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ED BIO SORBONNE PARIS CITE Proposition de sujet de thèse à l’appui d’une demande de contrat doctoral 2017-2018 Renseignements relatifs à l’Unité de Recherche : Label et intitulé : USR 2000 Spectrométrie de Masse pour la Biologie Nom et prénom du Directeur : Chamot-Rooke Julia Téléphone : + 33 1 40 61 38 59 Télécopie : Courriel: [email protected] Renseignements relatifs à l’Equipe : Nom de l’Equipe d’Accueil : USR 2000 Spectrométrie de Masse pour la Biologie Nom et prénom du responsable : Chamot-Rooke Julia Qualité du responsable : Directeur de Recherche CNRS Téléphone : + 33 1 40 61 38 59 Télécopie : Courriel : [email protected] Renseignements relatifs au sujet de thèse : Nom et prénom du Directeur de thèse (HDR) : Chamot-Rooke Julia Qualité : Directeur de Recherche CNRS Téléphone : + 33 1 40 61 38 59 Télécopie : Courriel : [email protected] Titre du sujet proposé : (En français) Détermination de la structure de complexes protéiques impliqués dans la virulence bactérienne par de nouvelles approches de spectrométrie de masse à haute résolution (En anglais) Development of innovative mass spectrometry approaches for the structural analysis of protein complexes involved in bacterial virulence. Département (cocher le département correspondant au sujet de thèse qui n’est pas obligatoirement le vôtre) : Biologie Cellulaire et moléculaire, Physiologie et Physiopathologie Immunologie Développement Génétique Neurobiologie et Vieillissement Infectiologie, Microbiologie Summary (5 lines maximum) : This PhD project proposes the development of new Mass Spectrometry (MS) based approaches for the structural analysis of challenging protein complexes. Two main axes will be developed: covalent crosslinking combined to MS and native MS. These developments will be used to decipher the structure of bacterial secretion systems, which are sophisticated nanomachineries involved in bacterial virulence. Proposition de sujet de thèse à l’appui d’une demande de contrat doctoral 2017-2087 Nom, prénom du directeur de l'unité de recherche : Chamot-Rooke Julia Numéro de l'unité de recherche (et établissement de rattachement) : USR 2000 Nom, prénom du responsable de l'équipe d'accueil (EAD) : Chamot-Rooke Julia Nom, prénom du directeur de thèse : Chamot-Rooke Julia Titre du sujet de thèse proposé (en anglais): Development of innovative mass spectrometry approaches for the structural analysis of protein complexes involved in bacterial virulence. 5 key words: mass spectrometry, cross-linking, structural biology, protein complexes, bacterial virulence Candidat pressenti : OUI NON Contenu scientifique du programme de la thèse (en anglais) Physical interactions between proteins form the basis of most biological functions. Therefore, a critical step toward understanding cellular processes is to determine the structure of protein complexes and reveal how their structural organization can relate to their function. However, studying the threedimensional organization of large protein assemblies represents a major challenge for structural biologists. In the last decade, Mass Spectrometry (MS) has become a very valuable tool for analyzing such complexes offering an unrivaled sensitivity and the possibility to analyze very large complexes in their biological state. For instance, cross-linking combined to MS leads to the identification of aminoacids that are in close proximity and thus provide spatial constraints for the modeling1. Our group recently designed a new trifunctional cross-linker (named NNP9) dedicated to MS analysis, which outperforms all commercial ones and allows the analysis of large membrane systems2. MS can also be used to analyze protein complexes in their native state (native MS). For these experiments, the noncovalent interactions between subunits are kept intact and thus the stoichiometry of the complex can be determined by the accurate measurement of its molecular mass. Additional fragmentation steps can also be performed to obtain information on the complex topology. Our goal, with this PhD project is to combine both cross-linking MS and native MS to decipher the structure of bacterial secretion systems that are challenging protein complexes involved in bacterial virulence. In a first part, the PhD student will evaluate the efficiency of several newly synthetized crosslinkers. These developments will be performed on model protein complexes. Native mass spectrometry experiments will be optimized in parallel. In a second part, he/she will work on elucidating the structure of several bacterial secretion systems in collaboration with different teams of biologists. These protein complexes are sophisticated nanomachineries anchored in the bacterial membrane and used by many bacterial pathogens to deliver toxins or other effectors into target cells. The newly developed MS-based approaches will be used to obtain high quality structural information. All data will finally be combined to build structural models for the complexes of interest. The PhD student will have full access to state-of-the-art high-resolution LC-MS mass spectrometers present in the lab (Orbitrap Lumos, Orbitrap Q-Exactive). He will also participate to high-level instrumental developments. References: (1) Petrotchenko, E. V.; Borchers, C. H. Mass Spectrom. Rev. 2010, 29, 862−876. (2) Nury, C. et al. 2015, 87, 1853−1860 Indiquez les cinq meilleures publications récentes de l’équipe : Canul-Tec, J.C. et al. Nature (in press). Vorontsov, E.A., et al. Mol Cell Proteomics 15, 3388-3404 (2016). Nury, C., et al. Anal Chem 87, 1853-1860 (2015). Gault, J., et al. Plos Pathog 11 (2015). Smith, L.M., et al. Nat Methods 10, 186-187 (2013).