No description available.

No description available.

No description available.

L'une des infections parasitaires les plus courantes chez les animaux à travers le monde est la babésiose ou piroplasmose. Causée par le développement intraérythrocytaire d'un parasite du genre Babesia, elle présente de nombreux signes cliniques semblables à ceux du paludisme. Ce parasite, du phylum des Apicomplexes, est transmis via le vecteur tique et effectue son cycle de reproduction dans les cellules rouges du sang de l'hôte vertébré. En Europe B. divergens et B. canis sont les espèces majoritairement responsables respectivement de la babésiose bovine et la babésiose canine. Dans une stratégie de recherche vaccinale, l'étude de protéines parasitaires en contact avec la circulation sanguine est primordiale pour comprendre les interactions hôte-parasite et identifier des candidats vaccins à haut potentiel. Les protéines à ancrage GPI (glycosylphosphatidylinositol) font partie de ces protéines. La première protéine à ancrage GPI décrite chez B. divergens est Bd37.1. Elle induit une protection totale contre une infection à B. divergens à la condition qu'une séquence hydrophobe soit ajoutée en C-terminale. La résolution de la structure RMN de cette protéine a permis de mettre en évidence un probable mécanisme de changement conformationnel en fonction du pH. La structure composée de 3 sous domaines montre que celle-ci n'est maintenue que par des ponts salins qui peuvent se rompre en milieu acide. Or l'environnement membranaire dans lequel évolue Bd37.1 ancrée à la surface du parasite et/ou à l'approche du globule rouge lors de l'invasion est acide. Cette dynamique conformationnelle de la protéine Δ-Bd37, liée à l'environnement membranaire, pourrait être à l'origine du mécanisme qui confère une immunité en fonction de la présence ou non de la séquence hydrophobe en C-terminale de Bd37.1. Nous avons cherché à estimer les implications d'une telle dynamique dans les interactions hôtes-parasites à travers l'étude structurale de 2 protéines parasitaires (Bd37.1 et Bc28.1). Dans le premier cas nous étudions la dynamique conformationnelle de la protéine d'adhésion Bd37.1. Nous avons exploré les différentes conformations que pourrait adopter la protéine Bd37.1 par une approche de biophysique et nous avons stabilisé ces différentes conformations en solution par le biais de mutations pour les étudier. Parmi ces mutants, le mutant EDK-Δ-Bd37 dont les ponts salins ont été rompus montre des caractéristiques différentes de Δ-Bd37. Les données enregistrées sur ce mutant nous ont amené à résoudre sa structure et à tester son pouvoir vaccinant. Dans une seconde partie, nous caractérisons biochimiquement et fonctionnellement une autre protéine Bc28.1, l'orthologue de Bd37.1. chez B. canis, accompagnée de la résolution de sa structure. Nous montrons que Bc28.1 est une protéine d'adhésion localisée à la surface du parasite et nous comparons les structures de Bd37.1 et Bc28.1. Ces deux structures sont finalement très différentes tandis que localisation et fonction sont similaires. / One of the most common parasitic infections in animals worldwide is babesiosis or piroplasmosis. Caused by the intraerythrocytic development of Babesia parasite, it has many clinical signs similar to those of malaria. This parasite of the phylum Apicomplexa, is transmitted via the tick vector and performs its reproductive cycle in red blood cells of the vertebrate host. B. In Europe divergens and B. canis species are mainly responsible respectively for bovine babesiosis and canine babesiosis. A strategy of vaccine research, the study of parasite proteins in contact with the bloodstream is essential for understanding host-parasite interactions and identify vaccine candidates with high potential. Anchored protein GPI (glycosylphosphatidylinositol) are part of these proteins. The first protein GPI anchors described in B. divergens is Bd37.1. It induces complete protection against infection with B. divergens provided a hydrophobic sequence is added at the C-terminus. Resolution NMR structure of this protein has highlighted a probable mechanism of conformational change as a function of pH. The structure consists of three sub areas shows that it is only maintained by salt bridges which can break in acidic medium. However, the environment within which Bd37.1 membrane anchored to the surface of the parasite and / or approach the red blood cell during the invasion is acidic. This conformational dynamics of the protein-Δ Bd37 linked to the membrane environment, could be at the origin of the mechanism that confers immunity depending on the presence or absence of the hydrophobic sequence at the C-terminus of Bd37.1. We sought to assess the implications of such dynamics in host-parasite interactions through structural study of two parasite proteins (Bd37.1 and Bc28.1). In the first case we study the conformational dynamics of the adhesion protein Bd37.1. We explored the different conformations that may be adopted by a protein Bd37.1 biophysical approach and we have stabilized in different conformations in solution through mutations to study. Among these mutants, the mutant Δ-Bd37-EDK including salt bridges were broken shows different characteristics Δ-Bd37. The data on this mutant led us to solve the structure and to test its power vaccinating. In a second part, we characterize biochemically and functionally Bc28.1 another protein, the ortholog Bd37.1. in B. canis, accompanied with the resolution of its structure. We show that Bc28.1 is an adhesion protein localized to the parasite surface and compare the structures and Bd37.1 Bc28.1. These two structures are ultimately very different while location and function are similar.

