Synthesis of a Stable Analog of the Phosphorylated Form of CheY: Phosphono-CheY
CheY is a response regulator of bacterial chemotaxis that is activated by phosphorylation of a conserved aspartate residue. However, studies of CheY-phosphate have proven challenging due to rapid hydrolysis of the aspartyl-phosphate in vitro. To combat this issue, we have designed a stable analog suitable for structural and functional studies. Herein, we describe a method for the chemical modification of Thermotoga maritima CheY to produce a phospho-analog designated as phosphono-CheY. Our modification produces a stable analog in the constitutively active form that enables the study of signal transfer to the downstream target.
KeywordsPhosphonomethyl trifluoromethanesulfonate Two-component system Cysteine modification Phosphate analog Chemical modification Response regulator Chemotaxis Phosphono-CheY
The authors acknowledge support from National Institutes of Health Grant R15 GM 063514-02A2 (C.J.H.) and R01 GM066775 (R.S. and B.C.). Instrumentation for the mass spectrometry facility in the Department of Chemistry and Biochemistry at UNCW was provided by a grant from the National Science Foundation Division of Chemistry (CHE-1039784). Special thanks to Dr. Brian Crane, Cory Bottone, R. Matthew Haas, Lindsey Boroughs, and Michael Harrington.
- 17.Beyersdorf MS, Sircar R, Lookadoo DB, Bottone CJ, Lynch MJ et al (2017) Production, characterization, and assessment of a stable analog of the response regulator CheY-phosphate from Thermotoga maritima. Protein Sci 26(8):1547–1554Google Scholar