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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 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 sixfold more tightly than unmodified CheY. Taken together these results suggest that Tm phosphono-CheY is a useful and unique analog of Tm CheY-phosphate.

SUBMITTER: Beyersdorf MS 

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

REPOSITORIES: biostudies-literature

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

Beyersdorf Matthew S MS   Sircar Ria R   Lookadoo Daniel B DB   Bottone Cory J CJ   Lynch Michael J MJ   Crane Brian R BR   Halkides Christopher J CJ  

Protein science : a publication of the Protein Society 20170514 8


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 ma  ...[more]

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