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A class-A GPCR solubilized under high hydrostatic pressure retains its ligand binding ability

Author:
Katayama, Yukie, Suzuki, Tatsuya, Ebisawa, Tatsuki, Ohtsuka, Jun, Wang, Shipen, Natsume, Ryo, Lo, Yu-Hua, Senda, Toshiya, Nagamine, Toshihiro, Hull, J. Joe, Matsumoto, Shogo, Nagasawa, Hiromichi, Nagata, Koji, Tanokura, Masaru
Source:
Biochimica et Biophysica Acta (BBA) - Biomembranes 2016 v.1858 no.9 pp. 2145-2151
ISSN:
0005-2736
Subject:
G-protein coupled receptors, ambient temperature, binding capacity, biosynthesis, cell membranes, denaturation, gel chromatography, high pressure treatment, insects, ligands, neuropeptide receptors, pheromones, solubilization
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 sub- jected to HHP treatment (200 MPa) at room temperature for 1–16 h in the presence of 0–2.0% (w/v) n-dodecyl- ß-D-maltopyranoside (DDM). The solubilization yield of PBANR-EGFP in the presence of 0.6% (w/v) DDM in- creased to ~1.5-fold after 1 h HHP treatment. Fluorescence-detection size-exclusion chromatography demon- strated 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 6 h retained ligand binding ability, whereas solubilization in the absence of HHP treatment resulted in denaturation.
Agid:
62961
Handle:
10113/62961