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

• Published in: Protein science Vol. 26; no. 8; pp. 1547 - 1554
• Main Authors: Beyersdorf, Matthew S., Sircar, Ria, Lookadoo, Daniel B., Bottone, Cory J., Lynch, Michael J., Crane, Brian R., Halkides, Christopher J.
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.08.2017; John Wiley and Sons Inc
• Subjects: Amino Acid Sequence; Aspartic Acid - analogs & derivatives; Aspartic Acid - chemistry; Aspartic Acid - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Calorimetry; Cation exchanging; Cation-exchange chromatography; Chemical modification; Chemical synthesis; chemotaxis; Chemotaxis - physiology; Cloning, Molecular; Crystal structure; Crystallography, X-Ray; Crystals; cysteine modification; Electron density; Escherichia coli - genetics; Escherichia coli - metabolism; Flagella; Flagella - chemistry; Flagella - metabolism; Fluorescence; Fluorescence polarization; Gene Expression; Heat measurement; High performance liquid chromatography; Hydrolysis; In vitro methods and tests; Kinetics; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Methyl-Accepting Chemotaxis Proteins - chemistry; Methyl-Accepting Chemotaxis Proteins - genetics; Methyl-Accepting Chemotaxis Proteins - metabolism; Models, Molecular; Motor task performance; Organophosphonates - chemistry; Organophosphonates - metabolism; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Phosphate; Phosphates; phosphate‐analog; Phosphorylation; Polarization; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Engineering; Protein Interaction Domains and Motifs; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Thermotoga maritima - chemistry; Thermotoga maritima - genetics; Thermotoga maritima - metabolism; Titration; Titration calorimetry; two‐component system; phosphate-analog; cysteine modification; chemotaxis; two-component system
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.3180

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