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In Vitro Alkylation Methods for Assessing the Protein Redox State

Abstract : Cysteines are important residues for protein structure, function, and regulation. Owing to their modified reactivity, some cysteines can undergo very diverse redox posttranslational modifications, including the reversible formation of disulfide bonds, a widespread protein regulatory process as well exemplified in plant chloroplasts for Calvin-Benson cycle enzymes. Both core- and peripheral-photorespiratory enzymes possess conserved cysteines, some of which have been identified as being subject to oxidative modifications. This is not surprising considering their presence in subcellular compartments where the production of reactive species can be important. However, in most cases, the types of modifications and their biochemical effect on protein activity have not been validated, meaning that the possible impact of these modifications in a complex physiological context, such as photorespiration, remains obscure. We here describe a detailed set of protocols for alkylation methods that have been used so far to (1) study the protein cysteine redox state either in vitro by submitting purified recombinant proteins to reducing/oxidation treatments or in vivo by western blots on protein extracts from plants subject to environmental constraints, and its dependency on the two major reducing systems in the cell, i.e., the thioredoxin and glutathione/glutaredoxin systems, and (2) determine two key redox parameters, i.e., the cysteine pKa and the redox midpoint potential.
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Flavien Zannini, Jérémy Couturier, Olivier Keech, Nicolas Rouhier. In Vitro Alkylation Methods for Assessing the Protein Redox State. Photorespiration : Methods and Protocols, pp.51-64, 2017, ⟨10.1007/978-1-4939-7225-8_4⟩. ⟨hal-02913009⟩



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