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La reconnaissance moléculaire joue un rôle central dans tous les processus biologiques ainsi que dans le développement de nouvelles biotechnologies. Depuis les 60 dernières années, deux mécanismes de reconnaissance moléculaire ont permis de décrire le couplage entre la liaison et le changement conformationnel observé chez les biomolécules. Le mécanisme par ajustement induit a lieu lorsque le ligand se lie à l'état inactif de la biomolécule et induit un changement de conformation vers la forme active. Le mécanisme par sélection conformationnelle, quant à lui, a lieu lorsque le ligand se lie directement à l'état spontanément actif de plus faible population et le stabilise, déplaçant ainsi la population de biomolécule vers cet état. Bien que nous connaissons des exemples de protéines qui fonctionnent selon chacun de ces mécanismes, nous ne comprenons pas encore les différences entre les performances de ces mécanismes ni les déterminants moléculaires qui leur donnent lieu. Une compréhension approfondie de ces mécanismes nous permettrait de mieux comprendre pourquoi certaines protéines ont évolué selon un mécanisme en particulier ainsi que de s'inspirer de ces mécanismes pour le développement de biotechnologies finement régulées. Jusqu'à aujourd'hui, ces deux mécanismes ont exclusivement été étudiés dans le contexte des biomolécules naturelles, principalement des protéines, dont la complexité dynamique et structurale rend difficile la comparaison et la manipulation individuelle des différents paramètres thermodynamiques. Il est donc particulièrement ardu de caractériser le rôle de chacun de ces paramètres quant à la sélection et la performance de ces mécanismes. Pour contourner ces limitations expérimentales, nos travaux de recherche se sont intéressés à recréer ces mécanismes à l'aide d'interrupteurs d'ADN fluorescents pour lesquels il est possible de prédire et de modifier la structure et les propriétés thermodynamiques ainsi que d'en mesurer l'activation en temps réel. Ce faisant, il a été possible d'observer que le mécanisme par ajustement induit est obtenu lorsque le site de liaison est partiellement accessible dans l'état inactif. Nous avons aussi observé que ce mécanisme permet une activation et une désactivation jusqu'à 10 000 fois plus rapide que la sélection conformationnelle, qui par contraste, donne lieu à une activation plus lente ainsi qu'à un maintien prolongé de l'activation. Ces différences cinétiques suggèrent ainsi un rôle évolutif distinct pour chacun et laissent envisager des applications en biotechnologies pour l'optimisation de la cinétique. / Molecular recognition plays a central role in almost every biological and biotechnological process. Over the last 60 years, two molecular recognition mechanisms have been used to appropriately describe the coupling between binding and conformational change in biomolecules. The induced fit mechanism takes place when ligand binding to the inactive state of the biomolecule induces the conformational change leading to the active state. On the other hand, in the conformational selection mechanism, where active and inactive states exist in equilibrium, the ligand binds selectively to the active state of the biomolecule and shifts the equilibrium towards this state by stabilizing it. Even though these mechanisms have been widely studied, it is still unclear if they differ in performance or how each mechanism can be modulated. Such a fundamental understanding of the differences between these mechanisms would shed light on the reasons for an apparent selective pressure driving the use of a specific mechanism for a given biomolecule and would also allow us to engineer new biomolecules which would benefit from the strengths of these mechanisms. To date, both mechanisms have been exclusively studied in the context of naturally occurring biomolecules, mainly proteins, whose structural and dynamic complexity as well as diversity seem to prevent comparison and manipulation of specific and individual thermodynamic parameters. Consequently, only little progress has been made towards characterizing the role of certain key thermodynamic parameters on the selection and performance of the mechanism. To circumvent this limitation, we have reproduced these mechanisms using simple fluorescent DNA constructs allowing for reliable prediction and variation of both structure and thermodynamics as well as real time monitoring of the activation process in presence of a DNA target. These DNA "switches" allowed us to determine that an induced fit mechanism occurs when the binding site is partially available in the inactive state and that this mechanism allows for a faster activation and deactivation (up to four orders of magnitude) compared to a conformational selection mechanism, which in contrast corresponds to a slower activation and deactivation, leading to a longer activation period. The observed kinetic differences between these mechanisms points towards potential uses for both in different areas of biotechnology as well as some rationale behind evolution favoring one mechanism over the other for a given protein.

