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 side chain in a sea of proteins of unknown function. Now, researchers led by Benjamin F. Cravatt III at Scripps Research Institute, La Jolla, Calif., have reported an approach to obtain a proteome-wide assessment of cysteine reactivity.

The identification of hyperreactive 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, comments Cristina M. Furdui of Wake Forest University School of Medicine.

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 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, such as for sifting through proteomes and identifying proteins or sites of special reactivity,” comments Leslie B. Poole, a biochemist at Wake Forest University.

Next up, Cravatt’s team aims to better understand the function of hyperreactive cysteines in proteins of unknown function or implicated in diseases such as cancer and neurodegeneration.