Synthesis and biochemical characterization of a phosphorylated analogue ofthe response regulator CheB

CheB is a response regulator protein in the bacterial chemotaxis two-compon ent signal transduction pathway. Methylesterase CheB functions together wit h methyltransferase CheR to modulate the level of glutamate methylation in transmembrane chemoreceptors in response to environmental stimuli. The leve l of glutamate methylation in turn indirectly controls the direction of fla gellar rotation. Like most two-component response regulators, CheB is activ ated in vivo by phosphorylation of a single aspartate, Asp 56, in its regul atory domain. Extensive biochemical and crystallographic studies have been completed on them inactive, unphosphorylated form of CheB. Because of the i nherent lability of aspartyl phosphate bonds and the intrinsic phosphatase activity of CheB, the activated, phosphorylated form of CheB cannot be isol ated for further characterization. We present a synthetic scheme to prepare an analogue of phosphorylated CheB using site-specific mutagenesis and che mical modification strategies. Initially, the two native cysteines found in CheB were substituted by serines and a cysteine was substituted for Asp 56 to yield D56C/C207S/C309S CheB. The unique cysteine in the substituted for m of CheB, was modified by sodium thiophosphate, Na3SPO3, Wing two sequenti al disulfide bond exchange reactions. The analogue, D56C/C207S/C309S CheB-S PO3, contained a thiophosphate group covalently bonded to the protein throu gh a disulfide linkage at residue 56. Mass spectrometry showed that the pro tein was singly modified. Reverse phase chromatography showed that greater than 95% of the protein was modified under optimized conditions and that th e analogue had a half-life of 28 days. In in vitro methylesterase assays in the presence of Mg2+, the analogue exhibited activity equivalent to that o f fully phosphorylated C207S/C309S CheB. Thus, D56C/C207S/C309S CheB-SPO3 i s a stable analogue that may be useful for characterization of the active f orm of CheB.