STRUCTURE AND FUNCTION IN RHODOPSIN - SEPARATION AND CHARACTERIZATIONOF THE CORRECTLY FOLDED AND MISFOLDED OPSINS PRODUCED ON EXPRESSION OF AN OPSIN MUTANT-GENE CONTAINING ONLY THE NATIVE INTRADISCAL CYSTEINECODONS

Previous mutagenesis studies have indicated the requirement of a terti ary structure in the intradiscal region with a disulfide bond between Cys-110 and Cys-187 for the correct assembly and/or function of rhodop sin. We have now studied a rhodopsin mutant in which only the natural intradiscal cysteines at positions 110, 185, and 187 are present while all the remaining seven cysteines in the wildtype bovine rhodopsin ha ve been replaced by serines. The proteins formed on expression of this mutant in COS-1 cells bind 11-cis-retinal only partially to form the rhodopsin chromophore. We show that this is due to the presence of bot h correctly folded chromophore-forming opsin and misfolded opsins. Met hods have been devised for the separation of the correctly folded and misfolded forms by selective elution from immunoaffinity adsorbants, U sing several criteria, which include SDS-PAGE as well as UV/visible an d CD spectroscopy, we find that the correctly folded mutant protein is indistinguishable in its spectral properties from the wild-type rhodo psin. Further, reaction of sulfhydryl groups in the correctly folded m utant pigment with N-ethylmaleimide indicates that alkylation of a sin gle sulfhydryl requires denaturation or illumination, while reaction w ith an additional two sulfhydryl groups occurs only after reduction. T he misfolded mutant opsins are characterized by reduced cl-helical con tent, sulfhydryl reactivity under native conditions in the dark, and a lso the presence of a disulfide bond. We conclude that the failure to bury Cys-185, or the formation of a disulfide between an alternative p air of cysteine residues, precludes establishment of the correct intra discal structure required for the binding of retinal.