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Dissertation Truebestein Final Structural and Biochemical Characterization of the Human Serine Protease HtrA1 Inaugural-Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Biologie und Geografie an der Universität Duisburg-Essen vorgelegt von Linda Trübestein aus Berlin April 2010 Gutachter: Prof. Michael Ehrmann, Prof. Peter Bayer Prüfungsdatum: 18.06.2010 III Parts of this work are included in the following publications: Tennstaedt, A., Truebestein, L., Hauske, P., Schmidt, N., Irle, I., Kaiser, M., Brandt, R., Sickmann, A., Egensperger, R., Baldi, A., Dehmelt, L. und Ehrmann M., „HtrA1 is a tau protease.“ (in revision in PNAS) Truebestein, L., Tennstaedt, A., Krojer, T., Hauske, P., Kaiser, M., Clausen, T., and Ehrmann, M., „Substrate binding remodels active site of HtrA1 and regulates protease activity.” (submitted to Nat Struc & Mol Biol) V Acknowledgements First of all I would like to thank Michael Ehrmann for his constant support and supervision during my PhD. Thanks for giving me the chance to work on this very interesting project and for all the trust you had in me. I am thankful to all the members in the Ehrmann Lab for creating a very nice working atmosphere, for all the discussions and the nice time we had. I want to thank Tim Clausen for giving me the great chance to work in his lab and for always giving me the feeling that I am very welcome. Thanks for all the motivation and the supervision during my PhD. Furthermore I would like to thank all the members of the Clausen Lab in Vienna. You were such fantastic colleges! I can hardly imaging to find something like this again. I want to thank Linn, Doris, Justyna, Flavia, Juliane, Sonja, Becky, Bastian, Jakob, Tobias, Robert, Anita and Alex for the warm welcome and for sharing lab space, equipment and knowledge so generously with me. I will never forget all the hours of chatting and laughing. It was a wonderful time. Especially I want to thank Tobias for all his support and his patience. You became never tired of discussing all my questions again and again and to keep me on the right track. I am very thankful to Justyna, Linn and Tobias for discussions and correcting my thesis. I want to thank Evi and Nina for always listening to my problems inside and outside the lab and for all their optimism and support during the ups and downs in my life. It makes life so much easier to have such good friends like you. I want to thank Roger for stepping into my life. You showed me what is important and how to keep the right balance. Nothing better than you could have happened to me. Finally I would like to thank my sister Nora, my parents and my step-parents with all my heart. Thanks for always believing in me and giving me the strength to go on. I can not imagine a happy life without you. VII Table of Contents ACKNOWLEDGEMENTS.................................................................................................................V TABLE OF CONTENTS .................................................................................................................. VII LIST OF FIGURES........................................................................................................................... X LIST OF TABLES............................................................................................................................ XI ABBREVATIONS.......................................................................................................................... XII 1 INTRODUCTION.................................................................................................................. 13 1.1 Protein quality control...................................................................................................................... 13 1.2 Proteolysis and proteolytic enzymes ................................................................................................ 14 1.3 Serine proteases............................................................................................................................... 16 1.3.1 The catalytic mechanism.................................................................................................................... 16 1.3.2 S1 specificity pocket........................................................................................................................... 18 1.4 PDZ domains .................................................................................................................................... 19 1.5 The HtrA-family of serine proteases ................................................................................................. 21 1.5.1 Family of bacterial HtrAs.................................................................................................................... 22 1.5.2 Family of human HtrAs ...................................................................................................................... 23 1.5.3 HtrA1.................................................................................................................................................. 23 1.5.4 HtrA2.................................................................................................................................................. 25 1.5.5 HtrA3.................................................................................................................................................. 26 1.6 Serine protease structures of the HtrA family .................................................................................. 27 1.6.1 Crystal structures of bacterial DegP................................................................................................... 27 1.6.2 Crystal structure of human HtrA2...................................................................................................... 31 2 AIMS.................................................................................................................................. 32 3 MATERIALS AND METHODS ................................................................................................ 33 VIII 3.1 Materials .......................................................................................................................................... 33 3.1.1 Chemicals and their suppliers ............................................................................................................ 33 3.1.2 Bacterial strains.................................................................................................................................. 33 3.1.3 Plasmids and vectors.......................................................................................................................... 34 3.1.4 Oligonucleotides for Cloning.............................................................................................................. 35 3.1.5 Media ................................................................................................................................................. 36 3.1.6 Antibiotics .......................................................................................................................................... 38 3.1.7 Proteins and enzymes........................................................................................................................ 38 3.1.8 Protein and nucleic acid standards .................................................................................................... 39 3.2 Methods........................................................................................................................................... 39 3.2.1 Microbiological methods ................................................................................................................... 39 3.2.2 Biochemical methods......................................................................................................................... 45 3.2.3 Crystallographic methods .................................................................................................................. 55 4 RESULTS............................................................................................................................. 60 4.1 Purification of HtrA1......................................................................................................................... 60 4.1.1 Purification of full length HtrA1......................................................................................................... 60 4.1.2 Purification of ∆N-HtrA1 and ∆N-HtrA1 S328A ...................................................................................... 63 4.1.3 Purification of the protease domain of HtrA1 (HtrA1-prot) .............................................................. 67 4.1.4 Purification of selenomethionine substituted ∆N-HtrA1................................................................... 67 4.2 Oligomeric state of HtrA1 in solution ............................................................................................... 69 4.2.1 Oligomeric state of HtrA1-prot .......................................................................................................... 69 4.2.2 Dynamic light scattering .................................................................................................................... 72 4.3 Isothermal Titration Calorimetry (ITC).............................................................................................. 74 4.4 Proteolytic activity of HtrA1 ............................................................................................................
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