title: Optimized assay for the quantification of histidine kinase autophosphorylation.
abstract: 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.
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, NY, 11794-3400, USA.
authors.forename: Takahiro B
authors.initials: TB
authors.lastname: Ueno
authors.affiliation: Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY, 11794-8661, USA.
authors.forename: Roger A
authors.initials: RA
authors.lastname: Johnson
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, NY, 11794-3400, USA. Electronic address: elizabeth.boon@stonybrook.edu.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Boon
mesh_terms: D001345:Autoradiography / Q000379:methods*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001681:Biological Assay / Q000379:methods*; D004789:Enzyme Activation; D000071677:Histidine Kinase; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011494:Protein Kinases; D000071677:Histidine Kinase
keywords: Autophosphorylation; Autoradiography; Filter binding assay; His-Asp phosphorelay; Histidine kinase; Phospho-His; TCS; Two-component signaling