The effects of amino acid replacements of glycine 121 on transmembrane helix 3 of rhodopsin.
Rhodopsin is a member of a family of G protein-coupled receptors with seven transmembrane (TM) helices. In rhodopsin, Gly121 is a highly conserved amino acid residue near the middle of TM helix 3. TM helix 3 is known to be involved in chromophore-protein interactions and contains the chromophore Schiff base counterion at position 113. We prepared a set of seven single amino acid replacement mutants of rhodopsin at position 121 (G121A, Ser, Thr, Val, Ile, Leu, and Trp) and control mutants with replacements of Gly114 or Ala117. The mutant opsins were expressed in COS cells and reconstituted with either 11-cis-retinal, the ground-state chromophore of rhodopsin, or all-trans-retinal, the isomer formed upon receptor photoactivation. The replacement of Gly121 resulted in a relative reversal in the selectivity of the opsin apoprotein for reconstitution with 11-cis-retinal over all-trans-retinal in COS cell membranes. The mutant pigments also were found to be thermally unstable to varying degrees and reactive to hydroxylamine in the dark. In addition, the size of the residue substituted at position 121 correlated directly to the degree of blue-shift in the lambdamax value of the pigment. These results suggest that Gly121 is an important and specific component of the 11-cis-retinal binding pocket in rhodopsin.
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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Lin
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Smith
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Sakmar
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D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D005998:Glycine; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D017299:Rod Opsins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D015293:Transducin / Q000378:metabolism; D014212:Tretinoin / Q000378:metabolism
D006898:Hydroxylamines; D017299:Rod Opsins; D019811:Hydroxylamine; D014212:Tretinoin; D012243:Rhodopsin; D015293:Transducin; D005998:Glycine