The nucleocapsid protein (N) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for genome replication, encapsidating the viral genome and regulating gene transcription. The protein is highly disordered, comprising two disordered termini and a central disordered domain that are essential to its function. The central domain contains a number of important mutations that are responsible for enhanced viral fitness, and comprises a region that is hyperphosphorylated during the viral cycle. NMR spectroscopy is the tool of choice for studying the conformational behaviour of intrinsically disordered proteins, an abundant class of proteins that are functional in their disordered form. They represent 40% of the proteome and are too dynamic to be studied by crystallography or electron microscopy. The host lab has developed a large number of unique NMR-based tools to help understand the function of this class of proteins at atomic resolution. We will use NMR, paramagnetic NMR, small angle scattering, single molecule FRET and electron microscopy, in combination with molecular dynamics simulation, to describe the interactions of N with viral partner proteins and viral RNA to describe the process of encapsidation of the viral genome by the nucleocapsid protein, as well as the impact of mutations present in variants of concern. The results will be correlated with light and electron microscopy, carried out in collaboration.

The Synchrotron Group at the Institut de Biologie Structurale in Grenoble is currently developing an innovative method called TR-FOX (Time-Resolved Functional Oscillation Crystallography). This technique aims to elucidate, firstly, the global dynamics of biological macromolecules in action and, secondly, their fine catalytic mechanism. It relies on the use of an injector capable of depositing onto the crystal, during the course of the X-ray diffraction data collection, a nanoliter droplet containing the substrate and cofactor of the studied reaction. This triggers the enzymatic reaction within the crystal. The approach can be combined with UV-Visible absorption spectroscopy to characterize the reaction kinetics more precisely. The goal is to obtain a series of structures during the catalytic cycle in order to make a molecular movie depicting the functioning of the enzyme. This thesis has two objectives: (i) improve and validate the TR-FOX method and, (ii) study the catalytic mechanism of two enzymes involved in carbon fixation either by capture or conversion of CO2.