Synthesis of a stable analog of the phosphorylated form of CheY: phosphono-CheY.

The chemical modification of a cysteinyl residue of D57C CheY by the addition of a phosphonomethyl group, (HO)(2)P(O)-CH(2)-, is described. This modification produces a nonlabile analog of an aspartyl phosphate residue in the active form of CheY. The chemically modified protein, phosphono-CheY, is suitable for structural and functional studies. An extensive discussion of the synthetic methodology and purification strategy is presented. A detailed protocol is given.