Credit illustration: Jeanne Le Peillet @jeannedart
Photosynthesis is the main energy and carbon source for the biosphere. The Calvin-Benson cycle fixes atmospheric CO2 into organic trioses in the stroma of chloroplasts. While the 11 enzymes individually catalyzing the chemical steps of the cycle are known, little has been reported about their supra-molecular organization. Hence, the CALVINTERACT project ambitions to redefine the molecular basis for Calvin-Benson photosynthetic carbon fixation, in the light of the regulatory interactions that emerged between its protein components. We propose to determine the crystal structures of three multi-protein complexes, to measure their protein-protein interactions in vitro and to detect native complexes from cell extracts. The study will be completed with the design of artificial interactions in a synthetic biochemistry perspective. Physiological effects will be assayed on the model unicellular alga Chlamydomonas reinhardtii.
The project ambitions to define in a model unicellular organism, the green alga Chlamydomonas reinhardtii, the structures of all Calvin-Benson enzymes, a pathway that ensures biological carbon fixation. Besides the determination of novel individual structures for chloroplastic enzymes, the project aims at describing homo-oligomerization of these proteins, their interactions with thioredoxin redox regulators, and protein-protein interactions that enzymes establish together. In a synthetic biology approach, artificial interactions are designed and their effects tested on enzymatic efficiency and metabolism.
The project mainly depends on the determination of high-resolution structures by X ray crystallography, with recombinant proteins overexpressed in the bacterial host Escherichia coli. During the first months of the project, we submitted one of the enzymes to electron microscopy analysis. Complexed states of Calvin Benson enzymes are searched for in algae extracts, by chromatographic means to begin with, then by co-immunoprecipitation in the future. An in-silico strategy of protein-protein interaction prediction was set up recently in collaboration with experts in computational biology.
Structures of all remaining enzymes, except one, were obtained. A novel redox regulator was revealed by the identification of surface residues in the crystallographic model. High molecular weight species of several enzymes were observed from size-exclusion chromatography, justifying further validation and characterization. The in vitro isolation of multi-enzymatic complexes and the de novo design of multi-protein assemblies are the prominent objectives of the second phase of the project.