Synthesis and Biochemical Characterization of an Analogue of CheY-Phosphate, a Signal Transduction Protein in Bacterial Chemotaxis

• Published in: Biochemistry (Easton) 1998-09, Vol.37 (39), p.13674-13680
• Main Authors: Halkides, Christopher J, Zhu, Xiangyang, Phillion, Dennis P, Matsumura, Philip, Dahlquist, Frederick W
• Format: Article
• Language: English
• Subjects: Alkylation; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Bacterial Proteins - physiology; Binding Sites; Chemotaxis; Chromatography, High Pressure Liquid; Magnesium - physiology; Membrane Proteins - biosynthesis; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Membrane Proteins - physiology; Methyl-Accepting Chemotaxis Proteins; Phosphates - metabolism; Phosphoproteins - biosynthesis; Phosphoproteins - chemistry; Phosphoproteins - metabolism; Phosphoproteins - physiology; Phosphoric Monoester Hydrolases - metabolism; Phosphorylation; Signal Transduction; Spectrometry, Fluorescence
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9806293

CheY is a signal transduction protein of the bacterial chemotaxis system that acts as a molecular switch to alter the swimming behavior of the bacterium. When CheY becomes phosphorylated at Asp57, CheY-Pi interacts with flagellar motor proteins, including FliM, to increase the likelihood that the flagellar motor will change its sense of rotation, increasing the frequency of tumbling. The structure of CheY in its dephosphorylated (inactive) state has been intensively investigated. The short lifetime (∼20 s) of the aspartyl phosphate has precluded the complete structural determination of CheY-Pi. We have synthesized an analogue of CheY-Pi by alkylating an aspartate-to-cysteine mutant at position 57 of CheY to add a phosphonomethyl group at Cys57. This analogue, phosphono-CheY, is stable for months. Phosphono-CheY binds to two of the targets of CheY-Pi, FliM and CheZ, in a manner similar to that of CheY-Pi and much better than either unphosphorylated CheY or the unmodified form of D57C CheY. Phosphono-CheY also binds Mg(II) with a dissociation constant of ∼6 mM at neutral pH and moderate salt level. These observations indicate that phosphono-CheY is a good biochemical analogue of CheY-Pi.