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Expanding the Genetic Code to Study the Structure and Interactions of Proteins

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Expanding the Genetic Code to Study the Structure and Interactions of Proteins Expanding the Genetic Code to Study the Structure and Interactions of Proteins Dissertation submitted for the degree of Doctor of Natural Sciences (Dr. rer. nat.) Presented by Moritz Johannes Schmidt at the Faculty of Sciences Department of Chemistry Date of the oral examination: 13.11.2015 First referee: Prof. Dr. Daniel Summerer Second referee: Prof. Dr. Malte Drescher Third referee: Prof. Dr. Karin Hauser Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-311789 This work was prepared from April 2012 to July 2015 in the group of Dr. Daniel Summerer (Chemical Biology of the Genetic Code) at the university of Konstanz. It was funded by the „Graduiertenschule Chemical Biology“ and the Aventis Foundation and the „Stiftung Stipendien-Fonds des Verbandes der Chemischen Industrie (VCI)“ with a „Hoechst Doktorandenstipendium“. [1] Reproduced in part with the permission of M. J. Schmidt, J. Borbas, M. Drescher and D. Summerer, J. Am. Chem. Soc., 2014, 136, 1238-41. Copyright 2014 American Chemical Society. [2] Reproduced in part with the permission of M. J. Schmidt, A. Weber, M. Pott, W. Welte and D. Summerer, ChemBioChem, 2014, 15, 1755-1760. Copyright 2014 Wiley-VCH. [3] Reproduced in part with the permission of M. J. Schmidt and D. Summerer, Angew. Chem. Int. Ed., 2013, 52, 4690-4693. Copyright 2013 Wiley-VCH. [4] Parts of this work are published in: [1] M. J. Schmidt, A. Fedoseev, D. Summerer and M. Drescher, „Genetically Encoded Spin Labels for in vitro and in-cell EPR studies of native Proteins” Methods Enzymol., 2015, in press. [2] M. J. Schmidt, J. Borbas, M. Drescher and D. Summerer, „A Genetically Encoded Spin Label for Electron Paramagnetic Resonance Distance Measurements” J. Am. Chem. Soc., 2014, 136, 1238-41. [3] M. J. Schmidt, A. Weber, M. Pott, W. Welte and D. Summerer, „Structural Basis of Furan-Amino Acid Recognition by a Polyspecific Aminoacyl-tRNA-Synthetase for its Genetic Encoding in Human Cells” ChemBioChem, 2014, 15, 1755-1760. [4] M. J. Schmidt, D. Summerer, „Red-Light-Controlled Protein-RNA Crosslinking with a Genetically Encoded Furan“ Angew. Chem. Int. Ed., 2013, 52, 4690-4693. Other Publications: [5] M. Pott, M. J. Schmidt, D. Summerer, „Evolved sequence contexts for highly efficient amber suppression with noncanonical amino acids” ACS Chem. Biol., 2014 , 12, 2815-2822.[5] [6] G. Kubik, M. J. Schmidt, J. E. Penner, D. Summerer, „Programmable and Highly Resolved in vitro Detection of 5-Methylcytosine by TALEs” Angew. Chem. Int. Ed. 2014 , 53, 6002-6006.[6] [7] M. J. Schmidt and D. Summerer, „Genetic Code Expansion as a Tool to Study Regulatory Processes of Transcription” Front. Chem., 2014, 2 (7), 1-11.[7] [8] M. J. Schmidt, D. Summerer, „A Need for Speed: Genetic Encoding of Rapid Cycloaddition Chemistries for Protein Labelling in Living Cells” ChemBioChem, 2012, 13, 1553-57.[8] Danksagung Ich möchte mich zuallererst bei Dr. Daniel Summerer für die Aufnahme in seine Arbeitsgruppe und das spannende Promotionsthema bedanken. Die ausgezeichnete Betreuung, das in mich gesetzte Vertrauen und der Freiraum für kreative Problemlösungen und Projektideen haben mich während meiner Doktorarbeit ungemein motiviert und mir Freude an der Arbeit bereitet. Für jede wissenschaftliche und nicht- wissenschaftliche Unterstützung möchte ich mich herzlichst bedanken. Ich möchte mich bei Prof. Dr. Malte Drescher für die Übernahme des Zweitgutachtens bedanken. Darüber hinaus möchte ich mich für die spannende und erfolgreiche Kollaboration, die Diskussisonsrunden und für jegliche Unterstützung bedanken. Ich danke Prof. Dr. Andreas Marx für die Übernahme des Prüfungsvorsitzes und zusammen mit Prof. Dr. Martin Scheffner für die wissenschaftlichen Diskussionsrunden im Rahmen meines Promotionskomitees. Ich danke Julia Borbas, Christian Hintze, Artem Fedoseev und Martin Spitzbarth, sowie allen Mitgliedern der AG Drescher, sowie für Prof. Dr. Christine Peter und Dennis Bücker die tolle Zusammenarbeit. Ich bedanke mich bei Prof. Dr. Wolfram Welte und Anne-Marie Weber für die tolle Zusammenarbeit. Ich danke der Graduiertenschule „Chemical Biology“ für finanzielle Unterstützung und Fortbildungskurse, sowie der Aventis Foundation und der Stiftung Stipendien-Fonds des Verbandes der Chemischen Industrie (VCI) für die finanzielle Unterstützung. Ich möchte mich bei allen Mitarbeitern der Arbeitsgruppe Summerer für die tolle Zeit, entspannte und lustige Arbeitsatmosphäre und jegliche Hilfsbereitschaft bedanken. Im Speziellen danke ich Grzegorz Kubik, Sarah Flade, Preeti Rathi, Sara Maurer, Sabrina Batke, Anna Burrichter und Moritz Pott für die einmalige Zeit die wir zusammen hatten. Ein großes Dankeschön