title: Opsin stability and folding: the role of Cys185 and abnormal disulfide bond formation in the intradiscal domain.
abstract: The structure in the extracellular, intradiscal domain of rhodopsin surrounding the Cys110-Cys187 disulfide bond has been shown to be important for correct folding of this receptor in vivo. Retinitis pigmentosa misfolding mutants of the apoprotein opsin (such as P23H) misfold, as defined by a deficiency in ability to bind 11-cis retinal and form rhodopsin. These mutants also possess an abnormal Cys185-Cys187 disulfide bond in the intradiscal domain. Here, by mutating Cys185 to alanine, we eliminate the possibility of forming this abnormal disulfide bond and investigate the effect of combining the C185A mutation with the retinitis pigmentosa mutation P23H. Both the P23H and P23H/C185A double mutant suffer from low expression and poor 11-cis retinal binding. Our data suggest that misfolding events occur that do not have an absolute requirement for abnormal Cys185-Cys187 disulfide bond formation. In the detergent-solubilised, purified state, the C185A mutation allows formation of rhodopsin at wild-type (WT) levels, but has interesting effects on protein stability. C185A rhodopsin is less thermally stable than WT, whereas C185A opsin shows the same ability to regenerate rhodopsin in detergent as WT. Purified C185A and WT opsins, however, have contrasting 11-cis retinal binding kinetics. A high proportion of C185A opsin binds 11-cis retinal with a slow rate that reflects a denatured state of opsin reverting to a fast-binding, open-pocket conformation. This slower rate is not observed in a stabilising lipid/detergent system, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Chaps, in which C185A exhibits WT (fast) retinal binding. We propose that the C185A mutation destabilises the open-pocket conformation of opsin in detergent resulting in an equilibrium between correctly folded and denatured states of the protein. This equilibrium can be driven towards the correctly folded rhodopsin state by the binding of 11-cis retinal.
authors.affiliation: Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK. craig.mckibbin@manchester.ac.uk
authors.forename: Craig
authors.initials: C
authors.lastname: McKibbin
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Toye
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Reeves
authors.forename: H Gobind
authors.initials: HG
authors.lastname: Khorana
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Edwards
authors.forename: Claudio
authors.initials: C
authors.lastname: Villa
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Booth
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003902:Detergents / Q000494:pharmacology; D016296:Mutagenesis; D017510:Protein Folding; D017299:Rod Opsins / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D003902:Detergents; D017299:Rod Opsins