Large-scale production of a disulfide-stabilized constitutively active mutant opsin.
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.
Min
K Christopher
KC
Department of Neurology, Columbia University, New York, NY 10032, USA. km369@columbia.edu
Jin
Yan
Y
Hendrickson
Wayne A
WA
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
D003902:Detergents; D004220:Disulfides; D055355:Opsins; D011994:Recombinant Proteins; D015293:Transducin; D003545:Cysteine; D012172:Retinaldehyde