gilt Eugenia Hoffmann und Moritz Pott für das Korrekturlesen meiner Arbeit. Moritz Pott danke ich zudem für die tolle Kollaboration und Zellkulturexperimente. Ich bedanke mich bei bei allen Bachelor- und Masterstudenden sowie Praktikanten, die mich in meiner Zeit begleitet haben. Im Speziellen Annika Röcker, Matthias Mack und Irina Helmle. Ich danke all meinen Freunden für die tolle und unvergessliche Zeit in Konstanz. Der Stammelf der WG in der Werner-Sombart Straße: Matthias, Timo und Linda. Herzlichst danke ich meiner Familie und meinen lieben Eltern für ihre grenzenlose Unterstützung in allen Lebenslagen. Vielen Dank, dass ihr immer für mich da wart. Ich danke Anja für die wunderschöne gemeinsame Zeit und deine Unterstützung! Table of Contents 1. Introduction ............................................................................................................ 1 1.1 Expansion of the Genetic Code ......................................................................... 1 1.1.1 Protein Biosynthesis ................................................................................... 1 1.1.2 Artificial Expansion of the Genetic Code .................................................... 1 1.1.3 Evolution of Orthogonal Aminoacyl-tRNA Synthetases .............................. 4 1.1.4 Pyrrolysyl-tRNA Synthetase ....................................................................... 7 1.2 Photocrosslinking Chemistries .......................................................................... 9 1.2.1 Genetically Encoded Photocrosslinkers ..................................................... 9 1.2.2 Furan-based Photocrosslinking Chemistry ............................................... 12 1.2.3 HIV-1 TAR-TAT Interaction ...................................................................... 14 1.3 Towards in Cell EPR Spectroscopy of Biomolecules ....................................... 17 1.3.1 Site-Directed Spin-Labeling of Proteins .................................................... 18 1.3.2 Thioredoxin .............................................................................................. 20 2. Aim of This Work .................................................................................................. 22 3. Results and Discussion ....................................................................................... 23 3.1 A Genetically Encoded Furan: A Red-Light-Controlled Photocrosslinker ......... 23 3.1.1 Introduction .............................................................................................. 23 3.1.2 Results and Discussion ............................................................................ 24 3.2 A Genetically Encoded Spin-Label .................................................................. 41 3.2.1 Introduction .............................................................................................. 41 3.2.2 Results and Discussion ............................................................................ 42 4. Summary and Outlook ......................................................................................... 65 5. Zusammenfassung und Ausblick ....................................................................... 68 6. Materials and Methods ......................................................................................... 72 6.1 Chemical Synthesis ......................................................................................... 72 6.1.1 General Information ................................................................................. 72 6.1.2 Chemicals ................................................................................................ 72 6.1.3 Synthesis Overview of H-Lys(Furan)-OH 3............................................... 73 6.1.4 Synthesis of 2-Hydroxyethyl-furan 1 ......................................................... 73 6.1.5 Synthesis of Boc-Lys(Furan)-OH 2 ........................................................... 74 6.1.6 Synthesis of H-Lys(Furan)-OH 3 .............................................................. 75 6.1.7 Synthesis Overview of H-Lys(Nitroxide)-OH 6 .......................................... 76 6.1.8 Synthesis of 3-Hydroxymethyl-1-oxy-2,2,5,5-tetramethylpyrroline 4 ......... 76 6.1.9 Synthesis of Boc-Lys(Nitroxide)-OH 5 ...................................................... 77 6.1.10 Synthesis of H-Lys(Nitroxide)-OH 6 ......................................................... 78 6.1.11 Synthesis of H-Lys(Hydroxylamine)-OH 7 ................................................ 78 6.1.12 Alternative Synthesis of H-Lys(Nitroxide)-OH 6 ........................................ 79 6.1.13 Synthesis of 3-Hydroxymethyl-1-oxy-2,2,5,5-tetramethylpyrroline 4 ......... 79 6.1.14 Alternative Synthesis of Boc-Lys(Nitroxide)-OH 5 .................................... 80 6.1.15 Synthesis
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