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Development of ATP-Based Signaling Molecules Development of ATP-based Signaling Molecules Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr.rer.nat.) vorgelegt von Susanne Ermert aus Bad Neuenahr-Ahrweiler an der Mathematisch-Naturwissenschaftliche Sektion Fachbereich Chemie Konstanz, 2018 Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-fd0z6lhm2fea9 Tag der mündlichen Prüfung: 24. Juli 2018 1. Referent: Prof. Dr. Andreas Marx 2. Referent: Prof. Dr. Martin Scheffner “Was man zu verstehen gelernt hat, fürchtet man nicht mehr.“ Marie Curie I II This work was prepared from April 2015 to June 2018 in the group of Prof. Dr. Andreas Marx (Chair of Organic and Cellular Chemistry) at the University of Konstanz. It was funded by the SFB 969 (Chemical and Biological Principles of Cellular Proteostasis) and supported by the Konstanz Research School Chemical Biology. Parts of this work have been published and were reproduced with permission: S. Ermert, S. M. Hacker, A. Buntru, M. Scheffner, C. R. Hauck and A. Marx, ChemBioChem 2017, 18, 378-381. Copyright 2017 Wiley-VCH. S. Ermert, A. Marx and S. M. Hacker, Top Curr Chem 2017, 375: 28. Copyright 2017 Springer International Publishing Switzerland. III IV Publications and Communications Parts of this work have been published. Journal Publications: S. Ermert, S. M. Hacker, A. Buntru, M. Scheffner, C. R. Hauck and A. Marx, ChemBioChem 2017, 18, 378-381. “Different Enzymatic Processing of -Phosphoramidate and -Phosphoester-Modified ATP Analogues” S. Ermert, A. Marx and S. M. Hacker, Top Curr Chem 2017, 375: 28. “Phosphate-Modified Nucleotides for Monitoring Enzyme Activity” in “Phosphate Labeling in Chemical Biology” edited by Henning Jessen Poster Presentations: S. Ermert, S. M. Hacker and A. Marx at the Symposium on Molecular Architectures for Fluorescent Imaging of Cells, October 2017 in Karlsruhe, Germany S. Ermert, S. M. Hacker and A. Marx at the International Symposium on Bioorganic Chemistry (ISBOC-11), September 2017 in Konstanz, Germany S. Ermert, D. Hammler, S. M. Hacker and A. Marx at the XVIIth Symposium on Chemistry of Nucleic Acid Components, June 2017 in Český Krumlov, Czech Republic S. Ermert, S. M. Hacker and A. Marx at the CRC 969 Symposium – Two Days of Proteostasis, October 2016 in Konstanz, Germany S. Ermert, S. M. Hacker and A. Marx at the Biochemistry – Shaping the Molecules of Life: Chemical Biology of Nucleic Acid and Protein Modifications, July 2016 in Frankfurt, Germany S. Ermert, S. M. Hacker and A. Marx at the VII. Nucleinsäurechemie-Treffen, September 2015 in Berlin, Germany S. Ermert, S. M. Hacker and A. Marx at the Konstanz Research School Chemical Biology Annual Retreat and Poster Sessions, 2015, 2016 and 2017 in Gültstein, Rastatt, Bad Herrenalb and Konstanz, Germany V VI Danksagung Ich danke Prof. Dr. Andreas Marx für die Aufnahme in die Arbeitsgruppe, das vielfältige und interessante Themengebiet auf dem ich arbeiten durfte, das Ermutigen zur Promotion, das zahlreiche Feedback und die motivierenden Worte, wenn es einmal nicht so gut im Labor lief. Bei Prof. Dr. Christof R. Hauck und Prof. Dr. Martin Scheffner möchte ich mich für das Feedback im Rahmen des Thesis Commitees und die zielführenden Diskussionen bezüglich verschiedenster biologischer Fragestellungen bedanken. Desweiteren danke ich Prof. Dr. Martin Scheffner für die Übernahme des Zweitgutachtens und Prof. Dr. Jörg Hartig für die Übernahme des Prüfungsvorsitzes. Daniel Hammler danke ich für die gute Zusammenarbeit auf dem gemeinsamen Forschungsprojekt, die Bereitstellung der Phosphate-modifizierten ATP- und Ap4-Analoga, die Unterstützung bei der Synthese der Ap3A-Analoga, das Messen der HR-MS sowie zahlreiche und zielführende Diskussionen. Bei Dr. Stephan M. Hacker bedanke ich mich für jegliche Tipps, Tricks und Diskussionen während meiner gesamten Promotion. Bei sämtlichen Mitgliedern der AG Marx die mich während meiner Promotion begleitet haben möchte ich mich für die tolle Arbeitsatmosphäre, sämtliche Unterstützung und die schöne Zeit bedanken. Hierbei möchte ich besonders meine direkten Labor- und Bürokollegen Vanessa Radtke, Sandra Lange und Simon Geigges, sowie die „Ap3A-Leute“, als auch Alex Finke, Joachim Lutz, Heike Kropp und Maike Lehner hervorheben. Bei Anke Gerull und Meike Liebmann bedanke ich mich sehr für das Nachziehen der Sulfo-Cy Farbstoffe und verschiedener ATP-Analoga. Der AG Hauck und hier im speziellen Chris Paone und Timo Baade möchte ich für die gute Zusammenarbeit auf dem Projekt, die Hilfestellungen bei jeglichen biologischen Problemen und die tolle Arbeitsatmosphäre während meiner Experimente bei ihnen im Labor danken. Bei Yannik Leist, Verena Betzler und Chris Albrecht bedanke ich mich für die Mitarbeit an der Synthese verschiedener