FLUOPROLINE

Intrinsically disordered proteins (IDPs), comprising a third of the eukaryotic proteome, play key roles in regulatory and signalling processes within cells. The sequences of IDPs frequently contain proline residues, either as part of a polyproline domain or as part of a (Ser/Thr)-Pro phosphorylation site. Currently, the functional importance of proline rich motifs in the regulation of biological functions is not well understood. It is known that subtle conformational changes of the proline ring's structure, as well as changes in the isomerization rate between cis and trans isomers play a role in modulating protein-protein interactions. However, these parameters are difficult to characterize at the level of individual proline residues, let alone 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 poly-proline homopolymer's assignment. The FLUOPROLINE project's objective is to develop a novel way to solve this issue by the incorporation of fluorine atoms (19F) on selected prolines. 19F-based NMR is highly sensitive and bio-orthogonal which enables straightforward readout of proline's conformation and dynamics. Furthermore, by the introduction of various strereoisomeric fluoro-prolines, it is possible to modify these properties in a controllable way. As a test case, this approach will be applied to decipher the molecular mechanisms underlying the specific recognition of different proline rich motifs (PRM) by the SH3 domain of BIN1 (amphiphysin), a protein whose dysfunction is involved in myopathy. These interactions are involved in particular in regulating the multiple functions of dynamin (DNM2) in membrane remodeling, endocytosis and autophagy. This interdisciplinary project, at the interface between biophysics, chemistry and chemical biology, as well as translational medicine, gathers two teams at IGBMC involved in structural biology (NMR: Bruno Kieffer) and Translational medicine (Jocelyn Laporte) and two teams in chemistry: Vladimir Torbeev specialized in protein chemistry at ISIS, Strasbourg and Bruno Linclau at the University of Southampton specializing in organofluorine chemistry. The proposed work consists of four objectives: (i) The description of conformational and dynamical properties of non-commercial fluorinated prolines able to stabilize either the cis or the trans conformation, as well as the pucker preference using by 19F NMR; (ii) The impact of PRM chemical modifications (fluorination, phosphorylation) on intra and intermolecular interactions between BIN1 SH3 and PRMs from BIN1 and DNM2; (iii) the design of potent peptides able to modulate with the BIN1/ DNM2 interaction; (iv) the development of experimental approaches enabling the observation of BIN1-DNM2 interaction and its modulation in a cellular context. Beside a gain in our fundamental understanding of the specific roles of proline residues in IDPs, the project will lead to methodology developments in the fluorine chemistry, the semi-synthesis of large protein constructs and the studies of molecular interactions by 19F NMR. The characterization of novel fluoroprolines with tuneable molecular properties will extend the current repertoire of chemically modified peptides developed for therapeutic purposes. Peptides modulating the BIN1/DNM2 complex could have medical applications. The strong involvement of two partners of the project in start-up creation will favour an efficient technological transfer for the design of new drugs.