Flexibility of Oxidized and Reduced States of the Chloroplast Regulatory Protein CP12 in Isolation and in Cell Extracts.

Helene Launay,Francois-Xavier Cantrelle,Brigitte Gontero,Regine Lebrun,Veronique Receveur-Brechot,Hui Shao,Olivier Bornet

doi:10.3390/biom11050701

Abstract

In the chloroplast, Calvin–Benson–Bassham enzymes are active in the reducing environment created in the light by electrons from the photosystems. In the dark, these enzymes are inhibited, mainly caused by 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 is 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: a cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C66–C75 pair than for the C23–C31 pair. The redox mid-potentials for the two cysteine pairs differ and are similar to those found for glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase, consistent with the regulatory role of CP12.

Highlights

Redox regulation based on disulfide-dithiol exchanges constitutes a rapid and reversible post translational modification (PTM) that affects protein conformation
We first investigated the dynamics of CP12red by measuring the amide proton exchange rate with H2 O by nuclear magnetic resonance (NMR) on CP12red to probe for the protected and exposed backbone amide protons
Similar to what has been observed for A. thaliana, our results indicate that the N-terminal disulfide in C. reinhardtii requires fewer reducing conditions to dissociate than the C-terminal disulfide, i.e., in thermodynamic terms, the N-terminal disulfide is easier to reduce than the C terminal disulfide

Summary

Introduction

Redox regulation based on disulfide-dithiol exchanges constitutes a rapid and reversible post translational modification (PTM) that affects protein conformation. PTMs of CDPs can contribute to the diversification and functionality of proteomes, by regulating different properties of proteins, which is termed the “proteoform concept” [2]. Among these CDPs, some are sensitive to redox changes, and these have been termed redox-dependent CDPs [3]. In these CDPs, key cysteine residues can form disulfide bridges under oxidizing conditions. Many examples of proteins containing IDRs, or of intrinsically disordered proteins (IDPs) that contain a redox-sensitive cysteine residue pair, have been

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Conclusion