How an enzyme achieves such high rates of catalysis in comparison to its solution counterpart reaction has baffled scientists for many decades. Much of our understanding of enzyme function is derived from research devoted to enzyme chemical reactions and analysis of static three-dimensional images of individual enzyme molecules. However, more recently, a role of protein dynamics in facilitating enzyme catalysis has emerged. It is often challenging to probe how protein motions are correlated to and impact on the catalytic cycle of enzymes. Nevertheless, this subject must be addressed to further our understanding of the roots of enzyme catalysis. Herein, this research question is approached by studying the link between protein domain dynamics and electron transfer chemistry in the diflavin oxidoreductase family of enzymes. Previous studies conducted on the diflavin oxidoreductases have implied a role of protein domain dynamics in catalysing electron transfer chemistry. However, diflavin oxidoreductase motions have not been experimentally correlated with mechanistic steps in the reaction cycle. To address these shortcomings, a 'real-time' analysis of diflavin oxidoreductase domain dynamics that occur during enzyme catalysis was undertaken. The methodology involved specific labelling of diflavin oxidoreductases (cytochrome P450 reductase, CPR, and neuronal nitric oxide synthase, nNOS) with external donor-acceptor fluorophores that were further used for time-resolved stopped-flow Förster resonance energy transfer (FRET) spectroscopy measurements. This approach to study enzyme dynamics was further linked with traditional UV-visible stopped-flow approaches that probed enzymatic electron transfer chemistry. Results showed a tight coupling between the kinetics of electron transfer chemistry and domain dynamics in the two diflavin oxidoreductase systems studied. Moreover, through the use of a flavin analogue (5-deazaflavin mononucleotide) and isotopically labelled nicotinamide coenzymes (pro-S/R NADP2H), key steps in the reaction mechanism were correlated with dynamic events in calmodulin, the partner protein of nNOS.The approaches developed in this project should find wider application in related studies of complex electron-transfer enzymes. Altogether, this research emphasises the key link between protein domain motions and electron transfer chemistry and provides a framework to describe the relationship between domain dynamics and diflavin oxidoreductase function.

SMFD techniques offer genuine detection possibilities which are often inaccessible using ensemble methods. This was demonstrated in three projects investigating translocation activity of CHD4 protein, analysis of MS2 phage capsid assembly and in-cell characterization of DNA structure. In other projects, binding interactions between two fluorescent probes and a short oligonucleotide were characterized and all optical depth of focus extended microscope configuration for imaging of individual molecules inside bacterial cells was developed and tested.

Mechanoresponsive Systeme antworten auf mechanische Reize mit einer Eigenschaftsänderung. Diese Dissertation umfasst die Arbeiten mit zwei mechanoresponsiven Systemen, die optisch auf mechanische Reize antworten. Sie basieren auf polymeren Strukturen, einer Polymerbürste und einem Hydrogelnetzwerk. Ihr optischer Antwortmechanismus ermöglicht die Beobachtung wirkender Kräfte als ein Ansatz zur in situ-Kraftmessung. Im ersten Teil wird ein existierendes, mechanoresponsives System zur Anwendung gebracht, das auf einer mit Fluoreszenzfarbstoff markierten Polyelektrolytbürste basiert. Die Ladungen des Polyelektrolyts können die Fluoreszenz des Farbstoffs unterdrücken, sodass lokale Kompression und Zugspannung über die Fluoreszenzintensität unterschieden werden können. Die mechanoresponsive Polymerbürste wurde als mechanosensitive Oberflächenbeschichtung angewandt, um Unterschiede in der Kontaktspannungsverteilung von Gecko-inspirierten adhäsiven Mikrostempelstrukturen aufzuklären. Die erarbeiteten Ergebnisse und daraus abgeleiteten Ablösemechanismen der Mikrostempeltypen deckten sich qualitativ mit Vorhersagen aus theoretischen Ansätzen. Aufgrund geometrischer Einschränkungen einer planaren Oberflächenbeschichtung zielt der zweite Teil darauf ab, dieses mechanoresponsive Prinzip in ein dreidimensionales Netzwerk zu überführen und ein mechanoresponsives Hydrogelnetzwerk als Plattform zur Kraftmessung zu entwickeln. Konzeptionell besitzt ein homogenes Netzwerk vorhersagbare mechanische Eigenschaften, sodass lokale optische Antworten auf mechanische Kräfte ermöglichen könnten, die wirkenden Kräfte zu lokalisieren und quantifizieren. Basierend auf einer Gestaltung nach der Flory-Rehner-Theorie wurden Präkursoren mit vordefinierter Größe und Architektur für die Hydrogelherstellung eingesetzt, um auf ein homogenes Netzwerk abzuzielen. Zu diesem Zweck wurde das Mischungsvolumen durch Tropfenmikrofluidik reduziert. Für den optischen Antwortmechanismus wurden die Hydrogelnetzwerk-Präkursoren mit zwei verschiedenen Fluorophoren markiert, die sich durch abstandsabhängige Emission über Förster-Resonanzenergietransfer auszeichnen. Die Funktionalität des optischen Antwortmechanismus wurde auf globaler Ebene durch Kollabieren und kontrolliertes Quellen des Netzwerks, dann auf lokalisierter Ebene durch definierte mechanische Belastung mit Rasterkraftmikroskopie gezeigt. Durch ihre Anpassbarkeit könnte die Hydrogelplattform zukünftig verschiedenste Anwendungen im Bereich intrisischer Kraftmessung weicher Materie bedienen. / Mechanoresponsive systems respond to mechanical triggers by changes in a certain property. This thesis covers the work conducted with two mechanoresponsive systems that respond optically to mechanical triggers. These two systems are based on polymer structures, a polymer brush and a hydrogel network. Thus, the optical response mechanism allows observing acting forces as an approach to force sensing in situ. In the first part, an existing mechanoresponsive system based on a polyelectrolyte brush labeled with a fluorescent dye is engaged in application. The charges of the polyelectrolyte are able to quench the fluorescence of the dye so that local compression or tension can be distinguished from the local fluorescence intensity. The mechanoresponsive polymer brush was applied as mechanosensitive surface coating to elucidate differences in the contact stress distributions of gecko-inspired adhesive micropillar structures. The determined results and the derived detachment mechanisms of the micropillar types were in qualitative accordance with predictions from theoretical approaches. Overcoming the geometrical limitations of a planar surface coating, the second part aims at translating the mechanoresponse principle to a three-dimensional network and developing a mechanoresponsive hydrogel as a platform for force sensing. Conceptually, a homogeneous network allows to predict mechanical properties so that localized optical mechanoresponses could enable locating and quantifying acting forces. Based on network design principles from the Flory-Rehner theory, precursors with predefined size and architecture were utilized in hydrogel preparation, aiming for a homogeneous network. Further in this regard, the mixing volume was reduced by employing droplet microfluidics. As optical response mechanism, the hydrogel network precursors were labeled with two kinds of fluorophore, featuring distance-dependent emission from Förster Resonance Energy Transfer. The functionality of the optical response mechanism was demonstrated on global level by collapsing and controlled swelling of the network, and on a localized level by defined mechanical stress, applied with Atomic Force Microscopy. Owing to its adjustability, the hydrogel platform might be employed in various applications that require intrinsic force sensing of soft matter in future.

