Domain swapping is a wide spread phenomenon which involves the association between two or more protein subunits such that intra-molecular interactions between domains in each subunit are replaced by equivalent inter-molecular interactions between the same domains in different subunits. This thesis is devoted to the analysis of the factors that drive proteins to undergo such association modes. The specific system analyzed is the monomer to swapped dimer formation of the B1 domain of the immunoglobulin G binding protein (GB1). The formation of this dimer was shown to be fostered by 4 amino acid substitutions (L5V, F30V, Y33F, A34F) (Byeon et al. 2003). In this work, computational protein design and molecular dynamics simulations, both with detailed atomic models, were used to gain insight into how these 4 mutations may promote the domain swapping reaction.<p>The stability of the wt and quadruple mutant GB1 monomers was assessed using the software DESIGNER, a fully automatic procedure that selects amino acid sequences likely to stabilize a given backbone structure (Wernisch et al. 2000). Results suggest that 3 of the mutations (L5V, F30V, A34F) have a destabilizing effect. The first mutation (L5V) forms destabilizing interactions with surrounding residues, while the second (F30V) is engaged in unfavorable interactions with the protein backbone, consequently causing local strain. Although the A34F substitution itself is found to contribute favorably to the stability of the monomer, this is achieved only at the expense of forcing the wild type W43 into a highly strained conformation concomitant with the formation of unfavorable interactions with both W43 and V54.<p>Finally, we also provide evidence that A34F mutation stabilizes the swapped dimer structure. Although we were unable to perform detailed protein design calculations on the dimer, due to the lower accuracy of the model, inspection of its 3D structure reveals that the 34F side chains pack against one another in the core of the swapped structure, thereby forming extensive non-native interactions that have no counterparts in the individual monomers. Their replacement by the much smaller Ala residue is suggested to be significantly destabilizing by creating a large internal cavity, a phenomenon, well known to be destabilizing in other proteins. Our analysis hence proposes that the A34F mutation plays a dual role, that of destabilizing the GB1 monomer structure while stabilizing the swapped dimer conformation.<p>In addition to the above study, molecular dynamics simulations of the wild type and modeled quadruple mutant GB1 structures were carried out at room and elevated temperatures (450 K) in order to sample the conformational landscape of the protein near its native monomeric state, and to characterize the deformations that occur during early unfolding. This part of the study was aimed at investigating the influence of the amino acid sequence on the conformational properties of the GB1 monomer and the possible link between these properties and the swapping process. Analysis of the room temperature simulations indicates that the mutant GB1 monomer fluctuates more than its wild type counter part. In addition, we find that the C-terminal beta-hairpin is pushed away from the remainder of the structure, in agreement with the fact that this hairpin is the structural element that is exchanged upon domain swapping. The simulations at 450 K reveal that the mutant protein unfolds more readily than the wt, in agreement with its decreased stability. Also, among the regions that unfold early is the alpha-helix C-terminus, where 2 out of the 4 mutations reside. NMR experiments by our collaborators have shown this region to display increased flexibility in the monomeric state of the quadruple mutant.<p>Our atomic scale investigation has thus provided insights into how sequence modifications can foster domain swapping of GB1. Our findings indicate that the role of the amino acid substitutions is to decrease the stability of individual monomers while at the same time increase the stability of the swapped dimer, through the formation of non-native interactions. Both roles cooperate to foster swapping. / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Nuclear fuel performance is highly affected by the behaviour of fission gases, particularly<p>at elevated burnups, where large amounts of gas are produced and can<p>potentially be released. The importance of fission gas release was the motivation<p>for large efforts, both experimentally and theoretically, in order to increase our<p>understanding of the different steps of the process, and to continuously improve<p>our models.<p>Extensions to higher burnups, together with the growing interest in novel types<p>of fuels such as inert matrix fuels envisaged for the transmutation of minor actinides,<p>make that one is still looking for a permanently better modelling, based<p>on a physical understanding and description of all stages of the release mechanism.<p>Computer simulations are nowadays envisaged in order to provide a better<p>description and understanding of atomic-scale processes such as diffusion, but even<p>in order to gain insight on specific processes that are inaccessible by experimental<p>means, such as the fuel behaviour during thermal spikes.<p>In the present work simulation techniques based on empirical potentials have<p>been used, focusing in a first stage on pure uranium dioxide. The behaviour of<p>point defects was at the core of this part, but also the estimation of elastic and<p>melting properties.<p>Then, in a second stage, the study has been extended to the behaviour of helium<p>and xenon. For helium, the diffusion in different domains of stoichiometry<p>was considered. The simulations enabled to determine the diffusion coefficient and<p>the migration mechanism, using both molecular dynamics and static calculation<p>techniques. Xenon behaviour has been investigated with the additional intention<p>to model the behaviour of small intragranular bubbles, particularly their interaction<p>with thermal spikes accompanying the recoil of fission fragments. For that<p>purpose, a simplified description of these events has been proposed, which opens<p>perspectives for further work.<p>/<p>Les performances du combustible nucléaire sont fortement affectées par le comportement<p>des gaz de fission, et ce particulièrement lorsqu’un taux d’épuisement<p>élevé est atteint, puisque d’importantes quantités de gaz sont alors produites<p>et peuvent potentiellement être relâchées. Les enjeux, entre autre économiques,<p>liés au relâchement de gaz de fission ont donné lieu à d’importants efforts, tant<p>sur le plan expérimental que théorique, afin d’accroître notre compréhension des<p>différentes étapes du processus, et d’améliorer sans cesse les mod`eles. Les extensions<p>à des taux d’épuisements encore plus élevés ainsi que l’intérêt croissant pour<p>de nouveaux types de combustible tels que les matrices inertes, envisages en vue<p>de la transmutation des actinides mineures, font qu’à l’heure actuelle, le besoin<p>permanent d’une meilleure modélisation, basée sur une compréhension et une description<p>physique des différentes étapes du processus de relâchement de gaz de<p>fission, est toujours de mise.<p>Les simulations par ordinateur ont ainsi été considérée comme un nouvel angle<p>de recherche sur les processus élémentaires se produisant à l’échelle atomique, à la<p>fois afin d’obtenir une meilleure compréhension de processus tels que la diffusion<p>atomique ;mais aussi afin d’avoir accès à certains processus qui ne sont pas observables<p>par des voies expérimentales, tels que la le comportement du combustible<p>lors de pointes thermiques.<p>Dans ce travail, deux techniques, basées sur l’utilisation de potentiels interatomiques<p>empiriques, ont permis d’étudier le dioxyde d’uranium, dans un premier<p>temps en l’absence d’impuretés. Cette partie était principalement centrée sur le<p>comportement des défauts ponctuels, mais a aussi concerné différentes propriétés<p>élastiques, ainsi que le processus de fusion du composé.<p>Ensuite l’étude a été étendue aux comportements de l’hélium de du xénon. Pour<p>ce qui a trait à l’hélium, la diffusion dans différents domaines de stoechiométrie<p>a été considérée. Les simulations ont permis de déterminer le coefficient de diffusion<p>ainsi que le mécanisme de migration lui-même. Quant au xénon, outre les<p>propriétés de diffusion, l’intention fut de se diriger vers la modélisation des petites<p>bulles intragranulaires, et plus précisément vers leur interaction avec les pointes<p>thermiques, créées lors du recul des fragments de fission. Une description simplifiée de ce processus a été proposée, qui offre de nouvelles perspectives dans ce<p>domaine.<p><p> / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished