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

Yukie Katayama, Tatsuya Suzuki, Tatsuki Ebisawa, Jun Ohtsuka, Shipen Wang, Ryo Natsume, Yu-Hua Lo, Toshiya Senda, Toshihiro Nagamine, J. Joe Hull, Shogo Matsumoto, Hiromichi Nagasawa, Koji Nagata, Masaru Tanokura
Biochimica et Biophysica Acta (BBA) - Biomembranes 2016 v.1858 no.9 pp. 2145-2151
G-protein coupled receptors, ambient temperature, binding capacity, biosynthesis, cell membranes, denaturation, gel chromatography, high pressure treatment, insects, ligands, neuropeptide receptors, pheromones, solubilization
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.