Research - Interdisciplinarity- 2024

The bio-conversion of_CO2 by the Ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO) reaches 1014 kilograms per year. The carboxylation of Ribulose 1,5 bisphosphate (RuBP) activity of RuBisCO competes with its oxygenation activity. However, the diffusion of_CO2 in water is much less efficient than that of _O2, and the majority of inorganic carbon is in the form of bicarbonate in the aquatic environments. The success of photosynthetic microorganisms relies in their_CO2 concentration mechanisms (CCM) that induces a flux of inorganic carbon near RuBisCO. Contrary to higher plant, eukaryotic microalgae such as the model green algae Chlamydomonas reinhardtii possess biophysical CCMs which involves a peculiar supramolecular organisation and specific location of RuBisCO as well as bicarbonate transporters and carbonic anhydrases. RuBisCO is found in two distinct locations in C. reinhardtii: either diffused in the chloroplast stroma or condensed in a liquid-liquid separated phase (LLSP) named Pyrenoid. The proportion of diffused vs condensed RuBisCO varies from 60%/40% under condition of high partial pressure of_CO2 (_PCO2) to 20%/80% under low _PCO2. While numerous studies have investigated the carboxylation vs oxygenation activities of isolated RuBisCO or in vivo, the effect of condensation on the enzyme activity has been poorly investigated. The objective of this project is to correlate RuBisCO condensation to its activity. This is declined in two main strategies: 1- in-vitro reconstituted LLPS with recombinant RuBisCO and its scaffold protein EPYC1 and a structure-function investigation, and 2- in-vivo quantification of carboxylation and oxygenation activities under conditions in which RuBisCO is mainly diffused or mainly condensed.

Bacteria have evolved sophisticated membrane channels, called secretins, specifically adapted for the assembly or secretion of bulky extracellular structures such as type 4 pili (T4P), needle complexes of type 3 secretion systems (T3SS) or large fully folded effectors in type 2 secretion systems (T2SS) (Review by Barbat et al., 2023b). We have recently shown that T2SS secretins must adopt different conformations to allow the passage of large effectors while maintaining the integrity of the bacterial envelope (Barbat et al., 2023a). In this project, we plan to develop and implement an ambitious in vivo experimental approach aimed not only at demonstrating the need for flexibility of the XcpD secretin during the secretion process, but also at identifying the partners that trigger these conformational changes. Specifically, we will further investigate the protein dynamics that exist between two adjacent secretin subunits by performing in-cell EPR experiments on spin-labelled proteins in their native environment (Review by Pierro et al., 2024) as well as in different genetic backgrounds lacking potential partners. With this approach, we hope to gain a better understanding of the different chronological steps that occur during the secretion process mediated by this singular nanomachine.

References:

Barbat, B., Douzi, B., Ball, G., Tribout, M., El Karkouri, K., Kellenberger, C., and Voulhoux, R. (2023a). Insights into dynamics and gating properties of T2SS secretins. Sci Adv 9, eadg6996.

Barbat, B., Douzi, B., and Voulhoux, R. (2023b). Structural lessons on bacterial secretins. Biochimie 205, 110-116.

Pierro, A., Bonucci, A., Magalon, A., Belle, V., and Mileo, E. (2024). Impact of Cellular Crowding on Protein Structural Dynamics Investigated by EPR Spectroscopy. Chem Rev 124, 9873-9898.