Background: Mutations at Gly-90 in rhodopsin cause two different phenotypes: retinitis pigmentosa and congenital night blindness. Results: G90V retinitis pigmentosa mutant shows constitutive activity and very low thermal stability in the dark state. Conclusion: Low conformational stability can trigger retinitis pigmentosa associated with rhodopsin mutations. Significance: Retinoids can help to stabilize the conformation of retinitis pigmentosa mutants.

Surface plasmon resonance spectroscopy allows the study of protein interaction dynamics in real-time. Application of this technique to G-protein coupled receptors, the largest family of receptors involved in signal transduction, has been complicated by their low level of expression and the critical dependence of their native conformation on the hydrophobic transmembrane lipid environment. Here, we investigate and compare three different strategies to immobilize rhodopsin, a prototypical G-protein coupled receptor on a sensor chip surface using antibodies and a lectin for receptor capturing. By further probing of different experimental conditions (pH, detergent type) we identified the optimal factors to maintain rhodopsin in a functional conformation and extended this approach to recombinant rhodopsin that was heterologously expressed in COS cells. Functional operation of rhodopsin on the sensor chip surface was proven by its activation and subsequent light-stimulated G-protein coupling. The influence of these experimental parameters on the association and dissociation kinetics of G-protein receptor coupling was determined. Thereby, we found that the kinetics of G(t) interaction were not changed by the strategy of immobilization or the type of detergent. Regeneration of opsin directly on a chip allowed recycling of the immobilized native and recombinant receptor. Thus, the approach provides an experimental framework for choosing the most suitable conditions for the solubilization, immobilization, and for functional tests of rhodopsin on a biosensor surface.

The visual photoreceptor rhodopsin undergoes a series of conformational changes upon light activation, eventually leading to the active metarhodopsin II conformation, which is able to bind and activate the G‐protein, transducin. We have previously shown that mutant rhodopsins G51V and G89D, associated with retinitis pigmentosa, present photobleaching patterns characterized by the formation of altered photointermediates whose nature remained obscure. Our current detailed UV–visible spectroscopic analysis, together with functional characterization, indicate that these mutations influence the relative stability of the different metarhodopsin photointermediates by altering their equilibria and maintaining the receptor in a nonfunctional light‐induced conformation that may be toxic to photoreceptor cells. We propose that G51V and G89D shift the equilibrium from metarhodopsin I towards an intermediate, recently named as metarhodopsin Ib, proposed to interact with transducin without activating it. This may be one of the causes contributing to the molecular mechanisms underlying cell death associated with some retinitis pigmentosa mutations.

It was previously shown that opsin can be regenerated with the newly synthesized 11-cis-7-methyl-retinal forming an artificial visual pigment. We now extend this study to include mutants at positions close to the retinal to further dissect the interactions of native and artificial chromophores with opsin. Several mutants at M207, W265 and Y268 have been obtained and regenerated with 11-cis-retinal and the 7-methyl analog. M207 is the site of the point mutation M207R associated with the retinal degenerative disease retinitis pigmentosa. All the studied mutants regenerated with 11-cis-retinal except for M207C which proved to be completely misfolded. The naturally occurring M207R mutant formed a pigment with an unprotonated Schiff base linkage, altered photobleaching and low MetarhodopsinII stability. Mutants regenerated with the 7-methyl analog showed altered photobleaching reflecting a structural perturbation in the vicinity of M207. The newly obtained mutants at M207 also showed reduced levels of transducin activation with M207R showing essentially no transducin activation. Our results highlight the tight coupling of the vicinity of C7 of retinal and M207 and support the involvement of this amino acid residue in the conformational changes associated with rhodopsin photoactivation.

For the first time to our knowledge, X-ray absorption spectroscopy (XAS) has been used to investigate the environment of putative Zn(2+) binding sites in rhodopsin. We studied native purified nondeionized rhodopsin without any further addition of Zn(2+), as well as with 1.5 mol of Zn(2+)-as zinc chloride-per mole of protein. Three different binding sites in rhodopsin were considered based on computational chemistry studies, and a quantitative analysis of the XAS signal was performed by fitting the experimental data to their simulated XAS spectra. Our results demonstrate that Zn(2+) is intrinsically bound to rhodopsin and are compatible with the existence of an octahedral coordination involving six oxygen atoms in the first shell (average Zn-O distance of 2.08 A), and with a second coordination shell containing one or two phosphorus or sulfur atoms at an average distance of 2.81 A.