Rod Visual Pigment Optimizes Active State to Achieve Efficient G Protein Activation as Compared with Cone Visual Pigments

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...

Section: Significancesupporting

confidence: 49%

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

Gulati

Jastrzębska

Banerjee

et al. 2017

“…Given Ci-opsin1's unique counterion system, we expected Ci-opsin1 to show somewhat different molecular properties from those of vertebrate visual and invertebrate-type opsins. We first investigated G protein activation efficiency of Ci-opsin1 using a fluorescence assay (21,22). Because Ci-opsin1 is colocalized with Gi-type of G protein in the ocellus of ascidian larvae (23), we irradiated the mixture of purified Ci-opsin1, Gi, and GTPγS with yellow light and confirmed an increase in fluorescence due to Gi activation ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Gi activation was measured by a fluorescence assay (21,22,43). Fluorescence changes were monitored using a laboratoryconstructed photon counting system with some modifications (43).…”

Section: Methodsmentioning

confidence: 99%

Evolutionary steps involving counterion displacement in a tunicate opsin

Kojima

Yamashita

Imamoto

et al. 2017

Self Cite

Ci-opsin1 is a visible light-sensitive opsin present in the larval ocellus of an ascidian, Ciona intestinalis. This invertebrate opsin belongs to the vertebrate visual and nonvisual opsin groups in the opsin phylogenetic tree. Ci-opsin1 contains candidate counterions (glutamic acid residues) at positions 113 and 181; the former is a newly acquired position in the vertebrate visual opsin lineage, whereas the latter is an ancestral position widely conserved among invertebrate opsins. Here, we show that Glu113 and Glu181 in Ci-opsin1 act synergistically as counterions, which imparts molecular properties to Ci-opsin1 intermediate between those of vertebrate-and invertebrate-type opsins. Synergy between the counterions in Ci-opsin1 was demonstrated by E113Q and E181Q mutants that exhibit a pH-dependent spectral shift, whereas only the E113Q mutation in vertebrate rhodopsin yields this spectral shift. On absorbing light, Ci-opsin1 forms an equilibrium between two intermediates with protonated and deprotonated Schiff bases, namely the MIlike and MII-like intermediates, respectively. Adding G protein caused the equilibrium to shift toward the MI-like intermediate, indicating that Ci-opsin1 has a protonated Schiff base in its active state, like invertebrate-type opsins. Ci-opsin1's G protein activation efficiency is between the efficiencies of vertebrate-and invertebrate-type opsins. Interestingly, the E113Y and E181S mutations change the molecular properties of Ci-opsin1 into those resembling invertebrate-type or bistable opsins and vertebrate ancient/vertebrate ancient-long or monostable opsins, respectively. These results strongly suggest that acquisition of counterion Glu113 changed the molecular properties of visual opsin in a vertebrate/tunicate common ancestor as a crucial step in the evolution of vertebrate visual opsins.opsin | G protein-coupled receptors | ascidian | molecular evolution | counterion

“…Experimental data were fitted by a single exponential function, and the v dark was estimated by the difference between the initial rates between two samples ('pigment name-n' and 'pigment name-7mr'). The v light was measured by fluorescence assay as previously described (41,42). The assay mixture consisted of 20 nM pigments, 0 or 1 μM Gt, 5 μM GTPγS, 0.015% DDM, 50 mM Hepes (pH 6.5), 140 mM NaCl, 5.8 mM MgCl 2 , and 1 mM DTT.…”

Section: Methodsmentioning

confidence: 99%

“…The assay mixture consisted of 20 nM pigments, 0 or 1 μM Gt, 5 μM GTPγS, 0.015% DDM, 50 mM Hepes (pH 6.5), 140 mM NaCl, 5.8 mM MgCl 2 , and 1 mM DTT. Experimental data were fitted by a single exponential function, and the v light was estimated as previously described (41,42). The k d was measured by a fluorescence assay as previously described (42,43).…”

Section: Methodsmentioning

confidence: 99%

Adaptation of cone pigments found in green rods for scotopic vision through a single amino acid mutation

Kojima

Matsutani

Yamashita

et al. 2017

Self Cite

Most vertebrate retinas contain a single type of rod for scotopic vision and multiple types of cones for photopic and color vision. The retinas of certain amphibian species uniquely contain two types of rods: red rods, which express rhodopsin, and green rods, which express a blue-sensitive cone pigment (M1/SWS2 group). Spontaneous activation of rhodopsin induced by thermal isomerization of the retinal chromophore has been suggested to contribute to the rod's background noise, which limits the visual threshold for scotopic vision. Therefore, rhodopsin must exhibit low thermal isomerization rate compared with cone visual pigments to adapt to scotopic condition. In this study, we determined whether amphibian blue-sensitive cone pigments in green rods exhibit low thermal isomerization rates to act as rhodopsin-like pigments for scotopic vision. Anura blue-sensitive cone pigments exhibit low thermal isomerization rates similar to rhodopsin, whereas Urodela pigments exhibit high rates like other vertebrate cone pigments present in cones. Furthermore, by mutational analysis, we identified a key amino acid residue, Thr47, that is responsible for the low thermal isomerization rates of Anura blue-sensitive cone pigments. These results strongly suggest that, through this mutation, anurans acquired special blue-sensitive cone pigments in their green rods, which could form the molecular basis for scotopic color vision with normal red rods containing green-sensitive rhodopsin.photoreceptor cell | visual pigment | amphibian | molecular evolution | color discrimination