Structure and function in rhodopsin. Separation and characterization of the correctly folded and misfolded opsins produced on expression of an opsin mutant gene containing only the native intradiscal cysteine codons.
Previous mutagenesis studies have indicated the requirement of a tertiary structure in the intradiscal region with a disulfide bond between Cys-110 and Cys-187 for the correct assembly and/or function of rhodopsin. 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positions 110, 185, and 187 are present while all the remaining seven cysteines in the wild-type bovine rhodopsin have been replaced by serines.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 both correctly folded chromophore-forming opsin and misfolded opsins. Methods have been devised for the separation of the correctly folded and misfolded forms by selective elution from immunoaffinity adsorbants. Using several criteria, which include SDS-PAGE as well as UV/visible and CD spectroscopy, we find that the correctly folded mutant protein is indistinguishable in its spectral properties from the wild-type rhodopsin. Further, reaction of sulfhydryl groups in the correctly folded mutant pigment with N-ethylmaleimide indicates that alkylation of a single sulfhydryl requires denaturation or illumination, while reaction with an additional two sulfhydryl groups occurs only after reduction. The misfolded mutant opsins are characterized by reduced alpha-helical content, sulfhydryl reactivity under native conditions in the dark, and also the presence of a disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS)
Ridge
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KD
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Lu
Z
Z
Liu
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X
Khorana
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D002942:Circular Dichroism; D003062:Codon / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D003062:Codon; D012243:Rhodopsin; D003545:Cysteine