Deciphering Complex Energy Landscape at Atomic Resolution of Human HSP90 using NMR and AI-Enhanced tools.
HSP90 is a human chaperone involved in the folding of a wide variety of client proteins, including many oncogenic proteins. This complex molecular machinery is known to undergo massive conformational rearrangements throughout its functional cycle. X-ray crystallography and cryoEM have provided high-resolution snapshot structures of this human machinery in complex with cochaperones and client proteins, but have failed to provide the kinetic and time-resolved information needed for a full understanding of its mechanism. We plan to use NMR experiments combined with a new AI-enhanced analysis tool to obtain a detailed picture of the energy landscape of this important drug target. This project will provide structural information on the different excited states of HSP90 and the conformational dynamics between these states. In collaboration with the pharmaceutical industry, we will exploit this new approach to reveal how ligands can modulate the energy landscape and population of different functional states. This information will be particularly useful for the design of new drugs capable of blocking the HSP90 chaperone in a single state, an important step towards the development of more specific and effective drugs.
Molecular dynamics and disorder in the viral replication machinery of SARS CoV 2
The nucleoprotein (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 central disordered domain is essential to the function of this highly dynamic protein, containing a number of important mutations that are responsible for enhanced viral fitness, and comprising 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. Post-translational modification, in particular phosphorylation, is thought to play an important functional role, that remains poorly understood, we will investigate the impact of phosphorylation on conformational dynamics and relate this to modifications in function. The results will be correlated with light end electron microscopy, carried out in collaboration.
[FeFe]-hydrogenase active site assembly machinery
To tackle the climate crisis, humanity urgently needs renewable and decarbonized energy sources. A promising solution lies in harnessing dihydrogen (H2), and enzymes known as [FeFe] hydrogenases can play a vital role in its production. These enzymes catalyze the reversible oxidation of dihydrogen, employing an active site called the "H-cluster," a complex organometallic structure. The intricate biosynthesis of this cluster involves three maturation proteins: HydG, HydE, and HydF. Despite recent progress, a full understanding of this process remains elusive due to the complexity of the chemical reactions involved. Our goal is to conduct a structural study combined with step-by-step reaction monitoring using spectroscopy. This approach aims to identify and characterize various reaction intermediates of one key enzyme in the process. This collaborative project involves two leading CEA teams specializing in the study of oxygen-sensitive metalloproteins. The doctoral student will benefit from an ideal scientific and technical environment to achieve this objective, crucially important for advancing hydrogen economy development.