaCCs ............................................................. 7 1.4.1. TMEM16A and B: Ca2+-activated Cl channels ............................................................................... 9 1.4.2. TMEM16F: A Ca2+ activated channel and lipid scramblase ......................................................... 10 1.4.3. Other mammalian TMEM16s ...................................................................................................... 11 1.4.4. Fungal TMEM16s ......................................................................................................................... 11 1.5. Aim and Outline of Thesis .................................................................................................................. 13 2. Result ......................................................................................................................................................... 14 2.1 Selection of TMEM16 homologues for structural studies................................................................... 14 2.2 Expression, Purification and Crystallisation Screening towards a Structural Characterisation .......... 18 2.2.1 General Strategy using GFP-based expression and purification pipeline .................................... 18 2.2.2 Small scale expression screening of homologues in S. cerevisiae ............................................... 19 2.2.3 Scale-up and purification of positive hits from the expression screen ........................................ 22 2.2.4 Optimisation of nfTMEM16 ......................................................................................................... 23 2.2.5. Crystallization of nfTMEM16 ....................................................................................................... 26 2.2.6 Summary of Screening ................................................................................................................. 28 2.3 Functional study of TMEM16 .............................................................................................................. 30 2.3.1 Learning from Structure of nhTMEM16 ....................................................................................... 30 2.3.2. Investigating nhTMEM16 potential role as an ion channel ........................................................ 31 2.4 Functional investigations of TMEM16 proteins .................................................................................. 35 2.4.1 Learning from the Structure of nhTMEM16 ................................................................................ 35 2.4.2. Investigating a potential role of nhTMEM16 as ion channel ...................................................... 36 2.4.3 Structure-function relationships of the mouse CaCC mTMEM16A ............................................. 42 2.4.3.1 Characterisation of mTMEM16A by electrophysiology ........................................................ 42 2.4.3.2 Role of the conserved Ca2+-binding site in the activation of ion conduction ....................... 45 2.4.3.3 Investigations on the ion conduction pathway .................................................................... 52 Article: X-Ray structure of a calcium-activated TMEM16 lipid scramblase…………………………………….…..…….60 II Article: Independent activation of ion conduction pores in the double-barreled calcium activated chloride channel TMEM16A…………………………………………………………………………………………………………………………………..79 3. Method ...................................................................................................................................................... 96 4. Discussion ................................................................................................................................................ 101 4.1. Ca2+-binding site ............................................................................................................................... 102 4.2 Transport pathway in TMEM16 proteins .......................................................................................... 104 4.3 Potential mechanisms of channel activation .................................................................................... 106 4.4 Outlook .............................................................................................................................................. 107 CURRICULUM VITAE .................................................................................................................................... 108 References ................................................................................................................................................... 110 III List of Figures Figure 1 Cl− signaling. .............................................................................................................................................. 2 Figure 2 Scheme of construct Design for homologue expression screening in S. cerevisiae. ............................... 18 Figure 3 FSEC profiles of selected TMEM16 family members. .............................................................................. 21 Figure 4 Protein purification of selected homologues. ......................................................................................... 23 Figure 5 Truncation of nfTMEM16 terminal domains.
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