title: Purification and biophysical characterization of the CapA membrane protein FTT0807 from Francisella tularensis.
abstract: The capA gene (FTT0807) from Francisella tularensis subsp. tularensis SCHU S4 encodes a 44.4 kDa integral membrane protein composed of 403 amino acid residues that is part of an apparent operon that encodes at least two other membrane proteins, CapB, and CapC, which together play a critical role in the virulence and pathogenesis of this bacterium. The capA gene was overexpressed in Escherichia coli as a C-terminal His6-tagged fusion with a folding reporter green fluorescent protein (frGFP). Purification procedures using several detergents were developed for the fluorescing and membrane-bound product, yielding approximately 30 mg of pure protein per liter of bacterial culture. Dynamic light scattering indicated that CapA-frGFP was highly monodisperse, with a size that was dependent upon both the concentration and choice of detergent. Circular dichroism showed that CapA-frGFP was stable over the range of 3-9 for the pH, with approximately half of the protein having well-defined α-helical and β-sheet secondary structure. The addition of either sodium chloride or calcium chloride at concentrations producing ionic strengths above 0.1 M resulted in a small increase of the α-helical content and a corresponding decrease in the random-coil content. Secondary-structure predictions on the basis of the analysis of the sequence indicate that the CapA membrane protein has two transmembrane helices with a substantial hydrophilic domain. The hydrophilic domain is predicted to contain a long disordered region of 50-60 residues, suggesting that the increase of α-helical content at high ionic strength could arise because of electrostatic interactions involving the disordered region. CapA is shown to be an inner-membrane protein and is predicted to play a key cellular role in the assembly of polysaccharides.
authors.affiliation: Department of Chemistry and Biochemistry, Arizona State University , Tempe, Arizona 85287, United States.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Martin-Garcia
authors.forename: Debra T
authors.initials: DT
authors.lastname: Hansen
authors.forename: James
authors.initials: J
authors.lastname: Zook
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Loskutov
authors.forename: Mark D
authors.initials: MD
authors.lastname: Robida
authors.forename: Felicia M
authors.initials: FM
authors.lastname: Craciunescu
authors.forename: Kathryn F
authors.initials: KF
authors.lastname: Sykes
authors.forename: Rebekka M
authors.initials: RM
authors.lastname: Wachter
authors.forename: Petra
authors.initials: P
authors.lastname: Fromme
authors.forename: James P
authors.initials: JP
authors.lastname: Allen
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology*; D055592:Biophysical Phenomena / Q000502:physiology; D005604:Francisella tularensis / Q000737:chemistry* / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology*; D008969:Molecular Sequence Data; D011237:Predictive Value of Tests
chemical_list: D001426:Bacterial Proteins; C108302:CapA protein, bacteria; D006360:Heat-Shock Proteins