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Bacterial Chemosensing pp 337–343Cite as

Synthesis of a Stable Analog of the Phosphorylated Form of CheY: Phosphono-CheY

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

Abstract

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.

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Acknowledgments

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.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, The University of North Carolina Wilmington, Wilmington, NC, USA

    Daniel B. Lookadoo, Matthew S. Beyersdorf & Christopher J. Halkides

Authors
  1. Daniel B. Lookadoo
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  2. Matthew S. Beyersdorf
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  3. Christopher J. Halkides
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Corresponding author

Correspondence to Christopher J. Halkides .

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Editors and Affiliations

  1. Department of Biology, Texas A&M University, College Station, Texas, USA

    Michael D. Manson

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Lookadoo, D.B., Beyersdorf, M.S., Halkides, C.J. (2018). Synthesis of a Stable Analog of the Phosphorylated Form of CheY: Phosphono-CheY. In: Manson, M. (eds) Bacterial Chemosensing. Methods in Molecular Biology, vol 1729. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7577-8_26

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  • DOI: https://doi.org/10.1007/978-1-4939-7577-8_26

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7576-1

  • Online ISBN: 978-1-4939-7577-8

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