Photochemical Properties of Mammalian Melanopsin

Melanopsin is the photoreceptor molecule of intrinsically photosensitive retinal ganglion cells, which serve as the input for various nonvisual behavior and physiological functions fundamental to organisms. The retina, therefore, possess a melanopsin-based nonvisual system in addition to the visual system based on the classical visual photoreceptor molecules. To elucidate the molecular properties of melanopsin, we have exogenously expressed mouse melanopsin in cultured cells. We were able to obtain large amounts of purified mouse melanopsin and conducted a comprehensive spectroscopic study of its photochemical properties. Melanopsin has an absorption maximum at 467 nm, and it converts to a meta intermediate having an absorption maximum at 476 nm. The melanopsin photoreaction is similar to that of squid rhodopsin, exhibiting bistability that results in a photosteady mixture of a resting state (melanopsin containing 11-<i>cis</i>-retinal) and an excited state (metamelanopsin containing all-<i>trans</i>-retinal) upon sustained irradiation. The absorption coefficient of melanopsin is 33000 ± 1000 M^–1 cm^–1, and its quantum yield of isomerization is 0.52; these values are also typical of invertebrate bistable pigments. Thus, the nonvisual system in the retina relies on a type of photoreceptor molecule different from that of the visual system. Additionally, we found a new state of melanopsin, containing 7-<i>cis</i>-retinal (extramelanopsin), which forms readily upon long-wavelength irradiation (yellow to red light) and photoconverts to metamelanopsin with short-wavelength (blue light) irradiation. Although it is unclear whether extramelanopsin would have any physiological role, it could potentially allow wavelength-dependent regulation of melanopsin functions

Efficiencies of Activation of Transducin by Cone and Rod Visual Pigments

How the light-induced transducin (Gt) activation process differs biochemically between cone visual pigments and rod visual pigment (rhodopsin) has remained unclear, because the Gt-activating state (Meta-II) of cone visual pigment decays too fast to precisely measure the activation efficiency by conventional biochemical methods such as the GTPγS binding assay. Here we measured the activation efficiencies of chicken green-sensitive cone visual pigment (cG) and bovine rhodopsin (bRh) in real time by monitoring the intrinsic fluorescence of tryptophan residues in the pigments and Gt. Michaelis–Menten analysis of Gt activation showed that the initial velocity for cG was approximately half that for bRh, while their Michaelis constants were comparable. Gt activation by cG was immediately slowed because of the fast hydrolysis of the retinal Schiff base in Meta-II, but this hydrolysis was suppressed by forming the complex with Gt. Using mutants of cG and bRh for positions 122 and 189, which exhibit altered rates of chromophore hydrolysis in Meta-II, we found that the initial velocity of Gt activation is negatively correlated with the rate of chromophore hydrolysis. These results suggest that the amino acid residues at positions 122 and 189 account for not only the resistance to the chromophore hydrolysis in Meta-II but also the conformation of Meta-II for efficient Gt activation. The substantially longer lifetime of the Gt activating state of Rh would be necessary to suppress the spontaneous quenching by the stochastic decay of the Gt-activating state when a rod responds to a single photon

Absorption spectra and G protein activation abilities of medaka TMT1A opsin.

<p>(A) and (B) Photochemical reactions of TMT1A opsin at 0°C. TMT1A opsin sample (curve 1 in A) was irradiated with blue light (460 nm) for 40 sec (curve 2 in A), followed by irradiation with orange light (>540 nm) for 5 min (curves 3 in A and B). The sample was subsequently subjected to two more rounds of irradiation with blue and orange lights. That is, the sample was irradiated with blue light for 40 sec (curve 4 in B), with orange light for 5 min (curve 5 in B), with blue light for 40sec (curve 6 in B), and with orange light for 5 min (curve 7 in B). (C) Difference spectra calculated from the spectra shown in A and B. Curve 2 is the difference spectrum calculated by subtracting curve 1 in A from curve 2 in A. Curve 3 is that calculated by subtracting curve 2 in A from curve 3 in A. Curve 4 is that calculated by subtracting curve 3 in B from curve 4 in B. Curve 5 is that calculated by subtracting curve 4 in B from curve 5 in B. Curve 6 is that calculated by subtracting curve 5 in B from curve 6 in B. Curve 7 is that calculated by subtracting curve 6 in B from curve 7 in B. (D) Difference spectra calculated from the spectra recorded during experiments on photochemical reactions of TMT1A opsin in membrane fractions. Irradiation procedures performed during experiments on TMT1A opsin in membrane fractions, and the procedures for calculating difference spectra were the same as those described in A and B, and in C, respectively. (E) Analysis of retinal configurations of TMT1A opsin. Retinal composition changed after blue light irradiation, subsequent orange light irradiation, blue light re-irradiation, and orange light re-irradiation. Curve numbers indicate the absorption spectra shown in A and B. (F) Time courses of G protein activations by purified TMT1A opsin. The extents of Go and Gi activations were measured in the dark (closed circles), after blue light irradiation (open circles) and after subsequent orange light irradiation (open triangles). Experiments were performed using 50 nM pigment and 500 nM G proteins at 0°C. Data are presented as the means ± S.E.M. of three independent experiments.</p

