Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Proc Natl Acad Sci U S A. 1996 May 14; 93(10): 4560–4564.
doi: 10.1073/pnas.93.10.4560
PMCID: PMC39316
PMID: 8643443

Structure and function in rhodopsin: correct folding and misfolding in point mutants at and in proximity to the site of the retinitis pigmentosa mutation Leu-125-->Arg in the transmembrane helix C.

P Garriga, X Liu, and H G Khorana
Author information Copyright and License information PMC Disclaimer

Abstract

L125R is a mutation in the transmembrane helix C of rhodopsin that is associated with autosomal dominant retinitis pigmentosa. To probe the orientation of the helix and its packing in the transmembrane domain, we have prepared and studied the mutations E122R, I123R, A124R, S127R, L125F, and L125A at, and in proximity to, the above mutation site. Like L125R, the opsin expressed in COS-1 cells from E122R did not bind 11-cis-retinal, whereas those from I123R and S127R formed the rhodopsin chromophore partially. A124R opsin formed the rhodopsin chromophore (lambda max 495 nm) in the dark, but the metarhodopsin II formed on illumination decayed about 6.5 times faster than that of the wild type and was defective in transducin activation. The mutant opsins from L125F and L125A bound 11-cis-retinal only partially, and in both cases, the mixtures of the proteins produced were separated into retinal-binding and non-retinal-binding (misfolded) fractions. The purified mutant rhodopsin from L125F showed lambda max at 500 nm, whereas that from L125A showed lambda max at 503 nm. The mutant rhodopsin L125F showed abnormal bleaching behavior and both mutants on illumination showed destabilized metarhodopsin II species and reduced transducin activation. Because previous results have indicated that misfolding in rhodopsin is due to the formation of a disulfide bond other than the normal disulfide bond between Cys-110 and Cys-187 in the intradiscal domain, we conclude from the misfolding in mutants L125F and L125A that the folding in vivo in the transmembrane domain is coupled to that in the intradiscal domain.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

 
icon of scanned page 4560
4560
icon of scanned page 4561
4561
icon of scanned page 4562
4562
icon of scanned page 4563
4563
icon of scanned page 4564
4564
 