La NADPH cytochrome P450 réductase (CPR) est une flavoprotéine multidomaines appartenant à la famille des diflavines réductases et un des composants essentiels du système redox des cytochromes P450. La CPR est formée de deux domaines catalytiques contenant des groupements prosthétiques FAD et FMN et d'un domaine de connexion. Le domaine FAD reçoit deux électrons du NADPH et les transfère un par un au domaine FMN, qui, à son tour, les transfère aux accepteurs. Le transfert d’électron du FMN vers les accepteurs nécessite un déplacement du domaine FMN par rapport au reste de la molécule. Au fils des années, les études structurales menées sur la CPR ont mis en évidence la réorganisation structurale et l’arrangement des domaines dans cette protéine. Cependant, les résultats de ces analyses ne fournissent pas d’informations concernant la vitesse à laquelle les mouvements des domaines de la CPR s’effectuent et n’incluent toujours pas les paramètres qui induisent le changement conformationnel ainsi que l'influence de ces changements sur l’activité catalytique de la CPR.Le projet de cette thèse a consisté à apporter de nouveaux éléments de compréhension sur la relation entre les changements conformationnels de la CPR et son cycle catalytique. La première partie de ce travail a porté sur le développement de stratégies de préparation au marquage des domaines catalytiques de la CPR, destinés à l’étude dynamique de cette protéine par le FRET. Différentes stratégies ont été envisagées parmi lesquelles l’incorporation de p-acétyle phénylalanine sur des positions définies dans la CPR. La deuxième partie de ce travail est consacrée à l’étude dynamique de la CPR via des techniques de RMN et SAXS combinées à des approches biochimiques. Les expériences menées ont permis de caractériser en solution et en absence de NaCl, la présence d’un état rigide, globulaire en conformation fermée dans laquelle les domaines FMN et FAD sont maintenus « verrouillés » par des interactions à l’interface entre ces deux domaines. L’augmentation de la concentration en NaCl permet une transition de cet état « verrouillé » vers un état plus ouvert pour lequel il n’y a plus d’interface entre les domaines FAD et FMN. L’état « déverrouillé » de la CPR correspond à un équilibre dynamique entre un ensemble de conformations en échange rapide. Cet équilibre est contrôlé par la force ionique et l’activité catalytique de la CPR est maximale lorsque les états verrouillés et déverrouillés sont également peuplés. Le modèle cinétique proposé par nos études a permis de mettre en évidence un lien direct entre la dynamique des domaines et l’activité du transfert d’électron au cours de cycle catalytique de la CPR. / NADPH cytochrome P450 reductase (CPR) is a multidomain flavoprotein that belongs to the diflavines reductase family. It is an essential component of redox system delivering electrons for cytochrome P450. CPR is composed of two catalytic domains containing FAD and FMN prosthetic groups and a connecting domain. FAD domain receives two electrons from NADPH and transfers them one by one to the FMN domain, which in turn transfers them to the acceptor. The electron transfer from FMN to the acceptor requires a large domain movement. Over the years, structural studies of CPR have highlighted the reorganization and arrangement of domains in this protein. However, the results of these analyses do not provide any information about how fast the domains movements takes place in CPR, and do not always include the parameters that induce conformational change as well as influence of those changes on the catalytic activity of the CPR. This thesis aims to bring new elements of comprehension on the relationship between conformational changes in CPR and its catalytic cycle. The first part of the work concerned the development of strategies to label the catalytic domains of CPR, a prerequisite for the dynamic study of this protein by FRET. Different strategies have been proposed including the incorporation of p-acetyl phenylalanine at defined positions in CPR. The second part of this work is devoted to the dynamic study of CPR through a combined SAXS, NMR and biochemical approaches. The experiments conducted allowed to characterize the presence of a rigid and globular state of closed conformation for CPR, in solution and in the absence of NaCl. In this conformation the FMN and FAD domains are kept "locked" by interface interactions between these two domains. Increasing the NaCl concentration permits the transition from the "locked" stats to an open conformation in which there is no more interface between the FAD and FMN domains. The "unlocked" state of CPR is a dynamic equilibrium between ensembles of conformations in fast exchange. This equilibrium is controlled by the ionic strength and CPR presents its maximum catalytic activity when the locked and unlocked states are equally populated. We proposed a kinetic model which allows demonstrating a direct link between the domain movement and electron transfer activity during the catalytic cycle of CPR.

