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Technische Universität München TECHNISCHE UNIVERSITÄT MÜNCHEN Department Chemie Labor für Synthetische Biochemie Expanding the chemical biology toolbox: Site-specific incorporation of unnatural amino acids and bioorthogonal protein labeling to study structure and function of proteins Susanne Veronika Mayer Vollständiger Abdruck der, von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzende/-r: Prof. Dr. Matthias Feige Prüfende/-r der Dissertation: 1. Prof. Dr. Kathrin Lang 2. Dr. Stephan Hacker, Junior Group Leader Die Dissertation wurde am 16.07.2019 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 30.07.2019 angenommen. ABSTRACT The fundamental understanding of biological processes at a molecular level constitutes the basis for the development of new drugs and therapies against diseases. Endowing biomolecules such as proteins with physical probes and chemical reporters provides an opportunity to elucidate these molecular mechanisms, by providing information on structure, dynamics, function and localization of labeled proteins. An especially exciting area consists in the development of new tools for the introduction of chemical reporters into proteins in their native environment - the living cell. Combining biological and chemical methods has proven to be a powerful approach in developing such new tools. One example is genetic code expansion (GCE), which represents an elegant and potent approach to site-specifically and co-translationally install reporters or probes into a protein of interest. This is achieved by using orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that direct the incorporation of an unnatural amino acid (uAA) into a protein in response to an amber stop codon introduced in a gene of interest. An emerging area with enormous potential consists in the site-specific incorporation of uAAs that contain functional groups that allow subsequent labeling with biophysical probes at a single site in a protein of interest. These uAAs, due to their small size, only minimally perturb structure and function of the target protein and decorate the protein with a bioorthogonal handle for further chemical or biochemical modification. Upon exogenous addition of a reaction partner the so installed handle can be decorated chemoselectively in a modular manner. The Diels-Alder cycloaddition with inverse-electron demand (iEDDAC) between electron-poor tetrazines (dienes) and electron-rich strained alkenes or alkynes (dienophiles) has emerged as a very powerful representative of bioorthogonal labeling reactions. It occurs very rapidly under physiological conditions and generates stable, covalent linkages with the formation of gaseous nitrogen as the only, nontoxic byproduct. In this thesis, GCE in combination with iEDDAC was used as a basis for the development of new tools to study protein structure and dynamics and to induce site-specific protein labeling in living cells. We contribute to the broad spectrum of bioorthogonal protein modification reactions by designing a new, very rapid photo-inducible iEDDAC labeling reaction with spatio-temporal control. Hereby, a photo-labile cyclopropenone-caged dibenzoannulated bicyclononyne probe (photo-DMBO) specifically and chemoselectively reacts only upon decarbonylation, triggered at 365 nm, with novel tetrazine-based uAAs TetK and mTetK. Incorporation of these uAAs into proteins with a novel orthogonal aaRS/tRNA pair, allowed us to determine very fast on-protein rate-constants of ~50 M-1s-1 between photo-DMBO and mTetK-bearing sfGFP, which is significantly faster than other photo-induced labeling reactions. Protein labeling with fluorophore conjugates of photo-DMBO at low µM concentrations was performed on purified tetrazine-bearing proteins, within the E. coli proteome or on the surface of living E. coli in a photo-induced manner. i Furthermore, a new approach based on GCE and iEDDAC using a small, rigid tetrazine amino acid (TetF) to site-specifically introduce nitroxide spin labels bearing cyclopropene (Cp) moieties into proteins was developed during this thesis. In collaboration with the group of Michael Sattler (Department of Chemistry, TUM), we characterized the resulting spin- labeled protein by pulsed electron electron double resonance (PELDOR) and paramagnetic resonance enhancement (PRE) experiments, as well as molecular dynamics (MD) simulations to benchmark our site-specific spin labeling approach against existing cysteine-based site- directed spin labeling (SDSL) techniques. By introducing one or two spin labels into the double-stranded RNA (dsRNA)-binding protein Loquacious-PD (Loqs-PD), which recognizes and processes endogenous siRNA in D. melanogaster, we were able to determine