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Investigation of Protein-Ligand Complexes by Native Mass Spectrometry and Ion Mobility-Mass Spectrometry

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Investigation of Protein-Ligand Complexes by Native Mass Spectrometry and Ion Mobility-Mass Spectrometry Investigation of Protein-Ligand Complexes by Native Mass Spectrometry and Ion Mobility-Mass Spectrometry Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Melanie Göth from Amberg, Germany 2017 The work reported in this thesis was performed from September 2013 to October 2017, at the Freie Universität Berlin and at the Fritz Haber Institute of the Max Planck Society of Berlin, under the supervision of Prof. Dr. Kevin Pagel. 1st Reviewer: Prof. Dr. Kevin Pagel Freie Universität Berlin 2nd Reviewer: Prof. Dr. Christoph A. Schalley Freie Universität Berlin Date of Defense: 7.12.2017 i ii Danksagung Viele Leute haben mich in den letzten vier Jahren auf meinem Weg begleitet und in unterschiedlichster Form dazu beigetragen, dass ich diese Arbeit erstellen konnte. Zuallererst möchte ich mich bei meinem Erstgutachter und Betreuer Prof. Dr. Kevin Pagel bedanken. Danke, für die Aufnahme in deine Gruppe und für dieses sehr vielseitige und spannende Thema. Ich bin sehr dankbar für die Freiheiten, die du mir für meine Forschung gegeben hast, aber auch gleichzeitig für die Betreuung und Ratschläge, die immer genau zur richtigen Zeit kamen. Danke auch für die Möglichkeit, dass ich zahlreiche Konferenzen, sowohl im In- als auch im Ausland, besuchen durfte und somit meinen Erfahrungsschatz erweitern konnte. Weiterhin möchte ich Prof. Dr. Christoph Schalley danken für die Übernahme des Zweitgutachters meiner Dissertation. Natürlich wäre die heutige Arbeit nicht ohne die nötige Finanzierung möglich gewesen. Aus diesem Grund möchte ich mich bei der Bayer AG für die Finanzierung meiner Doktorandenstelle bedanken und bei allen Leuten, die daran beteiligt waren: Dr. Anke Müller- Fahrnow und Dr. Andreas Becker, die dieses Kooperationsprojekt ermöglicht haben. Dr. Volker Badock, mein direkter Ansprechpartner und Betreuer bei Bayer, der alle Hebel in Bewegung gesetzt hat, damit ich ein paar Monate im Rahmen eines Projekts direkt in den Laboren von Bayer forschen durfte. Danke für die Möglichkeit, einmal in die tägliche Routine der pharmazeutischen Industrie zu schnuppern. Ich fand die Zeit bei euch sehr spannend und habe viel gelernt! Danke auch für die schnellen Antworten und Ratschläge, das mehrmalige Korrekturlesen und natürlich auch für das ein oder andere Treffen im Biergarten, bei denen wir wissenschaftliche und nicht-wissenschaftliche Themen über einem Bier besprochen haben. Ein großes Dankeschön geht auch an Dr. Benno Kuropka, den ich zunächst also Postdoc bei Bayer kennengelernt habe. Es hat mir sehr gut gefallen mit dir zu arbeiten, danke für deine Geduld und ebenso danke für nette Mittagspausen, sei es bei Bayer oder jetzt an der FU. Danke auch, dass du so engagiert mit unserem Paper warst und auch für das Korrekturlesen meiner Arbeit. An dieser Stelle möchte ich auch Dr. Jörg Weiske, Dr. Hanna Meyer und der kompletten Massenspektrometrieabteilung danken für die freundliche Aufnahme, die netten Gespräche und für eure Zeit, Rat und Tat. Als nächstes möchte ich dem Fritz-Haber-Institut und allen Mitgliedern der Molekülphysik, insbesondere Prof. Gerard Meijer und Prof. Dr. Gert von Helden danken für die Finanzierung meines ersten Promotionsjahres und dass ich auch danach noch als Gast bleiben durfte und meinen wunderbaren Schreibtisch behalten durfte. Ein großes Dankeschön geht an alle Mitglieder der Pagel und von Helden Gruppe. Ihr habt diese Zeit zu einer unvergesslichen iii Schönen gemacht! Die freundschaftliche Atmosphäre, unser guter Zusammenhalt und der wissenschaftliche Input, aber auch abendliche Sitzungen auf der MP-Terrasse und unsere gemeinsamen Skikurztrips oder sonstige Unternehmungen haben unter anderem dazu beigetragen, dass ich mich von Anfang an sofort wohl gefühlt habe. Besonders möchte ich auch meinen „Office Buddies“ Jakob, Leo und Frank danken für die lustigen, die Aha- und auch die stillen Momente. Danke Johanna, beste Konferenzzimmerteilpartnerin überhaupt! Ich könnte jetzt weiter seitenweise zu Jedem von euch etwas schreiben, warum ich euch dankbar bin und froh bin euch kennen gelernt zu haben. Leider sprengt das den Rahmen und deswegen sag ich es euch einfach beim nächsten Bier persönlich. Tausend Dank auf jeden Fall für eure Unterstützung während der letzten Monate! Weiterhin möchte ich allen meinen Bachelorstudenten und Forschungspraktikanten für euren Fleiß und eure gute Arbeit danken. Danke auch an die Core-Facility der FU, insbesondere an Dr. Andreas Springer und Fabian Klautzsch fürs Ultima-sitten, fürs Troubleshooting und auch sonst für die netten Pläuschchen zwischendurch. Während meiner Zeit als