Fluorescence Measurement of Kinetics of CheY Autophosphorylation with Small Molecule Phosphodonors

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1729)

Abstract

The Escherichia coli chemotaxis protein CheY is a model receiver domain containing a native tryptophan residue that serves as a fluorescent probe for CheY autophosphorylation with small molecule phosphodonors. Here we describe fluorescence measurement of apparent bimolecular rate constants for reaction of wild type and mutant CheY with phosphodonors acetyl phosphate, phosphoramidate, or monophosphoimidazole. Step-by-step protocols to synthesize phosphoramidate (K+ salt) and monophosphoimidazole (Na+ salt), which are not commercially available, are provided. Key factors to consider in developing autophosphorylation assays for other response regulators are also discussed.

Keywords

CheY Tryptophan fluorescence Stop-flow kinetics Acetyl phosphate Phosphoramidate Monophosphoimidazole Autophosphorylation Receiver domain Response regulator 

Notes

Acknowledgments

This work was supported by National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM050860. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Department of Microbiology and ImmunologyUniversity of North CarolinaChapel HillUSA

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