Unraveling the mechanism of enzymatic carbon fixation
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.
Towards a better understanding of membrane proteins through AI
Despite the remarkable advances in artificial intelligence (AI), particularly with tools like AlphaFold, the prediction of membrane protein structures remains a major challenge in structural biology. These proteins, which represent 30% of the proteome and 60% of therapeutic targets, are still significantly underrepresented in the Protein Data Bank (PDB), with only 3% of their structures resolved. This rarity is due to the difficulty in maintaining their native state in an amphiphilic environment, which complicates their study, especially with classical structural techniques.
This PhD project aims to overcome these challenges by combining the predictive capabilities of AlphaFold with experimental small-angle scattering (SAXS/SANS) data obtained under physiological conditions. The study will focus on the translocator protein TSPO, a key marker in neuroimaging of several serious pathologies (cancers, neurodegenerative diseases) due to its strong affinity for various pharmacological ligands.
The work will involve predicting the structure of TSPO, both in the presence and absence of ligands, acquiring SAXS/SANS data of the TSPO/amphiphile complex, and refining the models using advanced modeling tools (MolPlay, Chai-1) and molecular dynamics simulations. By deepening the understanding of TSPO’s structure and function, this project could contribute to the design of new ligands for diagnostic and therapeutic purposes.
Dynamic interplay of Rad51 nucleoprotein filament-associated proteins - Involvement in the regulation of homologous recombination
Homologous recombination (HR) is an important repair mechanism for DNA double-strand breaks induced by ionizing radiation. A key step in HR is the formation of Rad51 nucleoprotein filaments on the single-stranded DNA that is generated from these breaks. We were the first to show, using yeast as a model, that a tight control of the formation of these filaments is essential for HR not to induce chromosomal rearrangements by itself (eLife 2018, Cells 2021). In humans, the functional homologs of the yeast control proteins are tumor suppressors. Thus, the control of HR seems to be as important as the mechanism of HR itself. Our project involves the use of new molecular tools that allow a breakthrough in the study of these controls. We will use a functional fluorescent version of the Rad51 protein, first developed by our collaborators A. Taddei (Institut Curie), R. Guérois and F. Ochsenbein (I2BC, Joliot, CEA). This major advance will allow us to observe the influence of regulatory proteins on DNA repair by microscopy in living cells. We have also developed highly accurate structural models of control protein complexes associated with Rad51 filaments. We will adopt a multidisciplinary approach based on genetics, molecular biology, biochemistry, and protein structure in collaboration with W.D. Heyer (University of California, Davis, USA), to understand the function of the regulators of Rad51 filament formation. The description of the organization of these proteins with Rad51 filaments will allow us to develop new therapeutic approaches.
Condensates and Chromatin: How Phase Separation Shapes Plant Temperature Responses
Plants must adapt their development to environmental conditions, including rising temperatures due to climate change. Heat stress significantly impacts plant physiology, and to mitigate these effects, plants have evolved heat shock responses (HSR), with Heat Shock Factor A1a (HSFA1a) serving as a master regulator in Arabidopsis thaliana. Under nonstress conditions, HSFA1a remains cytosolic and inactive, bound to heat shock proteins (HSPs). Heat stress triggers HSP dissociation, enabling HSFA1a nuclear translocation, trimerization, chromatin binding, and activation of stress-responsive genes. Recent studies reveal that HSFA1a might act as a pioneer transcription factor to access closed chromatin regions and initiate HSR. Additionally, preliminary findings also suggest that HSFA1a undergoes liquid-liquid phase separation (LLPS) to form nuclear condensates that regulate gene expression. This project aims to 1) explore how temperature affects HSFA1a structure and oligomerization, 2) investigate LLPS of HSFA1a with and without DNA, 3) characterize HSFA1a pioneer activity, and 4) determine the physiological importance of LLPS in HSR.
Nitrogenase Active Site Assembly: What Distinguishes a Nitrogenase from a Scaffold
The challenges posed by climate change and soil degradation call for urgent solutions to reduce greenhouse gas emissions and reliance on nitrogen fertilizers while ensuring sufficient crop yields to feed a growing global population. A natural solution lies in the use of nitrogenase, a bacterial enzyme capable of converting atmospheric nitrogen into ammonia, which can be directly assimilated by plants. However, the biosynthesis of its metal cofactor, FeMo-co, is a complex process that requires the coordinated action of numerous proteins.
This PhD project aims to streamline this complex process by studying simplified nitrogenase systems found in certain organisms, which use fewer proteins, notably by combining multiple functions into single proteins. By conducting comparative structural and functional studies, we seek to understand how these simplified systems work and how they can be adapted for use in crops like cereals, potentially allowing large-scale cultivation without heavy nitrogen fertilizer use.
This project is a collaboration between leading teams at CEA’s Institute of Structural Biology and CSIC Madrid, specializing in metalloprotein structure-function analysis and the biochemistry and genetics of nitrogenase assembly. The successful candidate will work in a cutting-edge research environment, gaining international experience and valuable skills for a future career in academic research or R&D.