title: Studying the S-nitrosylation of model peptides and eNOS protein by mass spectrometry.
abstract: Oxidative addition of a nitric oxide (NO) molecule to the thiol group of cysteine residues is a physiologically important post-translational modification that has been implicated in several metabolic and pathophysiological events. Our previous studies have indicated that S-nitrosylation can result in the disruption of the endothelial NO synthase (eNOS) dimer. It has been suggested that for S-nitrosylation to occur, the cysteine residue must be flanked by hydrophilic residues either in the primary structure or in the spatial proximity through appropriate conformation. However, this hypothesis has not been confirmed. Thus, the objective of this study was to determine if the nature of the amino acid residues that flank the cysteine in the primary structure has a significant effect on the rate and/or specificity of S-nitrosylation. To accomplish this, we utilized several model peptides based on the eNOS protein sequence. Some of these peptides contained point mutations to allow for different combinations of amino acid properties (acidic, basic, and hydrophobic) around the cysteine residue. To ensure that the results obtained were not dependent on the nitrosylation procedure, several common S-nitrosylation techniques were used and S-nitrosylation followed by mass spectrometric detection. Our data indicated that all peptides independent of the amino acids surrounding the cysteine residue underwent rapid S-nitrosylation. Thus, there does not appear to be a profound effect of the primary sequence of adjacent amino acid residues on the rate of cysteine S-nitrosylation at least at the peptide levels. Finally, our studies using recombinant human eNOS confirm that Cys98 undergoes S-nitrosylation. Thus, our data validate the importance of Cys98 in regulating eNOS dimerization and activity, and the utility of mass spectroscopy to identify cysteine residues susceptible to S-nitrosoylation.
authors.affiliation: Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, IL 60115, USA.
authors.forename: Frank S
authors.initials: FS
authors.lastname: Taldone
authors.forename: Monorama
authors.initials: M
authors.lastname: Tummala
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Goldstein
authors.forename: Victor
authors.initials: V
authors.lastname: Ryzhov
authors.forename: Kandasamy
authors.initials: K
authors.lastname: Ravi
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Black
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D020030:Nitric Oxide Donors / Q000737:chemistry* / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009589:Nitrogen Oxides / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D026423:S-Nitroso-N-Acetylpenicillamine / Q000737:chemistry / Q000494:pharmacology; D026422:S-Nitrosoglutathione / Q000737:chemistry / Q000494:pharmacology; D012977:Sodium Nitrite / Q000737:chemistry / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization; D013096:Spermine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; D010455:Peptides; D013439:Sulfhydryl Reagents; C091861:spermine nitric oxide complex; D013096:Spermine; D026422:S-Nitrosoglutathione; D026423:S-Nitroso-N-Acetylpenicillamine; D052250:Nitric Oxide Synthase Type III; D003545:Cysteine; D012977:Sodium Nitrite