title: Crystal structure of a thermally stable rhodopsin mutant.
abstract: We determined the structure of the rhodopsin mutant N2C/D282C expressed in mammalian cells; the first structure of a recombinantly produced G protein-coupled receptor (GPCR). The mutant was designed to form a disulfide bond between the N terminus and loop E3, which allows handling of opsin in detergent solution and increases thermal stability of rhodopsin by 10 deg.C. It allowed us to crystallize a fully deglycosylated rhodopsin (N2C/N15D/D282C). N15 mutations are normally misfolding and cause retinitis pigmentosa in humans. Microcrystallographic techniques and a 5 microm X-ray beam were used to collect data along a single needle measuring 5 microm x 5 microm x 90 microm. The disulfide introduces only minor changes but fixes the N-terminal cap over the beta-sheet lid covering the ligand-binding site, a likely explanation for the increased stability. This work allows structural investigation of rhodopsin mutants and shows the problems encountered during structure determination of GPCRs and other mammalian membrane proteins.
authors.affiliation: MRC Laboratory of Molecular Biology, Structural Studies, Hills Road, Cambridge CB2 2QH, UK.
authors.forename: Jörg
authors.initials: J
authors.lastname: Standfuss
authors.forename: Guifu
authors.initials: G
authors.lastname: Xie
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Edwards
authors.forename: Manfred
authors.initials: M
authors.lastname: Burghammer
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Oprian
authors.forename: Gebhard F X
authors.initials: GF
authors.lastname: Schertler
mesh_terms: D000818:Animals; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics; D013696:Temperature