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. Changes in HPLC retention times, chemical assays
for phosphate and free thiol, and mass spectrometry experiments demonstrate modification of
Cys54 with a phosphonomethyl group. Additionally, a crystal structure showed electron density
for the phosphonomethyl group at Cys54, consistent with a modif... More
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. Changes in HPLC retention times, chemical assays
for phosphate and free thiol, and mass spectrometry experiments demonstrate modification of
Cys54 with a phosphonomethyl group. Additionally, a crystal structure showed electron density
for the phosphonomethyl group at Cys54, consistent with a modification at that position.
Subsequent biochemical experiments confirmed that protein crystals were phosphono-CheY.
Isothermal titration calorimetry and fluorescence polarization binding assays demonstrated that
phosphono-CheY bound a peptide derived from FliM, a native partner of CheY-phosphate, with
a dissociation constant of ~29 µM, at least six-fold more tightly than unmodified CheY. Taken
together these results suggest that Tm phosphono-CheY is a useful and unique analog of Tm
CheY-phosphate.
