Intrinsically Disordered Proteins represent a third of the human proteome and are involved in countless biological mechanisms from cell signalling to the formation of vital membraneless organelles in the cells as well as playing a role in numerous diseases and viruses. Since they lack a stable 3D structure, their dynamic properties play an important role in their biological functions. While characterizing this class of proteins in vitro is well established, their behavior in more physiological conditions from the crowded cellular milieu to the highly concentrated liquid-liquid phase separated membraneless organelles is poorly understood. In this thesis, we use the C-terminal domain of the Nucleoprotein of Measles Virus as a model system to investigate the dynamic properties of this prototypical disordered protein in its liquid-liquid phase separated condensed phase as well as in crowded environments using Nuclear Magnetic Resonance and Molecular Dynamics Simulations.

We first show that liquid-liquid phase separation increases the rotational correlation times associated with the backbone dynamics of the protein and that the contribution of local and long-range motional modes is significantly redistributed. MD simulations of concentrated proteins suggest that this redistribution is correlated with the increase of non-specific intermolecular proximity or entanglement within the concentrated phase, leading to more restricted motions within the protein’s backbone. NMR spin relaxation also showed that the distribution of slower segmental motions is correlated with the position of charged and aromatic residues in some regions, suggesting that the phase separation of our protein is stabilized by electrostatic and cation-pi interactions, in agreement with previous proposals.

A shorter construct of the Measles C-terminal domain of the Nucleoprotein was further studied upon crowding with a high molecular weight PEG10000 polymer up to concentrations that correspond to those found in biomolecular condensates. NMR spin relaxation measurements showed that high levels of macromolecular proximity in super-crowded conditions redistributes the dynamic modes of the protein backbone in a way that is not observed at lower levels of crowding, suggesting that significantly crowded conditions can modify the dynamic properties of certain IDPs. Finally, a protein-protein interaction between this construct and its partner in the C-terminal domain of the Measles Virus Phosphoprotein was studied upon crowding and exhibited a significant slow down of the kinetic properties of the interaction. Further studies of this interactions by NMR should contribute to a better understanding of the atomic-resolution effects of crowding on protein-protein interactions, crucial for biological processes.

This study provides insight into the effect of crowded environments on the backbone dynamics of Intrinsically Disordered Proteins and is a step towards a better understanding of how this crucial class of proteins behave in more physiological environments including the currently actively studied membrane-less organelles, ubiquitous in eukariotic cells and viral machineries. ​ ​