No description available.

Les récepteurs métabotropes au glutamate (mGluR) sont des RCPG de classe C. Ils sont exprimés dans le système nerveux central où, suite à l'activation par le glutamate, ils participent à la modulation de la transmission nerveuse. En raison de leur rôle essentiel dans la régulation de l'activité synaptique, ils représentent des cibles potentielles pour le développement de médicaments contre les troubles neurologiques et psychiatriques telles que la schizophrénie, l'épilepsie, l'anxiété et la douleur. Mon projet de recherche de doctorat a porté sur l'étude du mécanisme d'activation du domaine extracellulaire de liaison au ligand du mGluR (ECD), avec un accent particulier sur ce qui différencie au niveau moléculaire un agoniste partiel d'un agoniste total. A cette fin, j'ai utilisé une méthode innovante à l'échelle de la molécule unique appelée Transfert d'Energie par Résonance de Forster, développé pour l'étude de la dynamique conformationnelle des molécules individuelles à l'échelle de la nanoseconde. J'ai réussi à montrer que le dimère d'ECD oscille entre une conformation active et une conformation de repos sur une échelle de temps de ~100μsec et que les ligands influencent les vitesses de transition entre ces états avec des vitesses intermédiaires pour les agonistes partiels. Ces résultats sont validés par l'utilisation de mutants spécifiques et indiquent clairement que le rôle des ligands n'est pas de stabiliser une conformation donnée mais de modifier le comportement dynamique du récepteur. L'ensemble de ces résultats contribuent à une meilleure description du mécanisme d'activation des mGluRs, et ouvrent potentiellement la voie à la compréhension des RCPG en général. / Metabotropic Glutamate Receptors (mGluRs) are class C GPCRs, expressed throughout the central nervous system. They participate in the long term modulation of neural transmission following activation by the excitatory neurotransmitter glutamate. This critical role in the regulation of synaptic activity makes them promising targets in the development of drugs for the treatment of various neurologic and psychiatric disorders such as schizophrenia, epilepsy, anxiety and pain relief. My Ph.D. research project has focused on the study of the activation mechanism of the mGluR extracellular ligand binding Venus-Flytrap domain (VFT), with particular emphasis on the differences between partial and full agonists on a molecular level. To this aim, I have used a state-of-the-art single molecule Förster Resonance Energy Transfer (smFRET) approach, developed for the study of conformational dynamics of single molecules on the nanosecond to millisecond timescale. I have managed to show that the VFT-dimer constantly oscillates between an active and a resting conformation on a ~100µsec timescale. I also discovered that the role of ligands is to influence the transition rate between these boundary states, and that partial agonists display intermediate transition rates. My results, supported by the use of specific mutants, clearly indicate that the role of ligands is not to stabilize a given conformation but to modify the overall dynamic of the receptor, which favors a conformational selection mechanism. Altogether, these results represent a most-valuable contribution to the better understanding of the activation mechanism of mGluRs, and potentially GPCRs in general.

Human immunodeficiency virus reverse transcriptase (HIV RT) is a virally encoded polymerase responsible for replicating the HIV genome. Most HIV treatments include nucleotide RT inhibitors (NRTIs) which inhibit HIV RT replication by serving as a substrate for the polymerase reaction but then blocks subsequent polymerization after incorporation. However, resistance to these NRTIs may occur through specific mutations in HIV RT that increase the discrimination of HIV RT for natural nucleotides over NRTIs. The role of enzyme conformational dynamics in specificity and substrate selection was studied using transient kinetic methods on HIV RT enzymes that have been site-specifically labeled with a conformationally sensitive fluorophore, to measure the rates of binding and catalysis. First, HIV RT with the mutation of lysine to arginine at the residue position 65 (K65R) was examined for its resistance against the NRTI tenofovir diphosphate (TFV), an acyclic deoxyadenosine triphosphate (dATP) analog. It was found that HIV RT K65R resistance to TFV was achieved through decreased rates of catalysis and increased rates of dissociation for TFV over dATP when compared with the kinetics of wild-type HIV RT. Moreover, global fitting analysis confirmed a mechanism where a large conformational change, after initial ground state binding of the substrate, contributed significantly to enzyme specificity. This led to our investigation of the molecular basis for enzyme specificity using HIV RT as a model system. Again, transient kinetic methods were applied with the addition of molecular dynamics simulations. The simulated results were substantiated by the corroborating experimental results. It was found that a substrate-induced conformational change in the transition of HIV RT from an open nucleotide-bound state to a closed nucleotide-bound state was the major determinant in enzyme specificity. The molecular basis for substrate selection resulted from the molecular alignments of the substrate in the active-site, which induced the conformational change. When the correct nucleotide was bound, optimal molecular interactions in the active-site yielded a stably closed complex, which promoted nucleotide incorporation. In contrast, when an incorrect nucleotide was bound, the molecular interactions at the active-site were not ideal, which yielded an unstable closed complex, which promoted substrate dissociation rather than incorporation. / text