Photochemical Properties of Mammalian Melanopsin

Melanopsin is the photoreceptor molecule of intrinsically photosensitive retinal ganglion cells, which serve as the input for various nonvisual behavior and physiological functions fundamental to organisms. The retina, therefore, possess a melanopsin-based nonvisual system in addition to the visual system based on the classical visual photoreceptor molecules. To elucidate the molecular properties of melanopsin, we have exogenously expressed mouse melanopsin in cultured cells. We were able to obtain large amounts of purified mouse melanopsin and conducted a comprehensive spectroscopic study of its photochemical properties. Melanopsin has an absorption maximum at 467 nm, and it converts to a meta intermediate having an absorption maximum at 476 nm. The melanopsin photoreaction is similar to that of squid rhodopsin, exhibiting bistability that results in a photosteady mixture of a resting state (melanopsin containing 11-<i>cis</i>-retinal) and an excited state (metamelanopsin containing all-<i>trans</i>-retinal) upon sustained irradiation. The absorption coefficient of melanopsin is 33000 ± 1000 M^–1 cm^–1, and its quantum yield of isomerization is 0.52; these values are also typical of invertebrate bistable pigments. Thus, the nonvisual system in the retina relies on a type of photoreceptor molecule different from that of the visual system. Additionally, we found a new state of melanopsin, containing 7-<i>cis</i>-retinal (extramelanopsin), which forms readily upon long-wavelength irradiation (yellow to red light) and photoconverts to metamelanopsin with short-wavelength (blue light) irradiation. Although it is unclear whether extramelanopsin would have any physiological role, it could potentially allow wavelength-dependent regulation of melanopsin functions

Phylogenetic analysis of vertebrate Opn3/TMTopsin group.

<p>The phylogenetic tree was constructed by the neighbor-joining method using MEGA 6 software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141238#pone.0141238.ref030" target="_blank">30</a>]. Bootstrap probability (%) based on 1000 replicates is shown at the nodes. We tried to express TMT opsins from western clawed frog, zebrafish and medaka (blue letters) in cultured cells and successfully characterized the molecular properties of medaka TMT1A and TMT2 opsins (red letters). NCBI accession numbers used in this tree are as follows: zebrafish (BC163681, XM_002666663, NM_001281505, NM_001282373, NM_001282374, NM_001282375, EF043381), pufferfish (AF402774, XM_003968127, XM_003961790, XM_003970179, XM_003971042, XM_003963666), medaka (JX293354, JX293355, JX293356, JX293357, JX293358, NM_001305403), clawed frog (XM_002933372, LC009374, LC009375), green anole (XM_008106312, XM_008107065, XM_003228008, XM_008123887), flycatcher (XM_005050732, XM_005037520, XM_005043793), chicken (XM_004938518, XM_426139), opossum (JX293366, XM_001377887), Tasmanian devil (XM_003765692, XM_003767787), mouse (AF140241), human (AF140242) and bovine rhodopsin (NM_001014890).</p

Absorption spectra and G protein activation abilities of medaka TMT2 opsin.

