SYNTHESIS AND BIOCHEMICAL-CHARACTERIZATION OF AN ANALOG OF CHEY-PHOSPHATE, A SIGNAL-TRANSDUCTION PROTEIN IN BACTERIAL CHEMOTAXIS

CheY is a signal transduction protein of the bacterial chemotaxis syst em that acts as a molecular switch to alter the swimming behavior of t he bacterium. When CheY becomes phosphorylated at Asp57, CheY-P-i inte racts with flagellar motor proteins, including FliM, to increase the l ikelihood that the flagellar motor will change its sense of rotation, increasing the frequency of tumbling. The structure of CheY in its dep hosphorylated (inactive) state has been intensively investigated. The short lifetime (similar to 20 s) of the aspartyl phosphate has preclud ed the complete structural determination of CheY-P-i. We have synthesi zed an analogue of CheY-P-i by alkylating an aspartate-to-cysteine mut ant at position 57 of CheY to add a phosphonomethyl group at Cys57. Th is analogue, phosphono-CheY, is stable for months. Phosphono-CheY bind s to two of the targets of CheY-P-i, FliM and CheZ, in a manner simila r to that of CheY-P-i and much better than either unphosphorylated Che Y or the unmodified form of D57C CheY, Phosphono-CheY also binds Mg(II ) with a dissociation constant of similar to 6 mM at neutral pH and mo derate salt level. These observations indicate that phosphono-CheY is a good biochemical analogue of CheY-P-i.