title: Purification and characterization of the periplasmic domain of EnvZ osmosensor in Escherichia coli.
abstract: The EnvZ-OmpR histidyl-aspartyl phosphorelay system in E. coli responds to osmolarity by differentially modulating the expression of the major outer membrane porins OmpF and OmpC. To date, the natural ligand that activates EnvZ, a transmembrane histidine kinase, has not been identified and the role of the periplasmic domain of EnvZ is unclear. We now report on the purification and characterization of the periplasmic domain of EnvZ (Lys48-Arg162) which has been expressed as a soluble protein in fusion with the maltose-binding protein. Overexpression of the fusion protein did not compete for a signal that activates EnvZ. By amylose affinity chromatography and affinity blotting, interacting proteins could not be detected. The periplasmic domain was released by factor Xa and purified to homogeneity. From circular dichroism analysis, the periplasmic domain was estimated to consist of 35% alpha-helices and 16% beta-sheets.
authors.affiliation: Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: Egger
authors.forename: M
authors.initials: M
authors.lastname: Inouye
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003593:Cytoplasm / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D009994:Osmolar Concentration; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C057843:envZ protein, E coli