<p>(A) Absorption spectra of purified TMT2 opsin after reconstitution with 11-<i>cis</i>-retinal. Spectra were recorded before irradiation (curve 1), after blue light (460 nm) irradiation (curve 2) for 1 min and after subsequent UV light (360 nm) irradiation (curve 3) for 2 min at 0°C. (Inset) Spectral changes caused by blue light irradiation (curve 1) and subsequent UV light irradiation (curve 2). (B) Photoreactions of TMT2-expressing membrane fractions after reconstitution with 11-<i>cis</i>-retinal. The sample was successively irradiated with blue light for 10, 20, 40, 80, and 160 sec, and the difference spectra (curves 1–5) were calculated by subtracting the spectrum recorded before irradiation from those recorded after respective irradiations. (C) Absorption spectra before and after acid denaturation of TMT2 photoproduct. Spectra were recorded before irradiation (curve 1), after blue light irradiation (curve 2) and after subsequent addition of 2N HCl (curve 3). (Inset) The difference spectrum calculated by subtracting the spectra recorded before acidification from that recorded after acidification. (D) Analysis of retinal configurations of TMT2 opsin. Retinal composition changed after blue light irradiation. (E) Time courses of G protein activation abilities of purified TMT2 opsin. Go and Gi activation efficiencies were measured in the dark (closed circles) and after blue light irradiation (open circles). Experiments were performed using 50 nM pigments and 500 nM G proteins at 0°C. Data are presented as the means ± S.E.M. of three independent experiments.</p

Shift in Conformational Equilibrium Induces Constitutive Activity of G‑Protein-Coupled Receptor, Rhodopsin

Constitutively active mutants (CAMs) of G-protein-coupled receptors (GPCRs) cause various kinds of diseases. Rhodopsin, a light-absorbing GPCR in animal retinas, has retinal as an endogenous ligand; only very low levels of activation of G-protein can be obtained with the ligand-free opsin. However, the CAM of opsin activates G-protein much more efficiently than the wild type, but the mechanism underlying this remains unclear. The present work revisits the constitutive activity of rhodopsin from the standpoint of conformational dynamics. Single-molecule observation of the M257Y mutant of bovine rhodopsin demonstrated that the switch between active and inactive conformations frequently occurred in M257Y opsin, and frequent generation of the active state results in the population shift toward the active state, which accounts for the constitutive activity of M257Y opsin. Our findings demonstrate that the protein function has a direct connection with the structural dynamics

Ultrafast Carbonyl Motion of the Photoactive Yellow Protein Chromophore Probed by Femtosecond Circular Dichroism

Motions of the <i>trans</i>-<i>p</i>-coumaric acid carbonyl group following the photoexcitation of the R52Q mutant of photoactive yellow protein (PYP) are investigated, for the first time, by ultrafast time-resolved circular dichroism (TRCD) spectroscopy. TRCD is monitored in the near-ultraviolet, over a time scale of 10 ps. Immediately after excitation, TRCD is found to exhibit a large negative peak, which decays within a few picoseconds. A quantitative analysis of the signals shows that, upon excitation, the carbonyl group undergoes a fast (≪0.8 ps) and unidirectional flipping motion in the excited state with an angle of <i>ca.</i> 17–53°. For the subset of proteins that do not enter the signaling photocycle, TRCD provides strong evidence that the carbonyl group moves back to its initial position, leading to the formation of a nonreactive ground-state intermediate of trans conformation. The initial ground state is then restored within <i>ca.</i> 3 ps. Comparative study of R52Q and wild-type PYP provides direct evidence that the absence of Arg52 has no effect on the conformational changes of the chromophore during those steps

Photochemical Nature of Parietopsin

Parietopsin is a nonvisual green light-sensitive opsin closely related to vertebrate visual opsins and was originally identified in lizard parietal eye photoreceptor cells. To obtain insight into the functional diversity of opsins, we investigated by UV–visible absorption spectroscopy the molecular properties of parietopsin and its mutants exogenously expressed in cultured cells and compared the properties to those of vertebrate and invertebrate visual opsins. Our mutational analysis revealed that the counterion in parietopsin is the glutamic acid (Glu) in the second extracellular loop, corresponding to Glu181 in bovine rhodopsin. This arrangement is characteristic of invertebrate rather than vertebrate visual opsins. The photosensitivity and the molar extinction coefficient of parietopsin were also lower than those of vertebrate visual opsins, features likewise characteristic of invertebrate visual opsins. On the other hand, irradiation of parietopsin yielded meta-I, meta-II, and meta-III intermediates after batho and lumi intermediates, similar to vertebrate visual opsins. The pH-dependent equilibrium profile between meta-I and meta-II intermediates was, however, similar to that between acid and alkaline metarhodopsins in invertebrate visual opsins. Thus, parietopsin behaves as an “evolutionary intermediate” between invertebrate and vertebrate visual opsins