Nitrosopeptide mapping: a novel methodology reveals s-nitrosylation of dexras1 on a single cysteine residue.
S-Nitrosylation of specific cysteine residues is a reversible signaling mechanism of nitric oxide (NO) generated by NO synthase (NOS) enzymes. In some proteins, evidence has accumulated that more than one cysteine can be S-nitrosylated; however, it is difficult to distinguish S-nitrosylation on separate cysteine residues. We report a novel simple, sensitive, and specific procedure for nitrosopeptide mapping. Dexras1 is a monomeric G protein whose guanine nucleotide exchange activity is augmented by NO; the identity and number of its S-nitrosylated cysteines is unknown. We describe the radiolabeling of S-nitrosylated cysteine residues in Dexras1. A nitrosopeptide map, generated by two-dimensional peptide chromatography, reveals that only a single cysteine is S-nitrosylated following NO exposure. Mutagenesis of Cys11 abolished the effect of NO donors on Dexras1, implicating this residue in the NO-mediated activation of Dexras1.
Jaffrey
Samie R
SR
Department of Pharmacology, Weill Medical College of Cornell University, New York, NY 10021, USA.
Fang
Ming
M
Snyder
Solomon H
SH
D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins; D006801:Humans; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide; D010449:Peptide Mapping / Q000379:methods*; D026403:S-Nitrosothiols / Q000032:analysis* / Q000737:chemistry; D013462:Sulfur Radioisotopes; D018631:ras Proteins
C400132:RASD1 protein, human; D026403:S-Nitrosothiols; D013462:Sulfur Radioisotopes; D009569:Nitric Oxide; C021315:S-nitrosocysteine; D019204:GTP-Binding Proteins; D020559:Monomeric GTP-Binding Proteins; D018631:ras Proteins; D003545:Cysteine