Proteins are important biomolecules. Knowledge of their conformations and conformational dynamics is essential to understand their biological functionality. While there exist established methods to investigate the structure of a protein, methods to explore the dynamics like protein folding, allosteric regulations and catalysis are relatively new and still in development. Processes which happen on a millisecond or slower time scale can be eciently studied by NMR spectroscopy. To investigate

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... r dynamical processes, other spectroscopic methods are needed. In this work, time-resolved infrared spectroscopy on small peptides reveals information about dynamics on the picosecond time scale. Twodimensional infrared spectroscopy (2D-IR) obtains structural information like coupling and relative dipole orientations between molecular groups in a protein/peptide in equilibrium. This method can also be applied to a system being not in equilibrium, e.g a peptide undergoing conformational change (transient 2D-IR). Transient (2D-)IR experiments require a trigger to induce structural changes at a well dened time. This trigger can be e.g. a temperature-jump or a molecular photoswitch in the peptide. In this work, the substitution of the carbonyl oxygen in one peptide bond by a sulfur atom is used to create a photoswitch in the peptide. The resulting thioamide bond then can be excited selectively by ultraviolet light ( ! excitation red-shifted in regard with an amide bond). This moiety then isomerizes and forces the peptide to change its structure. To investigate the functioning of the thioamide bond as photoswitch, N-Methylthioacetamide has been investigated by dierent methods revealing trans ! cis isomerization with a high yield which is completed within one nanosecond. In this work, a thioamide containing peptide, Boc-Ala-Pro-(SC-NH)-Aib-Ala-OMe, is synthesized. This thioxopeptide has well-resolved bands in the amide I region which makes the interpretation of transient spectra easier. Furthermore, it contains a -Pro-Aib-amino acid sequence which is known to form stable -turn structure, characterized by a i ! i+3 hydrogen-bond. The breaking of this hydrogen-bond, induced by the photoswitch, is investigated by transient infrared spectroscopy (UV-pump/IR-probe spectroscopy). The dynamics is compared to other -Aib-containing thioxopeptides which tend to form -turns as well. All thioxopeptides show very similar photoisomerization dynamics, while there are signicant dierences for the relaxation in the thermal ground state. Furthermore, structural properties are revealed from the time-resolved measurements, which show very rigid structure in the inner part of all the thioxopeptides (-(SC-NH)-Aib-), while the tails are exible. Finally, the investigation on the i ! i+3 hydrogen-bond breaking of thioxopeptide Boc-Ala-Pro-(SC-NH)-Aib-Ala-OMe is extended to transient 2D-IR spectroscopy. For detailed interpretation of the spectra, they are compared to additionally performed computer simulations. Abstract Proteins are important biomolecules. Knowledge of their conformations and conformational dynamics is essential to understand their biological functionality. While there exist established methods to investigate the structure of a protein, methods to explore the dynamics like protein folding, allosteric regulations and catalysis are relatively new and still in development. Processes which happen on a millisecond or slower time scale can be efficiently studied by NMR spectroscopy. To investigate faster dynamical processes, other spectroscopic methods are needed. In this work, time-resolved infrared spectroscopy on small peptides reveals information about dynamics on the picosecond time scale. Twodimensional infrared spectroscopy (2D-IR) obtains structural information like coupling and relative dipole orientations between molecular groups in a protein/peptide in equilibrium. This method can also be applied to a system being not in equilibrium, e.g a peptide undergoing conformational change (transient 2D-IR). Transient (2D-)IR experiments require a trigger to induce structural changes at a well defined time. This trigger can be e.g. a temperature-jump or a molecular photoswitch in the peptide. In this work, the substitution of the carbonyl oxygen in one peptide bond by a sulfur atom is used to create a photoswitch in the peptide. The resulting thioamide bond then can be excited selectively by ultraviolet light (π → π * excitation red-shifted in regard with an amide bond). This moiety then isomerizes and forces the peptide to change its structure. To investigate the functioning of the thioamide bond as photoswitch, N -Methylthioacetamide has been investigated by