Production, characterization, and assessment of a stable analog of the response regulator CheY-phosphate from Thermotoga maritima.

Phosphorylation of CheY promotes association with the flagellar motor and ultimately controls the directional bias of the motor. However, biochemical studies of activated CheY-phosphate have been challenging due to the rapid hydrolysis of the aspartyl-phosphate in vitro. An inert analog of Tm CheY-phosphate, phosphono-CheY, was synthesized by chemical modification and purified by cation-exchange chromatography.

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.