La pseudouridylation, l'isomérisation du résidu urine (U) en pseudouridine ([PSI]) est la modification post-transcriptionnelle la plus fréquemment retrouvée dans les ARN. Elle est catalysée par une enzyme ARN:PSI-synthase. Chez les archées et les eucaryotes, cette activité est également portée par des particules ribonucléoprotéiques à boîtes H/ACA (RNP H/ACA). Chez les archées, le complexe comprend quatre protéines invariables dont l'ARN:PSI-synthase aCBF5 et trois protéines partenaires L7Ae, aNOP10 et aGAR1, ainsi qu'un ARN guide qui cible par appariement de bases la position de l'uridine à modifier de l'ARN substrat. Le rôle des partenaires a pu être identifié par des analyses structure-fonction basées sur des approches biochimiques, biophysiques et radiocristallographiques. Au cours de ce travail, nous avons démontré l'existence de disparités fonctionnelles entre les ARN guides d'un même organisme, et l'importance de l'interaction entre L7Ae et aNOP10 pour le positionnement correct de l'ARN substrat. Nous avons testé in vitro l'assemblage et l'activité de particules reconstituées en présence d'ARN guides non conventionnels. L'étude sur la dynamique de l'ARN substrat lors de la pseudouridylation a également été abordée et a permis de déterminer que aGAR1 n'était pas nécessaire pour le mécanisme de turnover de la particule, que la température jouait un rôle crucial pour cette activité, et que la nature du nucléotide cible ainsi que la longueur de l'ARN substrat étaient des éléments importants pour la sélection de cet ARN. Nous avons également mis au point une nouvelle technique basée sur le phénomène de FRET permettant de suivre l'association de l'ARN substrat à la RNP H/ACA / Pseudouridylation reaction that consists in the isomerization of uridines (U) into pseudouridines (PSI) is the most frequent post-transcriptional modification found in RNAs. It is catalyzed by enzymes with RNA:PSI-synthase activity. In Archaea and Eukarya, ribonucleoprotein particles, the so-called box H/ACA RNPs, possess such activity. In Archaea, the box H/ACA complex comprises four invariable proteins namely the RNA:PSI-synthase aCBF5 and three protein partners L7Ae, aNOP10 and aGAR1, and specific to each RNP, an RNA acting as a guide to secure by base pairing the RNA substrate and define the position to be modify. During these last years, several crystal structures of components of archaeal H/ACA RNP and fully assembled RNP have been resolved. Complementary biochemical and biophysical studies allowed detailed structure-function analyses to identify the role of the different components. During this work we identified functional differences between two RNA guides expressed in the same archaea, and demonstrated that the interaction between L7Ae and aNOP10 is important for a correct positioning of substrate RNA. We also tested in vitro the assembly and activity of RNP reconstituted on H/ACA-like guide RNAs. We investigated dynamics of substrate RNA during the pseudouridylation. We found that aGAR1 was not necessary for the turnover of the particle, that the temperature was crucial for such activity, and that the chemical structure of the targeted residue and length of the substrate RNA were important determinants for substrate selectivity. Finally, we have also developed a new technic based on FRET adapted to monitor binding of the susbtrate RNA to the box H/ACA RNP enzyme