title: A class-A GPCR solubilized under high hydrostatic pressure retains its ligand binding ability.
abstract: The effect of high hydrostatic pressure (HHP) on the solubilization of a class-A G protein-coupled receptor, the silkmoth pheromone biosynthesis-activating neuropeptide receptor (PBANR), was investigated. PBANR was expressed in expresSF+ insect cells as a C-terminal fusion protein with EGFP. The membrane fraction was subjected to HHP treatment (200MPa) at room temperature for 1-16h in the presence of 0-2.0% (w/v) n-dodecyl-β-D-maltopyranoside (DDM). The solubilization yield of PBANR-EGFP in the presence of 0.6% (w/v) DDM increased to ~1.5-fold after 1h HHP treatment. Fluorescence-detection size-exclusion chromatography demonstrated that the PBANR-EGFP ligand binding ability was retained after HHP-mediated solubilization. The PBANR-EGFP solubilized with 1.0% DDM under HHP at room temperature for 6h retained ligand binding ability, whereas solubilization in the absence of HHP treatment resulted in denaturation.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Yukie
authors.initials: Y
authors.lastname: Katayama
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Ebisawa
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ohtsuka
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Shipeng
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Green and Sustainable Chemistry, School of Engineering, Tokyo Denki University, 5 Senju Asahi-cho, Adachi-ku, Tokyo 120-8551, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Natsume
authors.affiliation: Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan.
authors.forename: Yu-Hua
authors.initials: YH
authors.lastname: Lo
authors.affiliation: Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Senda
authors.affiliation: Molecular Entomology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Nagamine
authors.affiliation: USDA-ARS Arid Land Agricultural Research Center, Maricopa, AZ, USA.
authors.forename: J Joe
authors.initials: JJ
authors.lastname: Hull
authors.affiliation: Molecular Entomology Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan.
authors.forename: Shogo
authors.initials: S
authors.lastname: Matsumoto
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, PR China.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Nagasawa
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: aknagata@mail.ecc.u-tokyo.ac.jp.
authors.forename: Koji
authors.initials: K
authors.lastname: Nagata
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: amtanok@mail.ecc.u-tokyo.ac.jp.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
mesh_terms: D000818:Animals; D012831:Bombyx / Q000737:chemistry* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006874:Hydrostatic Pressure; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics; D055550:Protein Stability; D043563:Receptors, Pheromone / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D019476:Insect Proteins; D043563:Receptors, Pheromone; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: G protein-coupled receptor; High hydrostatic pressure; Ligand binding ability; Pheromone biosynthesis-activating neuropeptide receptor; Solubilization