Quantitative Analysis of Intracellular Response Regulator Phosphatase Activity of Histidine Kinases

Rong Gao, Ann Stock

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Scopus citations

Abstract

Quantitation of two-component protein activities is becoming increasingly important to understand the general design principles for this widely distributed prokaryotic signaling pathway. In many two-component systems (TCSs), phosphatase activity of the sensor histidine kinase (HK) plays a major role in controlling the system output and resetting the system to the prestimulus state. Quantitation of the phosphatase activity is often carried out in vitro, usually with truncated proteins that may not recapitulate the intact HK in the cellular environment. This chapter outlines a method for characterizing the intracellular phosphatase activity by investigating the TCS deactivation dynamics upon stimulus withdrawal. Two experimental approaches, the direct Phos-tag gel analysis and the indirect reporter assay, are described for measuring the TCS deactivation dynamics in cell. Combined with a mathematic model, the experimentally determined kinetics can lead to proper evaluation of the intracellular phosphatase activity.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsKaren N. Allen
PublisherAcademic Press Inc.
Pages301-319
Number of pages19
ISBN (Print)9780128138816
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Enzymology
Volume607
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Keywords

  • Fluorescence reporter assay
  • Mathematic modeling
  • Phos-tag
  • Signaling shut-off
  • Temporal dynamics
  • Two-component system

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