Measurement of response regulator autodephosphorylation rates spanning six orders of magnitude.

Two-component regulatory systems, comprising sensor kinase and response regulator proteins, carry out signal transduction in prokaryotic and eukaryotic microorganisms, as well as plants. Response regulators act as phosphorylation-mediated switches, turning on and off cellular responses to environmental stimuli. Self-catalyzed dephosphorylation is an important determinant of the duration of the response regulator activated state. Reported response regulator autodephosphorylation rates vary over almost a million-fold range, consistent with control of biological processes that occur on widely different timescales. We describe general considerations for the design and execution of in vitro assays to measure the autodephosphorylation rates of purified response regulator proteins, as well as specific methods that utilize loss of 32P, changes in fluorescence, or release of inorganic phosphate. The advantages and disadvantages of different methods are discussed, including suitability for different timescales. In addition to outlining established methods, an assay modification is proposed to measure fast autodephosphorylation rates with radioactivity, and optimization of the fluorescence/pH jump method is described.