Transducin activation by a violet cone opsin

Purpose: To characterize the light-dependent activation of transducin by violet cone opsin (V-opsin) and study changes in structure upon absorption of light. Methods: A Xenopus V-opsin cDNA (accession number U23463), containing an epitope for a monoclonal antibody to bovine rhodopsin, was expressed in COS1 cells and purified by immunoaffinity chromatography. Activation of purified bovine rod transducin (G_T) was assayed using a [³⁵S]GTPγS filter binding assay and [³H]NEM labeling was carried out in the dark and light. Results: The UV/Vis spectrum of V-opsin had a maximum absorbance at 425 nm with extinction coefficient of 39,400 M^-1cm^-1 and half bandwidth of 5300 cm^-1, bleached to 380 nm. V-opsin was sensitive to hydroxylamine in the dark, reacting with a half-time of 3-5 min at 22°C. Light-exposed V-opsin stimulated guanyl nucleotide exchange on bovine rod G_T in a time dependent manner. The t_1/2 for activation was 0.75 min, similar to that of bovine rhodopsin. In exhaustive binding assays, both V-opsin and rhodopsin had similar concentration dependence, although V-opsin was not able to activate to the same extent. When assays were performed with λ>420 nm light, V-opsin was able to photocycle during the assay, exhibiting rapid regeneration and high affinity for 11-cis-retinal. The decay of the active species formed following photobleaching was rapid, being complete in <5 min, ∼10-fold faster than rhodopsin. NEM labeling revealed a 2-fold increase in accessible cysteines following exposure to light, significantly greater than observed with bovine rhodopsin. Conclusion: In vitro, V-opsin activates rod transducin with kinetics similar to rhodopsin but with faster decay of the activated pigment. These pigment properties can explain much of the response kinetics of cones.