In Search Of Cysteines

Cysteine's high potential for nucleophilicity makes the amino acid a potent site of reactivity in proteins, but researchers find it hard to assess the reactivity of a given cysteine sidechain in a sea of proteins of unknown function. Now researchers led by Benjamin Cravatt at Scripps Research Institute, La Jolla, have reported an approach to obtain a proteome-wide assessment of cysteine reactivity.

The identification of hyper-reactive cysteine sites is an important accomplishment because modification of cysteine residues—by oxidation, for example—can override posttranslational cues such as phosphorylation that results in activation, inactivation, or changes in stability of some proteins.

Cravatt's technique first exposes proteins to a probe with an electrophilic iodoacetamide that reacts with non-disulfide bonded cysteines and an alkyne handle for easy isolation. After the probe has a chance to react with exposed cysteines, the protein sample is chopped up by enzymes (Nature, DOI: 10.1038/nature09472). The probes, now with a reactive cysteine attached, are recovered using azide-alkyne Huisgen cycloaddition by means of the probe's alkyne handle. Using mass spectrometry and an isotopically labeled sample, the team can thus predict functional, reactive cysteines in proteomes.

The team tested its technique on cancer cell proteomes, as well as a group of 12 proteins engineered by biochemists to employ reactive cysteines. The technique succeeded in picking out the only two proteins in this collection whose engineered cysteines are catalytically active.

"The potential utility for this approach is great," comments Leslie Poole, a biochemist at Wake Forest University who was not involved in the research. "It will have significant utility for sifting through [proteomes] and identifying proteins or sites of special reactivity."