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

ABSTRACT: 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 cry

SUBMITTER: Beyersdorf MS 

PROVIDER: S-EPMC5521581 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature