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

Abstract

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.

Flavonoid allosteric modulation of mutated visual rhodopsin associated with retinitis pigmentosa

“…The G90V mutant with 1 µM Q treatment (G90V 11CR-Q) showed a similar Amax as G90V 9CR. A slightly increased A 280 /A max ratio was previously reported for this mutant 15 , 16 that essentially agrees with the results obtained here for G90V 11CR. This increase could be due, at least partially, to the presence of a small fraction of misfolded (non-retinal binding) protein or to the lack of structural stability of the regenerated mutant protein.…”

“…For the WT 9CR, the presence of Q results in a slight decrease of hydroxylamine accessibility to the retinal binding site and hydrolysis of the SB linkage. In contrast, G90V 11CR showed a non-linear kinetics and a dramatic decrease due to the less compact structure in the SB linkage environment, as previously observed 15 , 16 (t 1/2 = 10.2 ± 0.3). Here, Q slightly increased the chemical stability of G90V 11CR-Q (t 1/2 = 13.3 ± 0.6).…”

New insights into the molecular mechanism of rhodopsin retinitis pigmentosa from the biochemical and functional characterization of G90V, Y102H and I307N mutations

“…The Y102H and I307N mutants showed a spectroscopic behavior similar to the WT in agreement with previous results [13], with a maximum absorption band in the visible region at 498 nm and 500 nm, respectively. In the case of the G90V mutant, a blue shift of 10 nm that had been previously reported, was observed [10,11]. A summary of the spectral parameters, including the absorbance value of the visible chromophoric band, ε and the spectral A 280 /A λmax ratio are shown in Table 1.…”

“…For Y102H and G90V, a clear band is observed below the Rho monomer band. The presence of a band around 27 kDa may correspond to a truncated protein form as previously described [ 10 , 16 , 27 ]. In the case of the I307N mutant, two minor bands can be detected below the 40 kDa main opsin band that could also correspond to a truncated protein.…”

“…The Rho mutant G90V was first identified in a Swiss family of three generations, which showed a typical phenotype of an autosomal dominant form of RP, with marked fundus changes developing in later stages of life [ 9 ]. Subsequent to this discovery, studies regarding the G90V mutant have used heterologous expression systems and immunopurification strategies to deepen the knowledge on the structural details underlying the molecular mechanisms of the disease [ 10 , 11 ]. Furthermore, the deleterious effect of the G90V mutation could be partially reversed in vitro by means of 9-cis-retinal alone [ 11 ] or in combination with other ligands like the flavonoid quercetin [ 12 ].…”