Structure and function in rhodopsin: further elucidation of the role of the intradiscal cysteines, Cys-110, -185, and -187, in rhodopsin folding and function.
The disulfide bond between Cys-110 and Cys-187 in the intradiscal domain is required for correct folding in vivo and function of mammalian rhodopsin. Misfolding in rhodopsin, characterized by the loss of ability to bind 11-cis-retinal, has been shown to be caused by an intradiscal disulfide bond different from the above native disulfide bond. Further, naturally occurring single mutations of the intradiscal cysteines (C110F, C110Y, and C187Y) are associated with retinitis pigmentosa (RP). To elucidate further the role of every one of the three intradiscal cysteines, mutants containing single-cysteine replacements by alanine residues and the above three RP mutants have been studied. We find that C110A, C110F, and C110Y all form a disulfide bond between C185 and C187 and cause loss of retinal binding. C185A allows the formation of a C110-C187 disulfide bond, with wild-type-like rhodopsin phenotype. C187A forms a disulfide bond between C110 and C185 and binds retinal, and the pigment formed has markedly altered bleaching behavior. However, the opsin from the RP mutant C187Y forms no rhodopsin chromophore.
Departments of Biology and Chemistry, Massachusetts Institute of Technology 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
J
J
Hwa
P J
PJ
Reeves
J
J
Klein-Seetharaman
F
F
Davidson
H G
HG
Khorana
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D003545:Cysteine; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000235:genetics; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017299:Rod Opsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry
D011994:Recombinant Proteins; D017299:Rod Opsins; D012243:Rhodopsin; D003545:Cysteine