Reference : PhD Bruno Kieffer
Nuclear receptors are hormone regulators regulating the expression of genes upon hormonal signal in numerous tissues such as brain, muscles or prostate. These receptors, which responds to hormones such as glucocorticoids, vitamin D or testosterone contains folded domains and disordered regions that are enriched in proline residues. These proline-rich regions (PRM) are involved in the regulation process through their interactions with proteins named co-regulators. The major difficulty encountered to gain a fundamental understanding of sequence-function relationships in PRM regions of disordered proteins is due to the intrinsic difficulty to provide a structural and dynamical description of these regions at atomic resolution. While molecular disorder precludes the use of X-ray and cryo-EM approaches, the use of Nuclear Magnetic Resonance (NMR) faces the difficult issue of proline homopolymer's assignment. The key problem is that the proton signals of prolines tend to overlap, preventing site-specific assignment and measurements.
The addition of fluorine atoms (19F) on selected prolines of PRM provides an elegant way to solve this issue on two levels. First, the electronic effect of fluorine results in a dispersion of the remaining proton signals on the same proline residue, thereby enabling their identification in the NMR spectrum. Second, the 19F is also NMR active, with an intrinsic sensitivity close to 1H, allowing its detection through a 19F-NMR spectrum. Furthermore, the absence of fluorine in biomolecules facilitates the observation of fluorinated compounds in complex biological media, a feature that opens the way to in-cell NMR approaches.
The aim of the proposed thesis project is to take advantage of the conformational and NMR properties of fluorinated prolines to setup experimental NMR approaches allowing to investigate, at the molecular level, the mechanisms of proline rich motifs presents in NTD of several nuclear receptors that have been described as important for their regulatory function.
The PhD work will consist in designing novel NMR experiments to measure the conformational behavior of protein fragments encompassing fluorinated prolines. He will synthetize these fragments using semi-synthesis approaches including ligation strategies, in collaboration with the laboratory of Vladimir Torbeev at ISIS and the group of Bruno Linclau, in Southampton (UK). The student will also perform phosphorylation assays and aggregation studies using 19F NMR. A long-term perspective of the work is the transfection of modified proteins in model cell systems to evaluate the functional consequences of proline modifications in vivo. The profile of the student will be at the interface between NMR and biochemistry.
Date limite de candidature : 1 novembre 2018