SUMMARY Rhodopsin, the photoreceptor pigment of the retina, initiates vision upon photon capture by its covalently linked chromophore 11-cis-retinal. In the absence of light, the chromophore serves as an inverse agonist locking the receptor in the inactive dark state. In the absence of chromophore, the apoprotein opsin shows low-level constitutive activity. Toward revealing insight into receptor properties controlled by the chromophore, we applied dynamic single-molecule force spectroscopy to quantify the kinetic, energetic, and mechanical differences between dark-state rhodopsin and opsin in native membranes from the retina of mice. Both rhodopsin and opsin are stabilized by ten structural segments. Compared to dark-state rhodopsin, the structural segments stabilizing opsin showed higher interaction strengths and mechanical rigidities and lower conformational variabilities, lifetimes, and free energies. These changes outline a common mechanism toward activating G-protein-coupled receptors. Additionally, we detected that opsin was more pliable and frequently stabilized alternate structural intermediates.

Background: A G90D point mutation in rhodopsin causes congenital stationary night blindness. Results: The G90D mutation alters the chromophore-binding pocket, mechanical rigidity, and energetic stability of dark state rhodopsin. Conclusion: Significant perturbations are promoted by the G90D mutation. Significance: Characterizing the effect of point mutations in rhodopsin allows for a better understanding of how these mutations lead to dysfunction in retinal diseases.

Rhodopsin is the light receptor that initiates phototransduction in rod photoreceptor cells. The structure and function of rhodopsin is tightly linked to molecular interactions that stabilize and determine the receptor's functional state. Single-molecule force spectroscopy (SMFS) was used to localize and quantify molecular interactions that structurally stabilize bovine and mouse rhodopsin from native disc membranes of rod photoreceptor cells. The mechanical unfolding of bovine and mouse rhodopsin revealed nine major unfolding intermediates, each intermediate defining a structurally stable segment in the receptor. These stable structural segments had similar localization and occurrence in both bovine and mouse samples. For each structural segment, parameters describing their unfolding energy barrier were determined by dynamic SMFS. No major differences were observed between bovine and mouse rhodopsin thereby implying that the structures of both rhodopsins are largely stabilized by similar molecular interactions.Rhodopsin is a G protein-coupled receptor (GPCR)1 residing in rod outer segments (ROS) of photoreceptor cells, where it initiates phototransduction upon light-activation. Several crystal structures are now available for a handful of GPCRs (1-5). These structures highlight the conservation of the general architecture of GPCRs, which display seven transmembrane α-helices. Some of the mechanisms underlying receptor activation and function are likely conserved across members of this family of membrane proteins (4,6,7). Despite low amino acid sequence similarities, comparison of GPCR crystal structures reveals only relatively small deviations in the position of transmembrane α-helices (2,5,8). Yet those small differences are significant enough to facilitate the specific roles and functions of those †