Etude structurale et fonctionnelle par RMN d'une chaperonine de 1 MDa en action

Abstract : Chaperonins are essential molecular chaperons for the refolding of proteins in the cells. Size and complexity of these biological machineries make complex the study of their structural and functional properties. NMR spectroscopy offers an unique ability to monitor structural and dynamic changes in real-time and at atomic resolution. However, the NMR studies of large proteins and complexes has been a real challenge for a long time. In the first part of this thesis, it has been shown that the combination of methyl specific labeling, optimized NMR spectroscopy for large assemblies and electron microscopy can be used to monitor the different states of the functional cycle of a 1 MDa chaperonin. To study this mechanism, the native chaperonin was reconstituted with a labeling of the methionines and valines methyl groups. Methionines residues have been used as probes to identify the NMR spectra corresponding to intermediates states and active species of the functional cycle. Thanks to theses probes, it has been possible to follow in real time the structural rearrangements corresponding to the different conformations of the chaperonin during its functional cycle. The second part deals with the characterization of the interaction between the chaperonin and an unfolded protein. Observation of the stabilization of the unfolded protein by the chaperonin allowed to identify the holdase activity of the chaperonin. Using a clever combination of a differential methyl labeling and optimized NMR spectroscopy for large assemblies, it has been possible to follow the refolding of the unfolded protein by the chaperonin and the effects of the unfolded protein on the functional cycle of the chaperonin in action.
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Guillaume Mas. Etude structurale et fonctionnelle par RMN d'une chaperonine de 1 MDa en action. Biochimie [q-bio.BM]. Université Grenoble Alpes, 2015. Français. ⟨NNT : 2015GREAV036⟩. ⟨tel-01484643⟩

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