Doktorandin durfte ich mit vielen hervorragenden Wissenschaftlern zusammenarbeiten: Thank you, Dr. Frederik Lermyte and Prof. Dr. Frank Sobott for interesting insights in ETD-MS and a (culinary) guided tour through Antwerp. Dankeschön, Dr. Oren Moscovitz for infinite hours of measuring and learning that some proteins are just as hard as stones and won’t break, not even when you put them under pressure ;). Measuring with you was so much fun (although a lot did not work) and who knows, someday we have our natce-paper together (don’t stop believing). Thank you for motivating me and being a friend. Danke Dr. Olaf Jahn, dass du Kevin und mich in die Calmodulin-Familie aufgenommen hast und uns damit die Tür zu einem sehr spannenden Projekt und einer tollen Zusammenarbeit geöffnet hast. Zuletzt möchte ich mich bei meiner Familie und meinen Freunden für eure bedingungslose Unterstützung bedanken. Ich bin so froh, dass ich euch hab und sehr sehr dankbar, sei es für Finanzspritzen während der Studienzeit, für offene Ohren und gutes Zureden oder einfach ab und zu eine Portion Ablenkung. Ich hätte das alles definitiv nicht ohne euch geschafft! Danke Peter, für deine Liebe, dein Verständnis und, dass du für mich da bist. iv Kurzzusammenfassung Intermolekulare Wechselwirkungen von Proteinen untereinander oder mit anderen Molekülen, nehmen eine Schlüsselrolle in allen Prozessen lebender Organismen ein. Aus diesem Grund stellen Proteine wichtige therapeutische Angriffspunkte (Targets) dar und ihre strukturelle Aufklärung ist essenziell für die Entwicklung neuer Wirkstoffe. Native Massenspektrometrie (MS) ist ein attraktives Werkzeug, um Proteine und deren Komplexe zu untersuchen. Moleküle werden sanft ionisiert und aus der Lösung in die Gasphase überführt mit dem Ziel, inter- und intramolekulare Wechselwirkungen und die dreidimensionale Struktur aufrechtzuerhalten. Die ionischen Spezies werden durch ihr Masse-zu-Ladungsverhältnis detektiert, was zu Informationen über Masse, Ladung und Stöchiometrie führt. Native MS ist mit anderen Gasphasentechniken, wie Ionenmobilitätsspektrometrie (IMS), gut kompatibel und die Kombination beider Methoden zu Ionenmobilitätsmassenspektrometrie (IM-MS) liefert weitere strukturelle Informationen über die Größe und Form eines Moleküls. In dieser Arbeit wurden Protein-Ligandkomplexe mittels nativer MS und nativer IM-MS mit dem Ziel untersucht, sie hinsichtlich ihres Potentials für die Hochdurchsatzanalyse der Wirkstoffentwicklung zu bewerten. Zuerst wurde native MS eingesetzt, um den Einfluss des Standardlösungsmittels Dimethylsulfoxid (DMSO) auf die Proteingasphasenstruktur, sowie die Protein-Ligandaffinität zu analysieren. Es wurde gezeigt, dass sich je nach DMSO Konzentration in der Probe die Ladungszustandsverteilung und somit vermutlich auch die Gasphasenstruktur eines Proteins ändert. Weiterhin verringerte sich die Affinität des Liganden mit steigender DMSO Konzentration. In einer zweiten Studie wurde das Potential von nativer MS für die fragmentbasierte Wirkstoffentwicklung getestet. In diesem Ansatz werden kleine Molekülfragmente gegen ein Target gescreent, wodurch im Vergleich zum Standard- hochdurchsatzverfahren höhere Erfolgsraten bei gleichzeitig geringerer Probenanzahl möglich sind. Ergebnisse von vier getesteten Targetproteinen zeigten, dass native MS im Moment noch keinen großen Durchsatz erlaubt, jedoch wichtige Einblicke in Protein-Ligandkomplexe liefern kann, welche durch andere Methoden nicht ohne Weiteres zugänglich sind. In einer weiteren Studie wurde in Kombination mit IMS der Einfluss der unmittelbaren Proteinumgebung auf dessen Gasphasenstruktur untersucht. Dafür wurden Kronenether- moleküle nichtkovalent gebunden, um positiv geladene Seitenketten zu mikrosolvatisieren und sie dadurch vor einem Zurückfalten auf das Proteinrückgrat zu bewahren. Mit Hilfe von Tandem MS und IM-MS wurden Bindungsstellen der Kronenether identifiziert und darüber hinaus gezeigt, dass bestimmte Proteinseitenketten die Gasphasenstruktur auch ohne Bindung von Kronenether stabilisieren können. In der letzten Studie wurde IM-MS erfolgreich als Methode zum Konformationsscreening von Protein-Peptidkomplexen eingesetzt. Die v Komplexe zeigten nur geringe strukturelle Unterschiede, weshalb sie mittels kollisions- induzierter Entfaltung und Dissoziation weiter untersucht und ihre Unterschiede in Stabilität und Entfaltungsverhalten analysiert wurden. Zusammenfassend stellen native MS und IM-MS wertvolle Werkzeuge zur Charakterisierung von Protein-Ligandwechselwirkungen dar. Gegenwärtig sind die Methoden zwar noch nicht für eine große Probenanzahl geeignet, aber durch ständige Entwicklungen werden sie in naher Zukunft sicherlich eine bedeutendere Rolle im Prozess des Wirkstoff- designs einnehmen können. vi Abstract Intermolecular interactions of proteins with each other or
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