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The mutual conversions of metarhodopsin I (MI) and metarhodopsin II (MII) upon changing the pH of the MI/MII mixture were examined to confirm the direct evidence for MII which is widely believed to form a pH-dependent equilibrium with MI. The pH-dependent formation of MII from MI and presence of another MII which is in equilibrium with MI in a pH-independent manner, with an ability to bind to the peptide suggested there is no need for proton uptake by the protein moiety of opsin for the binding to the peptide.

Stellettazole A is an antibacterial homosesquiterpene amide of 1-guanidinopropyl-3-methylhistamine that was isolated by scientists from a Stelletta sponge. This antibacterial compound was found to inhibit Ca2+/calmodulin-dependent phosphodiesterase during certain biological processes. Further investigations into the structure determination and biological features of these metabolites revealed the existence of two bioactive dimeric stellettadines, also known as bistelletadines A and B.

Most vertebrates have one type of rhodopsin and multiple types of cone visual pigments with different absorption maxima in their retinas. The spectral sensitivities of multiple cone visual pigments contribute to color discrimination in these animals. Vertebrate cone visual pigments are classified into four groups based on their amino acid sequences. Among these groups, many pigments in the longer wavelength-sensitive group (L-group) have a unique spectral tuning mechanism, that is, the red-shift of absorption maximum induced by the binding of chloride to His181 of the protein moiety (chloride effect). However, a few pigments such as mouse green and guinea pig green pigments in L-group have a tyrosine residue instead of a histidine at position 181. Interestingly, mouse green shows no chloride effect, whereas guinea pig green shows a significant chloride effect. In the present site-directed mutational analysis, we revealed that this difference in the chloride effect in rodent pigments is completely explained by the replacements of two residues at positions 289 and 292. In addition, mutations at positions 181, 289, and 292 abolished 80% of the chloride effect in monkey red and green. Further analysis with chimeras showed that the residual 20% of the chloride effect could be attributed to helical interactions within the pigments. Thus, we concluded that these three amino acid residues are the main determinants of the chloride-dependent spectral shift in L-group pigments. [ABSTRACT FROM AUTHOR]

Visual pigments in rod and cone photoreceptor cells of vertebrate retinas are highly diversified photoreceptive proteins that consist of a protein moiety opsin and a light-absorbing chromophore 11-cis-retinal. There are four types of cone visual pigments and a single type of rod visual pigment. The reaction process of the rod visual pigment, rhodopsin, has been extensively investigated, whereas there have been few studies of cone visual pigments. Here we comprehensively investigated the reaction processes of cone visual pigments on a time scale of milliseconds to minutes, using flash photolysis equipment optimized for cone visual pigment photochemistry. We used chicken violet (L-group), chicken blue (M1-group), chicken green (M2-group), and monkey green (L-group) visual pigments as representatives of the respective groups of the phylogenetic tree of cone pigments. The S, M1, and M2 pigments showed the formation of a pH-dependent mixture of meta intermediates, similar to that formed from rhodopsin. Although monkey green (L-group) also formed a mixture of meta intermediates, pH dependency of meta intermediates was not observed. However, meta intermediates of monkey green became pH dependent when the chloride ion bound to the monkey green was replaced with a nitrate ion. These results strongly suggest that rhodopsin and S, M1, and M2 cone visual pigments share a molecular mechanism for activation, whereas the L-group pigment may have a special reaction mechanism involving the chloride-binding site. [ABSTRACT FROM AUTHOR]

VA/VAL opsin is one of the four kinds of nonvisual opsins that are closely related to vertebrate visual pigments in the phylogenetic tree of opsins. Previous studies indicated that among these opsins, parapinopsin and pinopsin exhibit molecular properties similar to those of invertebrate bistable visual pigments and vertebrate visual pigments, respectively. Here we show that VA/VAL opsin exhibits molecular properties intermediate between those of parapinopsin and pinopsin. VAL opsin from Xenopus tropicalis was expressed in cultured cells, and the pigment with an absorption maximum at 501 nm was reconstituted by incubation with 11-cis-retinal. Light irradiation of this pigment caused cis-to-trans isomerization of the chromophore to form a state having an absorption maximum in the visible region. This state has the ability to activate Gi and Gt types of G proteins. Therefore, the active state of VAL opsin is a visible light-absorbing intermediate, which probably has a protonated retinylidene Schiff base as its chromophore, like the active state of parapinopsin. However, this state was apparently photoinsensitive and did not show reverse reaction to the original pigment, unlike the active state of parapinopsin, and instead similar to that of pinopsin. Furthermore, the Gi activation efficiency of VAL opsin was between those of pinopsin and parapinopsin. Thus, the molecular properties of VA/VAL opsin give insights into the mechanism of conversion of the molecular properties from invertebrate to vertebrate visual pigments. [ABSTRACT FROM AUTHOR]

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.

Rhodopsin is a G protein-coupled receptor specialized for photoreception and contains a light-absorbing chromophore retinal that binds to the lysine residue of opsin through a protonated Schiff base linkage. Light converts rhodopsin to an equilibrium mixture of the active state metarhodopsin II (MII) and its precursor, metarhodopsin I (MI), which have deprotonated and protonated Schiff base chromophores, respectively. This equilibrium was thought to depend on the pKa of not the Schiff base chromophore but glutamic acid E134 in the highly conserved E/DRY triad in helix III. We performed mutational analyses of E134 and nearby residues to examine whether the equilibrium is really dependent on the pKa of E134 and to obtain clues about the contribution of E134 to the G protein activation characteristics of rhodopsin. All the single mutants at position 134 except for E134D lost the characteristic pH-dependent equilibrium, indicating that the carboxyl group of E134 is responsible for the equilibrium. Interestingly, mutation at position 134 caused little change in the MI or MII spectra or G protein activation efficiency of MII, while it caused a shift of the MI-MII equilibrium. The mutants containing hydrophobic or amide-containing residues at position 134 formed an equilibrium in favor of MII, resulting in an increase in light-induced G protein activation efficiency. On the other hand, the wild type exhibited an opsin activity lower than those of the mutants, which exhibited reasonable light-dependent activities. These results strongly suggest that the evolutionary significance of E134 is not an increase in G protein activity but rather suppression of the opsin activity.

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-cis-retinal) and an excited state (metamelanopsin containing all-trans-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-cis-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.

The C-terminus of the G protein α subunit has a well-known role in determining the selective coupling with the cognate G protein-coupled receptor (GPCR). In fact, rhodopsin, a prototypical GPCR, exhibits active state [metarhodopsin II (MII)] stabilization by interacting with G protein [extra formation of MII (eMII)], and the extent of stabilization is affected by the C-terminal sequence of Gα. Here we examine the relationship between the amount of eMII and the activation efficiency of Gi mutants whose Giα forms have different lengths of the C-terminal sequence of Goα. The results show that both the activation efficiencies of Gi and the amounts of eMII were affected by mutations; however, there was no correlation between them. This finding suggested that the C-terminal region of Gα not only stabilizes MII (active state) but also affects the nucleotide-binding site of Gα. Therefore, we measured the activation efficiency of these mutants by MII at several concentrations of GDP and GTP and calculated the rate constants of GDP release, GDP uptake, and GTP uptake. These rate constants of the Gi mutants were substantially different from those of the wild type, indicating that the replacement of the amino acid residues in the C-terminus alters the affinity of nucleotides. The rate constants of GDP uptake and GTP uptake showed a strong correlation, suggesting that the C-terminus of Giα controls the accessibility of the nucleotide-binding site. Therefore, our results strongly suggest that there is a long-range interlink between the C-terminus of Giα and its nucleotide-binding site.