Single-cysteine substitution mutants at amino acid positions 306-321 in rhodopsin, the sequence between the cytoplasmic end of helix VII and the palmitoylation sites: sulfhydryl reactivity and transducin activation reveal a tertiary structure.
As sensors for structure at the cytoplasmic face of rhodopsin, single-cysteine substitution mutants have been previously studied in the regions connecting helices III and IV and helices V and VI. In this paper we report on single-cysteine substitution mutants at amino acid positions 306-321, comprising the cytoplasmic sequence between helix VII and the palmitoylation sites in rhodopsin. The cysteine opsin mutants were expressed in COS-1 cells and on treatment with 11-cis-retinal all formed the characteristic rhodopsin chromophore. Cysteines at positions 306-316 and 319 reacted in the dark with the thiol-specific reagent 4, 4'-dithiodipyridine (4-PDS) but showed a wide variation in reactivity. Cysteines at positions 317, 318, 320, and 321 showed no reaction with 4-PDS. The mutants on illumination also showed wide variations in activating GT. The mutant Y306C showed almost no GT activation, I307C and N310C were poor, and the activity of the mutants M309C, F313C, and M317C was also reduced relative to WT. The results suggest that the region comprising amino acids 306-321 is a part of a tertiary structure and that specific amino acids in this region on light-activation participate in the interaction with GT.
Cai
K
K
Department of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge 02139, USA.
Klein-Seetharaman
J
J
Farrens
D
D
Zhang
C
C
Altenbach
C
C
Hubbell
W L
WL
Khorana
H G
HG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002417:Cattle; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D003593:Cytoplasm / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D007930:Leucine / Q000235:genetics; D008027:Light; D008969:Molecular Sequence Data; D019308:Palmitic Acid / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000737:chemistry*; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics
D004220:Disulfides; D010446:Peptide Fragments; D011725:Pyridines; D013439:Sulfhydryl Reagents; C007542:4,4'-dipyridyl disulfide; D019308:Palmitic Acid; D014443:Tyrosine; D012243:Rhodopsin; D015293:Transducin; D007930:Leucine; D003545:Cysteine