Differential Light-induced Responses in Sectorial Inherited Retinal Degeneration

Ramon, Eva; Cordomí, Arnau; Aguilà, Mònica; Srinivasan, Sundaramoorthy; Dong, Xiaoyun; Moore, Anthony T.; Webster, Andrew R.; Cheetham, Michael E.; Garriga, Pere

doi:10.1074/jbc.m114.609958

Cited by 34 publications

(71 citation statements)

References 58 publications

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“…Because TM7 moves with activation, increasing the size of the cavity, only the active structures seem to allow binding of a second retinal in this region. This cavity is compatible with the proposed entry/exit channels for retinal in rhodopsin [ 33 ] and with the presence of hydrophobic molecules observed in the crystal structures of rhodopsin, as we previously noted [ 34 ].…”

Section: Figsupporting

confidence: 91%

Beyond spectral tuning: human cone visual pigments adopt different transient conformations for chromophore regeneration

Srinivasan

Cordomí

Ramon

et al. 2015

Cell. Mol. Life Sci.

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Human red and green visual pigments are seven transmembrane receptors of cone photoreceptor cells of the retina that mediate color vision. These pigments share a very high degree of homology and have been assumed to feature analogous structural and functional properties. We report on a different regeneration mechanism among red and green cone opsins with retinal analogs using UV-Vis/fluorescence spectroscopic analyses, molecular modeling and site-directed mutagenesis. We find that photoactivated green cone opsin adopts a transient conformation which regenerates via an unprotonated Schiff base linkage with its natural chromophore, whereas red cone opsin forms a typical protonated Schiff base. The chromophore regeneration kinetics is consistent with a secondary retinal uptake by the cone pigments. Overall, our findings reveal, for the

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Section: Figsupporting

confidence: 91%

Beyond spectral tuning: human cone visual pigments adopt different transient conformations for chromophore regeneration

Srinivasan

Cordomí

Ramon

et al. 2015

Cell. Mol. Life Sci.

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“…The visible bands of the WT and the mutants where blue-shifted, with regard to the 11-cis-retinal containing samples, due to the specific interaction of 9-cis-retinal with the amino acids in the binding pocket. N55K mutant showed higher A 280 /A λmax than WT in DM buffer, which could imply lower chromophore stability during the purification process, as previously described 30 . The two mutations studied, G90V and N55K, showed abnormal photobleaching behavior, in DM buffer, with incomplete conversion of the visible band upon illumination (Figure 2).…”

Section: Discussionsupporting

confidence: 64%

“…The other studied mutant, N55K associated to the peculiar sector RP phenotype 30 , shows also improved thermal stability but no improvement in its retinal regeneration ability. This would be due to the specificity imposed by the Lys introduced in the transmembrane domain of the photoreceptor protein 30 Table 2. The mean and error bars of three independent measurements are represented.…”

Section: Discussionmentioning

confidence: 92%

“…Because 9-cis-retinal, and not 11-cis-retinal, was used for the regeneration of the recombinant proteins, the WT, G90V and N55K mutants showed visible absorbance bands at 486 nm, 480 nm and 480 nm respectively. Upon illumination, G90V and N55K mutants showed incomplete conversion of the visible band to the 380 nm absorbing species with ~25% remaining absorbance at this wavelength indicating partial trapping of a photointermediate with a protonated SB linkage ( Figure 2) 21,30 . WT and G90V showed similar behavior upon illumination in bicelles buffer and in DM buffer.…”

Section: Uv-vis Spectral Characterization Of Purified Wt and Mutantsmentioning

confidence: 99%

“…Phospholipid bicelles have been shown to improve the stability of Rho [26][27][28] , and for this reason we have studied two Rho mutants, G90V and N55K, and compared their properties in DM detergent and in DMPC/DHPC bicelles ( Figure 1). G90V (causing RP phenotype) and N55K (associated with sector RP) show structural instability in the dark and thermal sensitivity 21,29,30 . 9-cis-retinal was used as an exogenous analog 31,32 for a detailed characterization of these two specific Rho mutants.…”

Section: Introductionmentioning

confidence: 99%

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Phospholipid Bicelles Improve the Conformational Stability of Rhodopsin Mutants Associated with Retinitis Pigmentosa

et al. 2015

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Mutations in the visual photoreceptor rhodopsin are the cause of the retinal degenerative disease retinitis pigmentosa. Some naturally occurring mutations can lead to protein conformational instability. Two such mutations, N55K and G90V, in the first and second transmembrane helices of the receptor, have been associated with sector and classical retinitis pigmentosa, respectively, and showed enhanced thermal sensitivity. We have carefully analyzed the effect of phospholipid bicelles on the stability and ligand binding properties of these two mutants and compared it with those of the detergent-solubilized samples. We have used a phospholipid bilayer consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2dihexanoyl-sn-glycero-3-phosphocholine (DHPC). We find that DMPC/DHPC bicelles dramatically increase the thermal stability of the rhodopsin mutants G90V and N55K. The chromophore stability and regeneration of the mutants were also increased in bicelles when compared to their behavior in a dodecyl maltoside detergent solution. The retinal release process was slowed in bicelles, and chromophore entry, after illumination, was improved for the G90V mutant but not for N55K. Furthermore, fluorescence spectroscopy measurements showed that bicelles allowed more exogenous retinal binding to the photoactivated G90V mutant than in a detergent solution. In contrast, N55K could not reposition any chromophore either in the detergent or in bicelles. The results demonstrate that DMPC/DHPC bicelles can counteract the destabilizing effect of the disease-causing mutations and can modulate the structural changes in the ensuing receptor photoactivation in a distinct specific manner for different retinitis pigmentosa mutant phenotypes.

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An experimental comparison of human and bovine rhodopsin provides insight into the molecular basis of retinal disease

et al. 2017

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Rhodopsin is the visual pigment that mediates dim-light vision in vertebrates and is a model system for the study of retinal disease. The majority of rhodopsin experiments are performed using bovine rhodopsin; however, recent evidence suggests that significant functional differences exist among mammalian rhodopsins. In this study, we identify differences in both thermal decay and light-activated retinal release rates between bovine and human rhodopsin and perform mutagenesis studies to highlight two clusters of substitutions that contribute to these differences. We also demonstrate that the retinitis pigmentosa-associated mutation G51A behaves differently in human rhodopsin compared to bovine rhodopsin and determine that the thermal decay rate of an ancestrally reconstructed mammalian rhodopsin displays an intermediate phenotype compared to the two extant pigments.

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Differential Light-induced Responses in Sectorial Inherited Retinal Degeneration

Cited by 34 publications

References 58 publications

Beyond spectral tuning: human cone visual pigments adopt different transient conformations for chromophore regeneration

Beyond spectral tuning: human cone visual pigments adopt different transient conformations for chromophore regeneration

Phospholipid Bicelles Improve the Conformational Stability of Rhodopsin Mutants Associated with Retinitis Pigmentosa

An experimental comparison of human and bovine rhodopsin provides insight into the molecular basis of retinal disease

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