title: Large-scale production of a disulfide-stabilized constitutively active mutant opsin.
abstract: Previous studies of constitutively activated mutants of opsin in the absence of chromophore were carried out in crude cell membranes because such mutants could not be recovered in a detergent-solubilized form in the active state. We employed a strategy in which a stabilizing disulfide bond allowed for successful purification of a constitutively activated mutant opsin, N2C/E113Q/M257Y/D282C, solubilized in nonionic detergent from mammalian cell culture. The purified mutant opsin is able to activate transducin to a higher degree than opsin and may prove useful for future structural studies of the active state of GPCRs.
authors.affiliation: Department of Neurology, Columbia University, New York, NY 10032, USA. km369@columbia.edu
authors.forename: K Christopher
authors.initials: KC
authors.lastname: Min
authors.forename: Yan
authors.initials: Y
authors.lastname: Jin
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Hendrickson
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D003902:Detergents / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D016297:Mutagenesis, Site-Directed; D055355:Opsins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012172:Retinaldehyde / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D015293:Transducin / Q000378:metabolism
chemical_list: D003902:Detergents; D004220:Disulfides; D055355:Opsins; D011994:Recombinant Proteins; D015293:Transducin; D003545:Cysteine; D012172:Retinaldehyde