title: Characteristics of halorhodopsin-bacterioruberin complex from Natronomonas pharaonis membrane in the solubilized system.
abstract: Halorhodopsin is a retinal protein with a seven-transmembrane helix and acts as an inward light-driven Cl(-) pump. In this study, structural state of the solubilized halorhodopsin (NpHR) from the biomembrane of mutant strain KM-1 of Natronomonas pharaonis in nonionic detergent was investigated. A gel filtration chromatography monitored absorbances at 280 and 504 nm corresponding to the protein and a lipid soluble pigment of bacterioruberin (BR), respectively, has clearly detected an oligomer formation of the NpHRs and a complex formation between the NpHR and BR in the solubilized system. A molar ratio of NpHR:BR in the solubilized complex was close to 1:1. Further SDS-PAGE analysis of the solubilized NpHR cross-linked by 1% glutaraldehyde has revealed that the NpHR forms homotrimer in detergent system. Although this trimeric structure was stable in the presence of NaCl, it was dissociated to the monomer by the heat treatment at 45 °C in the desalted condition. The same tendency has been reported in the case of trimeric NpHR expressed heterologously on the E. coli membrane, leading to a conclusion that the change of strength of the trimeric association dependent on the ion binding is a universal feature of the NpHR. Interestingly, the trimer dissociation on the NpHR was accompanied by the complete dissociation of the BR molecule from the protein, indicated that the cavity formed by the NpHR protomers in the trimeric conformation is important for tight binding of the BR. Because the binding affinity for Cl(-) and the resistance to hydroxylamine under light illumination showed only minor differences between the NpHR in the solubilized state and that on the biomembrane, the influences of solubilization to the tertiary structure and function of the protein are thought to be minor. This NpHR-BR complex in the solubilized system has a potential to be a good model system to investigate the intermolecular interaction between the membrane protein and lipid.
authors.affiliation: School of Science and Technology, Meiji University, Tama-ku, Kawasaki-shi, Kanagawa 214-8571, Japan. tsasaki@isc.meiji.ac.jp
authors.forename: Takanori
authors.initials: T
authors.lastname: Sasaki
authors.forename: Nur Wahida Abdul
authors.initials: NW
authors.lastname: Razak
authors.forename: Noritaka
authors.initials: N
authors.lastname: Kato
authors.forename: Yuri
authors.initials: Y
authors.lastname: Mukai
mesh_terms: D002338:Carotenoids / Q000737:chemistry*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D019605:Euryarchaeota / Q000737:chemistry*; D025602:Halorhodopsins / Q000737:chemistry*; D012995:Solubility
chemical_list: D025602:Halorhodopsins; C002951:bacterioruberin; D002338:Carotenoids