Identification of NO-sensitive cysteine residues using cysteine mutants of recombinant proteins

Abstract

Nitric oxide (NO) is a free radical gas regulating a wide range of biological processes in plants. Proteins are the main reaction target of NO inside the cells. The relevance of S-nitrosation as one of the NO-mediated protein posttranslational modifications has been studied in detail. S-nitrosylation causes alterations of the activity/function, sub-cellular localization or interaction partners of proteins. Up to present, a large number of S-nitrosation candidates have been detected in plants. Recombinant proteins are widely used to show or confirm the protein posttranslational modifications. Here, using recombinant proteins subjected to biotin switch assay, the S-nitrosation of some nuclear candidates of Arabidopsis is verified. Proteins usually contain several cysteine residues which each might involve in structure of protein active sites. So, an important question is: which cysteine residue is the target of S-nitrosation and does it belong to an active site? Here, using the approach of substitution of cysteines by serines on recombinant proteins, the NO-sensitive cysteine residue of an Arabidopsis nuclear protein is identified. The next step could be to investigate the effect of S-nitrosation on protein activity/function and further to test the role of target cysteines and S-nitrosation of them in protein activity/function.