Production, characterization, and assessment of a stable analog of the response regulator CheY‐phosphate from Thermotoga maritima
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‐p...
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Published in | Protein science Vol. 26; no. 8; pp. 1547 - 1554 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Wiley Subscription Services, Inc
01.08.2017
John Wiley and Sons Inc |
Subjects | |
Online Access | Get full text |
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Summary: | 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.
PDB Code(s): 4QYW |
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Bibliography: | Studies of bacterial chemotaxis have been hampered by the fact that CheY‐phosphate, the activated form of a protein which changes the directional rotation of the flagellar motor, is rapidly hydrolyzed. We synthesized an analog of CheY‐phosphate in which a labile phosphorus‐oxygen bond is replaced by an inert phosphorus‐carbon bond. This analog has been shown to share similar binding profiles to native CheY‐phosphate and is anticipated to continue to be of high value in future studies of CheY and its influence on bacterial chemotaxis. Statement of Impact ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Statement of Impact: Studies of bacterial chemotaxis have been hampered by the fact that CheY‐phosphate, the activated form of a protein which changes the directional rotation of the flagellar motor, is rapidly hydrolyzed. We synthesized an analog of CheY‐phosphate in which a labile phosphorus‐oxygen bond is replaced by an inert phosphorus‐carbon bond. This analog has been shown to share similar binding profiles to native CheY‐phosphate and is anticipated to continue to be of high value in future studies of CheY and its influence on bacterial chemotaxis. |
ISSN: | 0961-8368 1469-896X |
DOI: | 10.1002/pro.3180 |