distinct intra-protein distances by PELDOR upon binding to double-stranded RNA, indicating that this binding event induces an ordered orientation of the two otherwise independently acting dsRNA-binding domains of Loqs-PD. In collaboration with Henning Mootz (Institute for Biochemistry, Universität Münster), we have developed an enzyme-mediated approach for the deprotection of a masked natural amino acid to control and modulate enzymatic activity. The bulky phenylacetyl (Pac) group was chosen as protecting group for lysine (PacK) to locally perturb the structure of a target protein and thereby abolish its activity. Together with the Mootz group we have discovered two different enzymes, penicillin G acylase (PGA) and a sirtuin (SrtN) from Bacillus subtilis that are both able to facilitate the cleavage of the Pac group from model proteins as well as more complex multi-domain proteins, where PacK was installed via GCE. This is the first report of an artificial enzyme-triggered decaging approach. The mild conditions of our enzyme- mediated approach render it a valuable alternative to chemical or optical triggers for the modulation of protein function. In conclusion, we have added several novel and powerful approaches, based on a combination of GCE with bioorthogonal labeling and decaging reactions, to the chemical biology toolkit, which in the future will contribute to the elucidation and modulation of biological processes in in vivo cellular settings. ii ZUSAMMENFASSUNG Das grundlegende Verständnis von biologischen Prozessen auf molekularer Ebene bildet die Basis für die Entwicklung neuer Wirkstoffe und Therapien zur Bekämpfung von Krankheiten. Die Ausstattung von Biomolekülen wie Proteinen mit biophysikalischen und chemischen Sonden liefert Informationen über Struktur, Dynamik, Funktion und Ort des markierten Biomoleküls. Dies ermöglicht die Aufklärung dieser molekularen Mechanismen. Ein besonders interessantes Gebiet stellt dabei die Entwicklung neuer Methoden für die Einführung von chemischen Sonden in Proteine in ihrer natürlichen Umgebung - der lebenden Zelle - dar. Die Kombination biologischer und chemischer Methoden hat sich als wirkungsvolle Vorgehensweise für die Entwicklung solch neuer Methoden erwiesen. Ein Beispiel dafür ist die Erweiterung des genetischen Codes (GCE), die einen eleganten und effektiven Ansatz darstellt, um Sonden während der Proteinbiosynthese positionsspezifisch in ein Zielprotein einzubringen. Orthogonale Aminoacyl-RNA Synthetase (aaRS)/tRNA Paare dirigieren dabei den Einbau von unnatürlichen Aminosäuren (uAA) als Antwort auf ein genetisch installiertes Ambercodon in das Zielprotein. Der positionsspezifische Einbau von uAAs, die Funktionalitäten tragen, welche daraufhin an einer bestimmten Stelle im Protein mit biophysikalischen Sonden versehen werden können, beinhaltet dabei großes Potential. Wegen ihrer geringen Größe stören diese uAAs die Struktur und Funktion des Zielproteins nur geringfügig und statten dieses mit einer bioorthogonalen Gruppe aus, die durch Zugabe eines entsprechenden Reaktionspartners biochemisch oder chemoselektiv über das Baukastenprinzip weiter funktionalisiert werden kann. Die Diels-Alder Cycloaddition mit inversem Elektronenbedarf (iEDDAC) zwischen elektronenarmen Tetrazinen (Diene) und elektronenreichen, ringgespannten Alkenen oder Alkinen (Dienophile) hat sich dabei als äußerst wirkungsvoller Vertreter dieser Reaktionen zur bioorthogonalen Markierung herausgestellt. Sie verläuft sehr schnell unter physiologischen Bedingungen und erzeugt stabile, kovalente Verbindungen und als einziges, ungiftiges Nebenprodukt wird gasförmiger Stickstoff freigesetzt. In dieser Arbeit wurden auf der Basis von GCE und iEDDAC neue Werkzeuge für die Untersuchung von Proteinstruktur und -dynamik, sowie zur Aktivierung positionsspezifischer Markierung von Proteinen in lebenden Zellen entwickelt. Durch Konstruktion einer neuen, sehr schnellen, lichtinduzierbaren iEDDAC Markierungsreaktion mit räumlicher und zeitlicher Kontrolle haben wir zum breiten Spektrum bioorthogonaler Reaktionen für die Dekoration von Proteinen beigetragen. Dabei reagiert eine photolabile Cyclopropenon- geschützte, dibenzoannulierte Bicyclononin-Sonde (photo-DMBO) erst nach Decarbonylierung bei 365 nm spezifisch und chemoselektiv mit den neuen, auf Tetrazinen basierenden uAAs TetK und mTetK. Der Einbau dieser uAAs in Proteine mit einem neuen orthogonalen aaRS/tRNA Paar ermöglichte uns die Bestimmung einer sehr schnellen Geschwindigkeitskonstante am Protein von ~50 M-1s-1 zwischen photo-DMBO und mTetK- modifiziertem sfGFP, was deutlich schneller ist als andere lichtinduzierbare Modifikationsreaktionen. Die lichtaktivierte Markierung von Proteinen
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