No description available.

abstract: Proteins are essential for most biological processes that constitute life. The function of a protein is encoded within its 3D folded structure, which is determined by its sequence of amino acids. A variation of a single nucleotide in the DNA during transcription (nSNV) can alter the amino acid sequence (i.e., a mutation in the protein sequence), which can adversely impact protein function and sometimes cause disease. These mutations are the most prevalent form of variations in humans, and each individual genome harbors tens of thousands of nSNVs that can be benign (neutral) or lead to disease. The primary way to assess the impact of nSNVs on function is through evolutionary approaches based on positional amino acid conservation. These approaches are largely inadequate in the regime where positions evolve at a fast rate. We developed a method called dynamic flexibility index (DFI) that measures site-specific conformational dynamics of a protein, which is paramount in exploring mechanisms of the impact of nSNVs on function. In this thesis, we demonstrate that DFI can distinguish the disease-associated and neutral nSNVs, particularly for fast evolving positions where evolutionary approaches lack predictive power. We also describe an additional dynamics-based metric, dynamic coupling index (DCI), which measures the dynamic allosteric residue coupling of distal sites on the protein with the functionally critical (i.e., active) sites. Through DCI, we analyzed 200 disease mutations of a specific enzyme called GCase, and a proteome-wide analysis of 75 human enzymes containing 323 neutral and 362 disease mutations. In both cases we observed that sites with high dynamic allosteric residue coupling with the functional sites (i.e., DARC spots) have an increased susceptibility to harboring disease nSNVs. Overall, our comprehensive proteome-wide analysis suggests that incorporating these novel position-specific conformational dynamics based metrics into genomics can complement current approaches to increase the accuracy of diagnosing disease nSNVs. Furthermore, they provide mechanistic insights about disease development. Lastly, we introduce a new, purely sequence-based model that can estimate the dynamics profile of a protein by only utilizing coevolution information, eliminating the requirement of the 3D structure for determining dynamics. / Dissertation/Thesis / Doctoral Dissertation Physics 2016

abstract: This thesis explores a wide array of topics related to the protein folding problem, ranging from the folding mechanism, ab initio structure prediction and protein design, to the mechanism of protein functional evolution, using multi-scale approaches. To investigate the role of native topology on folding mechanism, the native topology is dissected into non-local and local contacts. The number of non-local contacts and non-local contact orders are both negatively correlated with folding rates, suggesting that the non-local contacts dominate the barrier-crossing process. However, local contact orders show positive correlation with folding rates, indicating the role of a diffusive search in the denatured basin. Additionally, the folding rate distribution of E. coli and Yeast proteomes are predicted from native topology. The distribution is fitted well by a diffusion-drift population model and also directly compared with experimentally measured half life. The results indicate that proteome folding kinetics is limited by protein half life. The crucial role of local contacts in protein folding is further explored by the simulations of WW domains using Zipping and Assembly Method. The correct formation of N-terminal β-turn turns out important for the folding of WW domains. A classification model based on contact probabilities of five critical local contacts is constructed to predict the foldability of WW domains with 81% accuracy. By introducing mutations to stabilize those critical local contacts, a new protein design approach is developed to re-design the unfoldable WW domains and make them foldable. After folding, proteins exhibit inherent conformational dynamics to be functional. Using molecular dynamics simulations in conjunction with Perturbation Response Scanning, it is demonstrated that the divergence of functions can occur through the modification of conformational dynamics within existing fold for β-lactmases and GFP-like proteins: i) the modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate, while the resurrected ancient lactamases indicate enhanced active-site flexibility, which likely allows for the binding and subsequent degradation of different antibiotic molecules; ii) the chromophore and attached peptides of photocoversion-competent GFP-like protein exhibits higher flexibility than the photocoversion-incompetent one, consistent with the evolution of photocoversion capacity. / Dissertation/Thesis / Ph.D. Physics 2014