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

Ridge, Kevin D.; Lu, Zhijian; Liu, Xun; Khorana, H G

doi:10.1021/bi00010a016

Cited by 75 publications

(81 citation statements)

References 34 publications

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“…We note that although the Arg-177 and Asp-190 residues point away from the helical bundle, it is also possible that mutations to this ion pair may affect the positioning or orientation of helices 5 and 6. However, there is some precedence for conformational changes in loop E-2, as Ridge et al (77) initially interpreted our results to mean that disrupting the Arg-177/Asp-190 ion pair causes a loosening of the loop E-2 structure, and thus we hypothesized the increased rate of Schiff base hydrolysis was due its increased exposure to external solvent. However, to our surprise, we found that the presence of 50 mM hydroxylamine had no effect on any of the mutants tested, even when tested at three different temperatures (Fig.…”

Section: Arrhenius Analysis Indicates the Thermal Decay In Rhodopsin mentioning

confidence: 82%

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

Janz

Fay

Farrens

2003

Journal of Biological Chemistry

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The rhodopsin crystal structure reveals that intradiscal loop E-2 covers the 11-cis-retinal, creating a "retinal plug." Recently, we noticed the ends of loop E-2 are linked by an ion pair between residues Arg-177 and Asp-190, near the highly conserved disulfide bond. This ion pair appears biologically significant; it is conserved in almost all vertebrate opsins and may occur in other G-protein-coupled receptors. We report here that the Arg-177/Asp-190 ion pair is critical for the folding and stability of dark state rhodopsin. We find ion pair mutants that regenerate with retinal are functionally and spectrally wild-type-like yet thermally unstable in their dark state because of rapid hydrolysis of the retinal Schiff base linkage. Surprisingly, Arrhenius analysis indicates that the activation energies for the hydrolysis process are similar between the ion pair mutants and wild-type rhodopsin. Furthermore, the ion pair mutants do not show increased reactivity toward hydroxylamine, suggesting that their instability is not caused by an increased exposure to bulk solvent. Our results indicate that the loop E-2 ion pair is important for rhodopsin stability and thus suggest that retinitis pigmentosa observed in patients with Asp-190 mutations may in part be the result of thermally unstable rhodopsin proteins.

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Section: Arrhenius Analysis Indicates the Thermal Decay In Rhodopsin mentioning

confidence: 82%

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

Janz

Fay

Farrens

2003

Journal of Biological Chemistry

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“…Forty-eight hours after tetracycline induction, HEK-293 cells expressing WT opsin were harvested and incubated with both 9-and 11-cis-retinals. The rhodopsin formed was purified by immunoaffinity chromatography under conditions that selectively release folded opsin (19), and the first eluted fraction was examined by UV-visible spectroscopy and HPLC. The pigment obtained in post-harvest regeneration of WT opsin with 9-cis-retinal has a shifted absorption spectrum with max ϭ 487 nm (Fig.…”

Section: Resultsmentioning

confidence: 99%

Retinoids Assist the Cellular Folding of the Autosomal Dominant Retinitis Pigmentosa Opsin Mutant P23H

Noorwez

Malhotra

McDowell

et al. 2004

Journal of Biological Chemistry

143

144

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The clinically common mutant opsin P23H, associated with autosomal dominant retinitis pigmentosa, yields low levels of rhodopsin when retinal is added following induction of the protein in stably transfected HEK-293 cells. We previously showed that P23H rhodopsin levels could be increased by providing a 7-membered ring, locked analog of 11-cis-retinal during expression of P23H opsin in vivo. Here we demonstrate that the mutant opsin is effectively rescued by 9-or 11-cis-retinal, the native chromophore. When retinal was added during expression, P23H rhodopsin levels were 5-fold (9-cis) and 6-fold (11-cis) higher than when retinal was added after opsin was expressed and cells were harvested. Levels of P23H opsin were increased ϳ3.5-fold with both compounds, but wild-type protein levels were only slightly increased. Addition of retinal during induction promoted the Golgi-specific glycosylation of P23H opsin and transport of the protein to the cell surface. P23H rhodopsins containing 9-or 11-cis-retinal had blueshifted absorption maxima and altered photo-bleaching properties compared with the corresponding wild-type proteins. Significantly, P23H rhodopsins were more thermally unstable than the wild-type proteins and more rapidly bleached by hydroxylamine in the dark. We suggest that P23H opsin is similarly unstable and that retinal binds and stabilizes the protein early in its biogenesis to promote its cellular folding and trafficking. The implications of this study for treating retinitis pigmentosa and other protein conformational disorders are discussed.

