Development of novel sample preparation strategies for in-cell NMR

NMR spectroscopy is offering increasing possibilities to obtain structural and dynamic information about macromolecules at atomic resolution. In recent years, it has been extended to the investigation of biological macromolecules in their physiological environment. In-cell NMR spectroscopy allows obtaining physiologically relevant structural and functional information inside living cells through the direct observation of several processes such as protein folding and interaction, metal ion binding, and drugs screening. This thesis aims to widen the application of in-cell NMR for the characterization of the structural and functional properties of proteins as well as their interactions. In a first study, we investigated the folding and the redox state of three human disulphidecontaining proteins (Mia40, Cox17, and SOD1) in the cytoplasm of human and bacterial cells. We successfully determined their redox-state distribution in isolation and with cofactors or redox partners, and found that it is controlled by specific proteins and pathways. In a second study, we employed in vitro and in-cell NMR to characterize the effect of a potential drug (ebselen) on SOD1 mutants. The results revealed that ebselen promotes the correct folding of destabilized SOD1 mutants in cells, and restores their dimerization towards the proper maturation pathway in vitro. Finally, we worked on sample preparation to increase the range of applications of in-cell NMR. On the one hand, we studied several protein systems (CytC, PFN1 and MNK6) in bacterial cells through MAS solid state NMR in order to detect intracellular soluble proteins that are not detectable with canonical experiments of solution NMR. On the other hand, we sought to expand the existing methods of solution in-cell NMR in order to study protein-protein interactions in human cells. In particular, we worked to combine DNA transfection and the delivery of an isotope-labelled recombinant protein to maximize the selectivity of protein labelling inside cells, and minimize the signals of cellular background in the NMR spectra.