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

  • Daniel B. Lookadoo
  • Matthew S. Beyersdorf
  • Christopher J. Halkides
Protocol
Part of the Methods in Molecular Biology book series (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.

Keywords

Phosphonomethyl trifluoromethanesulfonate Two-component system Cysteine modification Phosphate analog Chemical modification Response regulator Chemotaxis Phosphono-CheY 

Notes

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.

References

  1. 1.
    Bren A, Eisenbach M (1998) The N terminus of the flagellar switch protein, FliM, is the binding domain for the chemotactic response regulator, CheY. J Mol Biol 278:507–514CrossRefGoogle Scholar
  2. 2.
    Sarkar MK, Paul K, Blair D (2010) Chemotaxis signaling protein CheY binds to the rotor protein FliN to control the direction of flagellar rotation in Escherichia coli. Proc Natl Acad Sci U S A 107:9370–9375CrossRefGoogle Scholar
  3. 3.
    Zhou J, Lloyd SA, Blair DF (1998) Electrostatic interactions between rotor and stator in the bacterial flagellar motor. Proc Natl Acad Sci U S A 95:6436–6441CrossRefGoogle Scholar
  4. 4.
    Vartanian AS, Paz A, Fortgang EA, Abramson J, Dahlquist FW (2012) Structure of flagellar motor proteins in complex allows for insights into motor structure and switching. J Biol Chem 287:35779–35783CrossRefGoogle Scholar
  5. 5.
    Lowry DF, Roth AF, Rupert PB, Dahlquist FW, Moy FJ et al (1994) Signal transduction in chemotaxis. J Biol Chem 269:26358–26362PubMedGoogle Scholar
  6. 6.
    Shannon DA, Weerapana E (2015) Covalent protein modification: the current landscape of residue-specific electrophiles. Curr Opin Chem Biol 24:18–26CrossRefGoogle Scholar
  7. 7.
    Boutureira O, Bernardes GJ (2015) Advances in chemical protein modification. Chem Rev 115:2174–2195CrossRefGoogle Scholar
  8. 8.
    Halkides CJ, Zhu X, Phillion DP, Matsumura P, Dahlquist FW (1998) Synthesis and biochemical characterization of an analogue of CheY-phosphate, a signal transduction protein in bacterial chemotaxis. Biochemistry 37:13674–13680CrossRefGoogle Scholar
  9. 9.
    Silversmith R, Bourret RB (1998) Synthesis and characterization of a stable analog of the phosphorylated form of the chemotaxis protein CheY. Protein Eng 11:205–212CrossRefGoogle Scholar
  10. 10.
    Sircar R, Borbat PP, Lynch MJ, Bhatnagar J, Beyersdorf MS et al (2015) Assembly states of FliM and FliG within the flagellar switch complex. J Mol Biol 427:867–886CrossRefGoogle Scholar
  11. 11.
    Halkides CJ, McEvoy MM, Casper E, Matsumura P, Volz K (2000) The 1.9 A resolution crystal structure of phosphono-CheY, an analogue of the active form of the response regulator, CheY. Biochemistry 39:5280–5286CrossRefGoogle Scholar
  12. 12.
    McAdams K, Casper ES, Haas RM, Santarsiero BD, Eggler AL et al (2008) The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides. Arch Biochem Biophys 479:105–113CrossRefGoogle Scholar
  13. 13.
    Halkides CJ, Bottone CJ, Casper ES, Haas RM, McAdams K (2007) Synthesis of a stable analog of the phosphorylated form of CheY: phosphono-CheY. Methods Enzymol 422:338–351CrossRefGoogle Scholar
  14. 14.
    Penefsky HS (1979) A centrifuged-column procedure for the measurement of ligand binding by beef heart F1. Methods Enzymol 56:527–530CrossRefGoogle Scholar
  15. 15.
    Riddles PW, Blakeley RL, Zerner B (1983) Reassessment of Ellman’s reagent. Methods Enzymol 91:49–60CrossRefGoogle Scholar
  16. 16.
    Bachman J (2013) Site-directed mutagenesis. Methods Enzymol 529:241–248CrossRefGoogle Scholar
  17. 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

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Daniel B. Lookadoo
    • 1
  • Matthew S. Beyersdorf
    • 1
  • Christopher J. Halkides
    • 1
  1. 1.Department of Chemistry and BiochemistryThe University of North Carolina WilmingtonWilmingtonUSA

Personalised recommendations