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“…For convenience, the numbering of the mutants has been changed from that in the original paper (8) as in Table 1. All the mutant genes were expressed in COS-1 cells and purified by absorption on antirhodopsin 1D4-Sepharose as described (11,13). In addition, the deletion and sequence replacement mutants in Table 1 were prepared by expression in stable mammalian cell lines (14).…”

Section: Methodsmentioning

confidence: 99%

Structure and function in rhodopsin: Peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase

Creuzenet

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Phosphorylation of light-activated rhodopsin by the retina-specific enzyme, rhodopsin kinase (RK), is the primary event in the initiation of desensitization in the visual system. RK binds to the cytoplasmic face of rhodopsin, and the binding results in activation of the enzyme which then phosphorylates rhodopsin at several serine and threonine residues near the carboxyl terminus. To map the RK binding sites, we prepared two sets of rhodopsin mutants in the cytoplasmic CD and EF loops. In the first set, peptide sequences in both loops were either deleted or replaced by indifferent sequences. In the second set of mutants, the charged amino acids (E134, R135, R147, E239, K245, E247, K248, and E249) were replaced by neutral amino acids in groups of 1-3 per mutant. The deletion and replacement mutants in the CD loop showed essentially no phosphorylation, and they appeared to be defective in binding of RK. Of the mutants in the EF loop, that with a deletion of 13 amino acids, was also defective in binding to RK while the second mutant containing a replacement sequence bound RK but showed a reduction of about 70% in V max for phosphorylation. The mutants containing charged to neutral amino acid replacements in the CD and EF loops were all phosphorylated but to different levels. The charge reversal mutant E134R͞ R135E showed a 50% reduction in V max relative to wild-type rhodopsin. Replacements of charged residues in the EF loop decreased the K m by 5-fold for E239Q and E247Q͞K248L͞ E239Q. In summary, both the CD and EF cytoplasmic loops are intimately involved in binding and interaction of RK with light-activated rhodopsin.Light activation of rhodopsin initiates two biochemical cascades, one leading to visual sensitization and the other to desensitization. The primary event in the first cascade is the binding of transducin to light-activated rhodopsin (metarhodopsin II; Meta II), and that in the second is the binding of rhodopsin kinase (RK) to the same photointermediate resulting in its phosphorylation (2-4). While the structural requirements for interaction between Meta II and transducin have been investigated extensively, the study of RK-rhodopsin interaction has begun only recently. Akhtar and coworkers (5) dissected the action of RK into two distinct steps. The first involved binding to Meta II, which resulted in activation of the enzyme. Catalysis of phosphorylation then followed as a second step. Palczweski et al. (6) compared the activation of RK by a number of rhodopsin derivatives and concluded that the cytoplasmic EF loop was involved in binding to RK. More recently, Weiss and coworkers (7) studied the effects of certain amino acid replacements in cytoplasmic loops AB, CD, and EF and suggested the involvement of all the three loops in phosphorylation by RK.We now report on further characterization of the requirements for the binding of RK to Meta II and catalysis of the phosphorylation reaction. Two sets of mutants in the cytoplasmic loops CD and EF ( Fig. 1) (8) have been studied. In one set, pepti...

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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

Cited by 75 publications

References 34 publications

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

Retinoids Assist the Cellular Folding of the Autosomal Dominant Retinitis Pigmentosa Opsin Mutant P23H

Structure and function in rhodopsin: Peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase

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