Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

Abstract

Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a oneway reaction that activates transducin (G t ) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C 11 =C 12 double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged G t activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates G t in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure-function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis-to-11,13-dicis isomerization.is the visual pigment found in rod outer segments of vertebrate and invertebrate photoreceptors that mediates the transformation of light into vision (1-5). By contrast, microbial Rhs mediate both the energy conversion and cell signaling required for cell survival (2, 6, 7). All classes of Rhs feature a heptahelical transmembrane structure that incorporates a covalently bound retinal chromophore, but they differ in their ability to interconvert between their two spectral absorption states driven by light exposure (2). Although vertebrate Rh undergoes a one-way photobleaching reaction of the retinal chromophore after light absorption (8), microbial Rhs exhibit an intrinsic photocyclic behavior with no chromophore release (2,8). This makes vertebrate Rhs unsuitable for optogenetic applications that require reversible control over effector ligands. Nevertheless, all Rhs require a cis-trans/trans-cis isomerization of their chromophores to trigger a protein conformational change that mediates the downstream signaling cascade or energy conversion (2,8). Previous studies on bovine Rh regenerated with sixcarbon-ring retinal chromophores (Rh6mr) featuring a locked C 11 =C 12 cis-trans isomerization (SI Appendix, Fig. S1) have implied their ability to activate the G protein transducin (G t ). These results suggest an alternative mechanism that can propagate the downstream visual cascades (9)(10)(11)(12)(13)(14). In this study, we provide direct evidence that Rh6mr can activate G t by an atypical cis-to-dicis isomerization that is also photocyclic, undergoing an 11,13-dicis-to-11-cis reisomerization. Even though it is believed that cis-trans isomerization is required to achieve the conformational change in opsin needed for G t activation, our results generalize this prerequisite to any isomerization that can achieve the same con...

A novel small molecule chaperone of rod opsin and its potential therapy for retinal degeneration

“…To confirm the inverse agonist and antagonist activity of YC-001 toward rod opsin, the initial rate of G t activation was measured by a fluorescence change due to GTPγS uptake leading to dissociation of the G t :opsin complex 39 , 40 . Bovine rod opsin has a basal activity for G t activation 41 .…”

“…G t was extracted and purified from frozen bovine ROS membranes as described in Preparation of Opsin Membranes. The intrinsic increase in the fluorescence from Gtα was measured with a L55 luminescence spectrophotometer (PerkinElmer Life Sciences) using an excitation and emission wavelengths of 300 and 345 nm, respectively 39 , 40 . To test the effect of YC-001 on the basal activity of rod opsin, opsin membranes were incubated for 15 min at 20 °C with a 40 µM concentration of either YC-001, the non-active analog-YC-014, 9- cis -retinal or a co-treatment with 9- cis -retinal and YC-001.…”

“…This step was followed by the addition of 300 μM GTPγS (Sigma-Aldrich) to determine the GTPγS-induced complex dissociation and corresponding fluorescence changes. G t activation rates were determined and plotted for the first 100 s of the G t activation assay 40 , 67 .…”

Targeting G protein-coupled receptor signaling at the G protein level with a selective nanobody inhibitor

“…ROS were washed with isotonic and hypotonic buffer to remove both soluble and membrane-associated ROS proteins 56 , 57 . Rh was purified by a protocol described previously 58 . Briefly, native Rh membranes were solubilized by a zinc/alkyl-glucoside extraction method and centrifuged at 100,000 g for 40 min to extract Rh.…”

Stereospecific modulation of dimeric rhodopsin

“…Bovine ROS were isolated and prepared as previously described in Papermaster (36). ROS washed with isotonic and hypotonic buffers were used for Rh purification (in the dark under a dim red light with .670 nm observance) as previously described (37)(38)(39). Briefly, for Rh extractions, native Rh membranes were solubilized with a zinc/alkyl-glucoside and centrifuged at 100,000 g for 40 min.…”