title: Ser-130 of Natronobacterium pharaonis halorhodopsin is important for the chloride binding.
abstract: Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump from Natronobacterium pharaonis. In order to clarify the role of Ser-130(phR) residue which corresponds to Ser-115(shR) for salinarum hR on the anion-binding affinity, the wild-type and Ser-130 mutants substituted with Thr, Cys and Ala were expressed in E. coli cells and solubilized with 0.1% n-dodecyl beta-D-maltopyranoside The absorption maximum (lambda(max)) of the S130T mutant indicated a blue shift from that of the wild type in the absence and presence of chloride. For S130A, a large red shift (12 nm) in the absence of chloride was observed. The wild-type and all mutants showed the blue-shift of lambda(max) upon Cl(-) addition, from which the dissociation constants of Cl(-) were determined. The dissociation constants were 5, 89, 153 and 159 mM for the wild-type, S130A, S130T and S130C, respectively, at pH 7.0 and 25 degrees C. Circular dichroic spectra of the wild-type and the Ser-130 mutants exhibited an oligomerization. The present study revealed that the Ser-130 of N. pharaonis halorhodopsin is important for the chloride binding.
authors.affiliation: Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: Maki
authors.initials: M
authors.lastname: Sato
authors.forename: Takashi
authors.initials: T
authors.lastname: Kikukawa
authors.forename: Tsunehisa
authors.initials: T
authors.lastname: Araiso
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Okita
authors.forename: Kazumi
authors.initials: K
authors.lastname: Shimono
authors.forename: Naoki
authors.initials: N
authors.lastname: Kamo
authors.forename: Makoto
authors.initials: M
authors.lastname: Demura
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Nitta
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002712:Chlorides / Q000737:chemistry; D025602:Halorhodopsins / Q000737:chemistry / Q000378:metabolism*; D008027:Light; D008969:Molecular Sequence Data; D019633:Natronobacterium / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D013053:Spectrophotometry
chemical_list: D002712:Chlorides; D025602:Halorhodopsins; D012694:Serine