Optimized assay for the quantification of histidine kinase autophosphorylation

• Published in: Biochemical and biophysical research communications Vol. 465; no. 3; pp. 331 - 337
• Main Authors: Ueno, Takahiro B., Johnson, Roger A., Boon, Elizabeth M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25-09-2015
• Subjects: Autophosphorylation; Autoradiography; Autoradiography - methods; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding Sites; Biological Assay - methods; Enzyme Activation; Filter binding assay; His-Asp phosphorelay; Histidine Kinase; Phospho-His; Phosphorylation; Protein Binding; Protein Interaction Mapping - methods; Protein Kinases - chemistry; Protein Kinases - metabolism; TCS; Two-component signaling; Autophosphorylation; Autoradiography; His-Asp phosphorelay; Phospho-His; TCS; Two-component signaling; Filter binding assay; Histidine kinase
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2015.07.121

Although two-component signaling systems, comprising a sensory histidine kinase and a response regulator, are a primary means by which bacteria detect and respond to environmental stimuli, they are poorly characterized. Here we report optimized conditions for detecting histidine phosphorylation using a facile medium-throughput filter paper-binding assay. Employing this assay we report the kinetic parameters of previously uncharacterized histidine kinases from Vibrio haveyi, Vibrio parahaemolytius, Shewanella oneidensis, and Legionella pneumophila. In characterizing these kinases, we effectively double the number of kinetically characterized histidine kinases that have been reported in the literature.
•Two-component signaling (TCS) is essential to bacteria, but it is poorly characterized.•Environment-responsive histidine phosphorylation is the initial step in TCS.•We report a facile medium-throughput assay for quantifying histidine phosphorylation.•Using this assay, we analyze the kinetics of many uncharacterized histidine kinases.