SYNTHESIS AND CHARACTERIZATION OF A STABLE ANALOG OF THE PHOSPHORYLATED FORM OF THE CHEMOTAXIS PROTEIN CHEY

The bacterial chemotaxis protein CheY is activated in vivo by the cova lent phosphorylation of a single aspartate residue at position 57. How ever, this phosphate linkage is unstable (t(1/2) approximate to 20 s a t room temperature), thereby precluding many biochemical analyses. Her e we present a synthetic scheme to prepare an analog of CheY-phosphate (Che Y-P) with chemical stability of the phosphate linkage enhanced b y several orders of magnitude relative to the native protein. Starting with CheY D57C, a site-specific mutant of CheY with a unique cysteine residue in place of the aspartate at position 57, two sequential disu lfide exchange reactions were performed to form the final product 'Che Y D57C-SPO3' with a thiophosphate moiety covalently bonded to the prot ein in a disulfide linkage. Mass spectral analysis showed that the des ired analog was present at 70-80 % of the total protein. The disulfide linkage had a t(1/2) of 8 days at 4 degrees C. Biochemical characteri zation of CheY D57C-SPO3 included assessment of conformational propert ies using tryptophan fluorescence, evaluation of metal binding propert ies and measurement of binding interactions with the chemotaxis protei ns CheZ and FliM. Despite possessing a phosphoryl group at a nearly id entical location as native CheY-phosphate, the analog was unable to em ulate CheY-phosphate function, thereby supporting the idea that there are very precise geometric requirements for successful CheY activation .