Structure and function in rhodopsin. Requirements of a specific structure for the intradiscal domain.
We concluded previously from mutagenesis in the intradiscal domain of bovine rhodopsin that the formation of a tertiary structure comprising the N-terminal tail and the three polypeptide loops is essential to the in vivo assembly of the functional rhodopsin. We now report on more comprehensive mutagenic studies in the intradiscal domain to determine more precisely the requirement for the formation of the above-proposed tertiary structure. Three large deletions, two consisting of groups of 10 amino acids each, and the third of 34 amino acids, were carried out in the N-terminal loop. All the three mutant opsins only poorly formed the rhodopsin chromophore. In the BC loop, we carried out five 2 amino acid deletions, 2 single amino acid deletions, and three mutations in which short sequences in the loop were reversed. All the resulting mutant opsins had lost the ability to bind 11-cis-retinal. In the DE loop, where extensive mutagenesis had previously been carried out, we carried out 3 amino acid replacements (Asn, Thr, Tyr) at Cys187. None of these mutants bound 11-cis-retinal. In loop FG, we carried out four 2 amino acid deletions, 1 single amino acid deletion, 3 amino acid replacements, and one mutation in which the sequence of the 7 amino acids was reversed. All the mutants in FG loop partially formed the rhodopsin chromophore. All the mutants now described appeared to be retained in the endoplasmic reticulum: several that were examined in detail were complexed with non-opsin proteins, the chaperonins. Treatment with ATP-MgCl2 released the latter from the mutant rhodopsins. Our overall conclusion is that the formation of the specific structure in the intradiscal domain has highly stringent spatial requirements.
Anukanth
A
A
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Khorana
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012172:Retinaldehyde / Q000494:pharmacology; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D017299:Rod Opsins / Q000235:genetics; D017384:Sequence Deletion; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013696:Temperature; D014508:Urea
D017299:Rod Opsins; D014508:Urea; D012243:Rhodopsin; D012172:Retinaldehyde