title: Oxygen-sensing histidine-protein kinases: assays of ligand binding and turnover of response-regulator substrates.
abstract: Heme-based sensors are a recently discovered functional class of heme proteins that serve to detect physiological fluctuations in oxygen (O(2)), carbon monoxide (CO), or nitric oxide (NO). Many of these modular sensors detect heme ligands by coupling a histidine-protein kinase to a heme-binding domain. They typically bind O2, CO, and NO but respond only to one of these ligands. Usually, they are active in the ferrous unliganded state but are switched off by saturation with O2. The heme-binding domains of these kinases are quite varied. They may feature a PAS fold, as in the Bradyrhizobium japonicum and Sinorhizobium melitoti FixL proteins, or a GAF fold, as in the Mycobacterium tuberculosis DevS and DosT proteins. Alternative folds, such as HNOB (also H-NOX), have also been noted for such signal-transducing kinases, although these classes are less well studied. Histidine-protein kinases function in partnership with cognate response-regulator substrate(s): usually transcription factors that they activate by phosphorylation. For example, FixL proteins specifically phosphorylate their FixJ partners, and DevS and DosT proteins phosphorylate DevR in response to hypoxia. We present methods for purifying these sensors and their protein substrates, verifying the quality of the preparations, determining the K(d) values for binding of ligand and preparing sensors of known saturation, and measuring the rates of turnover (k(cat)) of the protein substrate by sensors of known heme status.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
authors.forename: Marie-Alda
authors.initials: MA
authors.lastname: Gilles-Gonzalez
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Gonzalez
authors.forename: Eduardo Henrique Silva
authors.initials: EH
authors.lastname: Sousa
authors.forename: Jason
authors.initials: J
authors.lastname: Tuckerman
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D006420:Hemeproteins / Q000032:analysis / Q000502:physiology; D000071677:Histidine Kinase; D008024:Ligands; D010100:Oxygen / Q000032:analysis* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D011786:Quality Control; D017186:Titrimetry / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D006420:Hemeproteins; D008024:Ligands; D011494:Protein Kinases; D000071677:Histidine Kinase; D010744:Phosphoric Monoester Hydrolases; D010100:Oxygen