ATP-Analoga im Rahmen ihrer Bachelorarbeit bzw. ihres Mitarbeiterpraktikums. Luisa Huber danke ich für die Zusammenarbeit bei den Echtzeit- Fluoreszenzmessungen mit dem Ap3A-Analogon während ihrer Masterarbeit. Fabian Offensperger und der AG Scheffner danke ich für die Bereitstellung der Enzyme sowie die Hilfestellung bei der Durchführung der E6AP Autoubiquitinierungs-Assays. Dr. Alexander Buntru, Kathrin Kopp, Ann-Kathrin Fuchs und Susanne Feindler-Boeckh von der AG Hauck VII danke ich für das Bereitstellen des GST-FAK Substrates sowie der Enzyme für den v-Src Kinase Assay. Bei der „NMR Core Facility“ der Universität Konstanz, insbesondere bei Anke Friemel möchte ich mich für die durchgeführten Messungen bedanken. Dem SFB 969 „Chemische und biologische Prinzipien der zellulären Proteostase“ und der KoRS-CB danke ich für die finanzielle Unterstützung während meiner gesamten Promotion. Meinen Freunden in Konstanz danke ich nicht nur für die tolle Zeit während meines Studiums und der anschließenden Promotion, sondern auch, dass sie mir die Entscheidung für die Promotion in Konstanz sehr erleichtert haben. Zu guter Letzt danke ich meiner Familie, Christoph und meinen Freunden aus der Heimat. Ihr habt mich während meiner gesamten Zeit hier in Konstanz tatkräftig unterstützt und mich trotz der einigen Kilometer Entfernung regelmäßig besucht. Ohne euch wäre ich nicht so weit gekommen. VIII Table of Contents 1 General Introduction ..................................................................................................... 1 1.1 Adenosine Triphosphate (ATP) ........................................................................................ 1 1.2 Modified ATP Analogs to Study Enzyme Activity................................................................. 3 1.2.1 Phosphate-Modified ATP Analogs .............................................................................. 5 1.2.2 Doubly Labeled ATP Analogs for FRET Applications ...................................................... 7 1.2.3 Doubly Labeled Diadenosine Polyphosphate Analogs for FRET Applications ..................... 9 1.3 Theoretical Basis of Förster Resonance Energy Transfer (FRET) .......................................... 10 1.4 ATP-Consuming Enzymes .............................................................................................. 12 1.4.1 Ubiquitin-Activating Enzyme E1 (UBA1) .................................................................... 12 1.4.2 Focal Adhesion Kinase (FAK) ................................................................................... 13 1.4.3 Src Kinase ............................................................................................................ 15 2 Scope of the Thesis ..................................................................................................... 17 3 Synthesis of Phosphate-Modified ATP Analogs ─ Are they Substrates for UBA1, FAK or Src Kinase? ............................................................................................................................ 19 3.1 Synthesis of Phosphate-Modified ATP Analogs .................................................................. 19 3.1.1 Synthesis of -Phosphoramidate-Modified ATP Analogs ............................................... 21 3.1.2 Synthesis of Fluorescent Dyes ................................................................................. 23 3.2 Design and Selection of Assays to Detect Enzymatic Consumption of Different ATP Analogs ... 24 3.2.1 E6AP Autoubiquitination Assay to Study UBA1 Activity ............................................... 24 3.2.2 FAK In Vitro Assay ................................................................................................. 25 3.2.3 V-Src Kinase In Vitro Assay .................................................................................... 26 3.3 Stability of -Phosphate-Modified ATP Analogs ................................................................. 27 3.3.1 Stability of the Sulfo-Cy3-Labeled Phosphoramidate-Modified ATP Analog ..................... 27 3.3.2 Stability of the Atto488-Labeled Acyl Phosphate-Modified ATP Analog ............................ 28 3.4 Enzymatic Consumption of Different Phosphate-Modified ATP Analogs................................. 29 3.4.1 Consumption by UBA1 ............................................................................................ 29 3.4.2 Consumption by FAK-KD......................................................................................... 34 3.4.3 Consumption by v-Src-KD....................................................................................... 38 3.5 Conclusive Summary ...................................................................................................
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