title: Disassembling and bleaching of chloride-free pharaonis halorhodopsin by octyl-beta-glucoside.
abstract: Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is a transmembrane, seven-helix retinal protein of the archaeal bacterium and acts as an inward light-driven chloride ion pump in the membrane. The denaturation process of NpHR solubilized with n-octyl-beta-d-glucopyranoside (OG) was investigated to clarify the effects of the chloride ion and pH on the stability and bleaching of the NpHR chromophore. Initially, active NpHR solubilized with n-dodecyl-beta-d-maltopyranoside (DM) was obtained from the recombinant halo-opsin (NpHO), which was expressed in Escherichia coli cells, by adding all-trans retinal to the medium. Apparent molecular weight of the active NpHR solubilized with DM, which was determined by gel-filtration chromatography and dynamic light scattering, indicated the oligomeric state. The bleaching of NpHR in the dark by the addition of 50 mM OG in the presence and absence of chloride was investigated. In the presence of 256 mM NaCl, the bleaching of NpHR was strongly inhibited. On the other hand, in the absence of NaCl, an immediate decrease of absorbance at 600 nm was observed. Stopped-flow rapid-mixing analysis clarified the bleaching process in the absence of chloride as DM-NpHR (oligomeric) <--> OG-NpHR (disassembled) <--> intermediate --> NpHO and free retinal, and each rate constant were determined. The formation of an intermediate (450 nm) in the dark was found to be strongly dependent on pH, as well as anion and detergent concentrations. The disassembling and protonation of a Schiff base corresponding to the bleaching intermediate is also discussed.
authors.affiliation: Division of Biological Sciences, Graduate School of Science, Hokkaido University, 060-0810, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Kubo
authors.forename: Maki
authors.initials: M
authors.lastname: Sato
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Aizawa
authors.forename: Chojiro
authors.initials: C
authors.lastname: Kojima
authors.forename: Naoki
authors.initials: N
authors.lastname: Kamo
authors.forename: Mineyuki
authors.initials: M
authors.lastname: Mizuguchi
authors.forename: Keiichi
authors.initials: K
authors.lastname: Kawano
authors.forename: Makoto
authors.initials: M
authors.lastname: Demura
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry; D002713:Chlorine / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D005960:Glucosides / Q000494:pharmacology*; D025602:Halorhodopsins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019633:Natronobacterium / Q000737:chemistry*; D038761:Photobleaching; D011489:Protein Denaturation / Q000187:drug effects; D012545:Schiff Bases; D012995:Solubility; D013057:Spectrum Analysis; D014801:Vitamin A / Q000494:pharmacology
chemical_list: D019843:Archaeal Proteins; D003902:Detergents; D005960:Glucosides; D025602:Halorhodopsins; D012545:Schiff Bases; D014801:Vitamin A; C018619:octyl-beta-D-glucoside; D002713:Chlorine