different methods revealing trans → cis isomerization with a high yield which is completed within one nanosecond. In this work, a thioamide containing peptide, Boc-Ala-Pro-ψ(SC-NH)-Aib-Ala-OMe, is synthesized. This thioxopeptide has well-resolved bands in the amide I region which makes the interpretation of transient spectra easier. Furthermore, it contains a -Pro-Aib-amino acid sequence which is known to form stable β-turn structure, characterized by a i → i+3 hydrogen-bond. The breaking of this hydrogen-bond, induced by the photoswitch, is investigated by transient infrared spectroscopy (UV-pump/IR-probe spectroscopy). The dynamics is compared to other -Aib-containing thioxopeptides which tend to form β-turns as well. All thioxopeptides show very similar photoisomerization dynamics, while there are significant differences for the reix laxation in the thermal ground state. Furthermore, structural properties are revealed from the time-resolved measurements, which show very rigid structure in the inner part of all the thioxopeptides (-ψ(SC-NH)-Aib-), while the tails are flexible. Finally, the investigation on the i → i+3 hydrogen-bond breaking of thioxopeptide Boc-Ala-Pro-ψ(SC-NH)-Aib-Ala-OMe is extended to transient 2D-IR spectroscopy. For detailed interpretation of the spectra, they are compared to additionally performed computer simulations. x Zusammenfassung Proteine sind wichtige Biomoleküle. Kenntnisseüber deren Konformation und Konformationsdynamik ist essentiell, um ihre biologische Funktion zu verstehen. Während Methoden zur Strukturbestimmung von Proteinen etabliert sind, sind deren zur Untersuchung der Dynamik wie Proteinfaltung, allosterische Regulationen und Katalyse relativ neu und noch in Entwicklung. Prozesse, welche auf einer Zeitskala von Millisekunden oder langsamer stattfinden, können mit NMR-Spektroskopie untersucht werden. Um schnellere Prozesse zu untersuchen, werden andere spektroskopische Methoden gebraucht. In dieser Arbeit wird zeitaufgelöste Infrarotspektroskopie gebraucht, um Informationenüber die Dynamik von kleinen Peptiden auf einer Pikosekunden-Zeitskala zu erhalten. Zweidimensionale Infrarotspektroskopie (2D-IR) erlaubt es, Strukturinformationen wie Kopplungen und relative Dipolorientierungen zwischen molekularen Gruppen in Proteinen/ Peptiden im Gleichgewicht zu untersuchen. Diese Methode kann auf Systeme, welche nicht im Gleichgewicht sind, z.B. ein Peptid während einer Konformationsänderung, angewandt werden (Transiente 2D-IR). Diese Experimente benötigen einen Auslöser, um die Konformationsänderung zu einem definierten Zeitpunkt zu induzieren. Dieser Auslöser kann zum Beispiel ein Temperatursprung oder ein molekularer Fotoschalter in einem Peptid sein. In dieser Arbeit wird die Ersetzung eines Carbonyl-Sauerstoffatom in einer Peptidbindung mit einem Schwefelatom benutzt, um einen Fotoschalter zu erzeugen. Die so erhaltene Thioamidbindung kann dann mit ultraviolettem Licht selektiv angeregt werden (die π → π * Anregung is gegenüber einer Amidbindung rotverschoben). Dieser Teil des Moleküls isomerisiert dann und zwingt das Peptid, seine Struktur zuändern. Um die Funktionalität der Thioamidbindung zu untersuchen, wurde N-Methylthioacetamid mit verschiedenen Methoden untersucht, was eine trans → cis Isomerisierung mit hoher Ausbeute zeigte, die innerhalb einer Nanosekunde vollendet ist. In dieser Arbeit wird ein thioamidhaltiges Peptid, Boc-Ala-Pro-ψ(SC-NH)-Aib-Ala-OMe, synthetisiert. Dieses Thioxopeptid hat aufgelöste Banden in der Amid I-Region, was die Interpretation von transienten Spektren leichter macht. Zudem enthält es eine -Pro-Aib-Aminosäuresequenz, von welcher man weiss, dass sie stabile β-Turns bildet, welche mit einer i → i+3 Wasserstoffbrücke charakterisiert sind. Das Aufbrechen dieser Wasserstoffbrücke, welche mit xi dem Fotoschalter herbeigeführt wird, wird mit transienter Infrarotspektroskopie untersucht (UV-pump/IR-probe Spektroskopie). Die Dynamik wird mit anderen -Aib-haltigen Thioxopeptiden verglichen, welche auch zur Bildung von β-Turns tendieren. Währen alle Thioxopeptide vegleichbare Fotoisomerisierungsdynamik zeigen, gibt es signifikante Unterschiede für die Relaxation im thermischen Grundzustand. Des Weiteren können strukturelle Eigenschaften mit den zeitaufgelösten Messungen untersucht werden, welche für alle Thioxopeptide eine starre Struktur im inneren Teil des Moleküls zeigen (-ψ(SC-NH)-Aib-), während deren Enden flexibel sind. Schliesslich wird die Untersuchungüber das Aufbrechen der i → i+3 Wasserstoffbrücke in Boc-Ala-Pro-ψ(SC-NH)-Aib-Ala-OMe auf transiente 2D-IR Spektroskopie erweitert. Für die genaue Interpretation der Spektren werden sie mit zusätzlich durchgeführten Computersimulationen verglichen. xii Chapter 1