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Here, we report on a new temperature responsive polymer brush system with a terminal “click” functionality. Bifunctionalized poly(N-isopropylacrylamide) (PNiPAAm) with distinct functional end groups was synthesized by atom transfer radical polymerization (ATRP) and grafted to a modified silicon substrate. The presence of the active terminal alkyne functionality is validated using an azide-modified rhodamine B (N3-RhB) via copper(I) catalyzed alkyne–azide cycloaddition (CuAAC). The optical properties and swelling dynamics of an N3-RhB modified PNiPAAm brush are analyzed in dry state and in situ by VIS-spectroscopic ellipsometry (SE). The best-fit results are obtained using a Gaussian oscillator model and are confirmed by UV/VIS-spectroscopy. We observed evidence of interactions between the aromatic residues of the dye and the PNiPAAm amide groups, which significantly affect the swelling behavior of the modified polymer brush. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Molecular Biology has allowed the characterization and manipulation of the molecules of life in the wet lab. Also the structures of those macromolecules are being continuously elucidated. During the last decades of the past century, there was an increasing interest to study how the different genes are organized into different organisms (‘genomes’) and how those genes are expressed into proteins to achieve their functions. Currently the sequences for many genes over several genomes have been determined. In parallel, the efforts to have the structure of the proteins coded by those genes go on. However it is experimentally much harder to obtain the structure of a protein, rather than just its sequence. For this reason, the number of protein structures available in databases is an order of magnitude or so lower than protein sequences. Furthermore, in order to understand how living organisms work at molecular level we need the information about the interaction of those proteins. Elucidating the structure of protein macromolecular assemblies is still more difficult. To that end, the use of computers to predict the structure of these complexes has gained interest over the last decades.<p>The main subject of this thesis is the evaluation of current available computational methods to predict protein – protein interactions and build an atomic model of the complex. The core of the thesis is the evaluation protocol I have developed at Service de Conformation des Macromolécules Biologiques et de Bioinformatique, Université Libre de Bruxelles, and its computer implementation. This method has been massively used to evaluate the results on blind protein – protein interaction prediction in the context of the world-wide experiment CAPRI, which have been thoroughly reviewed in several publications [1-3]. In this experiment the structure of a protein complex (‘the target’) had to be modeled starting from the coordinates of the isolated molecules, prior to the release of the structure of the complex (this is commonly referred as ‘docking’).<p>The assessment protocol let us compute some parameters to rank docking models according to their quality, into 3 main categories: ‘Highly Accurate’, ‘Medium Accurate’, ‘Acceptable’ and ‘Incorrect’. The efficiency of our evaluation and ranking is clearly shown, even for borderline cases between categories. The correlation of the ranking parameters is analyzed further. In the same section where the evaluation protocol is presented, the ranking participants give to their predictions is also studied, since often, good solutions are not easily recognized among the pool of computer generated decoys.<p>An overview of the CAPRI results made per target structure and per participant regarding the computational method they used and the difficulty of the complex. Also in CAPRI there is a new ongoing experiment about scoring previously and anonymously generated models by other participants (the ‘Scoring’ experiment). Its promising results are also analyzed, in respect of the original CAPRI experiment. The Scoring experiment was a step towards the use of combine methods to predict the structure of protein – protein complexes. We discuss here its possible application to predict the structure of protein complexes, from a clustering study on the different results.<p>In the last chapter of the thesis, I present the preliminary results of an ongoing study on the conformational changes in protein structures upon complexation, as those rearrangements pose serious limitations to current computational methods predicting the structure protein complexes. Protein structures are classified according to the magnitude of its conformational re-arrangement and the involvement of interfaces and particular secondary structure elements is discussed. At the end of the chapter, some guidelines and future work is proposed to complete the survey. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Here, we report on a new temperature responsive polymer brush system with a terminal “click” functionality. Bifunctionalized poly(N-isopropylacrylamide) (PNiPAAm) with distinct functional end groups was synthesized by atom transfer radical polymerization (ATRP) and grafted to a modified silicon substrate. The presence of the active terminal alkyne functionality is validated using an azide-modified rhodamine B (N3-RhB) via copper(I) catalyzed alkyne–azide cycloaddition (CuAAC). The optical properties and swelling dynamics of an N3-RhB modified PNiPAAm brush are analyzed in dry state and in situ by VIS-spectroscopic ellipsometry (SE). The best-fit results are obtained using a Gaussian oscillator model and are confirmed by UV/VIS-spectroscopy. We observed evidence of interactions between the aromatic residues of the dye and the PNiPAAm amide groups, which significantly affect the swelling behavior of the modified polymer brush. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.