Images in this article

Fig. 2

Fig. 2
on p.4562

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  • Berson EL. Retinitis pigmentosa. The Friedenwald Lecture. Invest Ophthalmol Vis Sci. 1993 Apr;34(5):1659–1676. [PubMed] [Google Scholar]
  • Dryja TP, Berson EL. Retinitis pigmentosa and allied diseases. Implications of genetic heterogeneity. Invest Ophthalmol Vis Sci. 1995 Jun;36(7):1197–1200. [PubMed] [Google Scholar]
  • Sung CH, Davenport CM, Hennessey JC, Maumenee IH, Jacobson SG, Heckenlively JR, Nowakowski R, Fishman G, Gouras P, Nathans J. Rhodopsin mutations in autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6481–6485. [PMC free article] [PubMed] [Google Scholar]
  • Macke JP, Davenport CM, Jacobson SG, Hennessey JC, Gonzalez-Fernandez F, Conway BP, Heckenlively J, Palmer R, Maumenee IH, Sieving P, et al. Identification of novel rhodopsin mutations responsible for retinitis pigmentosa: implications for the structure and function of rhodopsin. Am J Hum Genet. 1993 Jul;53(1):80–89. [PMC free article] [PubMed] [Google Scholar]
  • Inglehearn CF, Keen TJ, Bashir R, Jay M, Fitzke F, Bird AC, Crombie A, Bhattacharya S. A completed screen for mutations of the rhodopsin gene in a panel of patients with autosomal dominant retinitis pigmentosa. Hum Mol Genet. 1992 Apr;1(1):41–45. [PubMed] [Google Scholar]
  • Doi T, Molday RS, Khorana HG. Role of the intradiscal domain in rhodopsin assembly and function. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4991–4995. [PMC free article] [PubMed] [Google Scholar]
  • Anukanth A, Khorana HG. Structure and function in rhodopsin. Requirements of a specific structure for the intradiscal domain. J Biol Chem. 1994 Aug 5;269(31):19738–19744. [PubMed] [Google Scholar]
  • Ridge KD, Lu Z, Liu X, Khorana HG. 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. Biochemistry. 1995 Mar 14;34(10):3261–3267. [PubMed] [Google Scholar]
  • Liu X, Garriga P, Khorana HG. Structure and function in rhodopsin: correct folding and misfolding in two point mutants in the intradiscal domain of rhodopsin identified in retinitis pigmentosa. Proc Natl Acad Sci U S A. 1996 May 14;93(10):4554–4559. [PMC free article] [PubMed] [Google Scholar]
  • Karnik SS, Sakmar TP, Chen HB, Khorana HG. Cysteine residues 110 and 187 are essential for the formation of correct structure in bovine rhodopsin. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8459–8463. [PMC free article] [PubMed] [Google Scholar]
  • Sakmar TP, Franke RR, Khorana HG. Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8309–8313. [PMC free article] [PubMed] [Google Scholar]
  • Zhukovsky EA, Oprian DD. Effect of carboxylic acid side chains on the absorption maximum of visual pigments. Science. 1989 Nov 17;246(4932):928–930. [PubMed] [Google Scholar]
  • Nathans J. Determinants of visual pigment absorbance: identification of the retinylidene Schiff's base counterion in bovine rhodopsin. Biochemistry. 1990 Oct 16;29(41):9746–9752. [PubMed] [Google Scholar]
  • Nakayama TA, Khorana HG. Mapping of the amino acids in membrane-embedded helices that interact with the retinal chromophore in bovine rhodopsin. J Biol Chem. 1991 Mar 5;266(7):4269–4275. [PubMed] [Google Scholar]
  • Jäger F, Fahmy K, Sakmar TP, Siebert F. Identification of glutamic acid 113 as the Schiff base proton acceptor in the metarhodopsin II photointermediate of rhodopsin. Biochemistry. 1994 Sep 13;33(36):10878–10882. [PubMed] [Google Scholar]
  • Cohen GB, Oprian DD, Robinson PR. Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296. Biochemistry. 1992 Dec 22;31(50):12592–12601. [PubMed] [Google Scholar]
  • Fahmy K, Siebert F, Sakmar TP. A mutant rhodopsin photoproduct with a protonated Schiff base displays an active-state conformation: a Fourier-transform infrared spectroscopy study. Biochemistry. 1994 Nov 22;33(46):13700–13705. [PubMed] [Google Scholar]
  • Arnis S, Fahmy K, Hofmann KP, Sakmar TP. A conserved carboxylic acid group mediates light-dependent proton uptake and signaling by rhodopsin. J Biol Chem. 1994 Sep 30;269(39):23879–23881. [PubMed] [Google Scholar]
  • Dryja TP, Hahn LB, Cowley GS, McGee TL, Berson EL. Mutation spectrum of the rhodopsin gene among patients with autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9370–9374. [PMC free article] [PubMed] [Google Scholar]
  • Sung CH, Davenport CM, Nathans J. Rhodopsin mutations responsible for autosomal dominant retinitis pigmentosa. Clustering of functional classes along the polypeptide chain. J Biol Chem. 1993 Dec 15;268(35):26645–26649. [PubMed] [Google Scholar]
  • Kaushal S, Khorana HG. Structure and function in rhodopsin. 7. Point mutations associated with autosomal dominant retinitis pigmentosa. Biochemistry. 1994 May 24;33(20):6121–6128. [PubMed] [Google Scholar]
  • Franke RR, Sakmar TP, Oprian DD, Khorana HG. A single amino acid substitution in rhodopsin (lysine 248----leucine) prevents activation of transducin. J Biol Chem. 1988 Feb 15;263(5):2119–2122. [PubMed] [Google Scholar]
  • Ferretti L, Karnik SS, Khorana HG, Nassal M, Oprian DD. Total synthesis of a gene for bovine rhodopsin. Proc Natl Acad Sci U S A. 1986 Feb;83(3):599–603. [PMC free article] [PubMed] [Google Scholar]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed] [Google Scholar]
  • Oprian DD, Molday RS, Kaufman RJ, Khorana HG. Expression of a synthetic bovine rhodopsin gene in monkey kidney cells. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8874–8878. [PMC free article] [PubMed] [Google Scholar]
  • WALD G, BROWN PK. The molar extinction of rhodopsin. J Gen Physiol. 1953 Nov 20;37(2):189–200. [PMC free article] [PubMed] [Google Scholar]
  • Farrens DL, Khorana HG. Structure and function in rhodopsin. Measurement of the rate of metarhodopsin II decay by fluorescence spectroscopy. J Biol Chem. 1995 Mar 10;270(10):5073–5076. [PubMed] [Google Scholar]
  • Fahmy K, Sakmar TP. Regulation of the rhodopsin-transducin interaction by a highly conserved carboxylic acid group. Biochemistry. 1993 Jul 20;32(28):7229–7236. [PubMed] [Google Scholar]
  • Wessling-Resnick M, Johnson GL. Allosteric behavior in transducin activation mediated by rhodopsin. Initial rate analysis of guanine nucleotide exchange. J Biol Chem. 1987 Mar 15;262(8):3697–3705. [PubMed] [Google Scholar]
  • Kaushal S, Ridge KD, Khorana HG. Structure and function in rhodopsin: the role of asparagine-linked glycosylation. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):4024–4028. [PMC free article] [PubMed] [Google Scholar]
  • Sung CH, Schneider BG, Agarwal N, Papermaster DS, Nathans J. Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8840–8844. [PMC free article] [PubMed] [Google Scholar]
  • Sakmar TP, Franke RR, Khorana HG. The role of the retinylidene Schiff base counterion in rhodopsin in determining wavelength absorbance and Schiff base pKa. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3079–3083. [PMC free article] [PubMed] [Google Scholar]
  • Davidson FF, Loewen PC, Khorana HG. Structure and function in rhodopsin: replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):4029–4033. [PMC free article] [PubMed] [Google Scholar]
  • Marti T, Rösselet SJ, Otto H, Heyn MP, Khorana HG. The retinylidene Schiff base counterion in bacteriorhodopsin. J Biol Chem. 1991 Oct 5;266(28):18674–18683. [PubMed] [Google Scholar]
Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
-