Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

In-Situ Flexibility of Both Redox-States of the Chloroplast Regulatory Protein CP12

Version 1 : Received: 31 March 2021 / Approved: 2 April 2021 / Online: 2 April 2021 (11:23:04 CEST)

How to cite: Launay, H.; Shao, H.; Bornet, O.; Cantrelle, F.; Lebrun, R.; Receveur-Brechot, V.; Gontero, B. In-Situ Flexibility of Both Redox-States of the Chloroplast Regulatory Protein CP12. Preprints 2021, 2021040048 (doi: 10.20944/preprints202104.0048.v1). Launay, H.; Shao, H.; Bornet, O.; Cantrelle, F.; Lebrun, R.; Receveur-Brechot, V.; Gontero, B. In-Situ Flexibility of Both Redox-States of the Chloroplast Regulatory Protein CP12. Preprints 2021, 2021040048 (doi: 10.20944/preprints202104.0048.v1).

Abstract

In the chloroplast, Calvin-Benson-Bassham enzymes are active in the reducing environment imposed in the light via the electrons from the photosystems. In the dark these enzymes are inhibited, and this regulation is mainly mediated via oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges are simultaneous. In oxidized CP12, the C23-C31 pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C66-C75 pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment that is, in cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C66-C75 pair compared to the C23-C31 pair. Finally, the redox mid-potentials for the two cysteine pairs differ and are similar to those found for phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase, that we relate to the regulatory role of CP12.

Subject Areas

Calvin-Benson-Bassham cycle; Conditionally disordered protein; Intrinsically disordered protein; photosynthesis regulation.

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