Take Matsuyama

Evolution of opsins and phototransduction.

Opsins are the universal photoreceptor molecules of all visual systems in the animal kingdom. They can change their conformation from a resting state to a signalling state upon light absorption, which activates the G protein, thereby resulting in a signalling cascade that produces physiological responses. This process of capturing a photon and transforming it into a physiological response is known as phototransduction. Recent cloning techniques have revealed the rich and diverse nature of these molecules, found in organisms ranging from jellyfish to humans, functioning in visual and non-visual phototransduction systems and photoisomerases. Here we describe the diversity of these proteins and their role in phototransduction. Then we explore the molecular properties of opsins, by analysing site-directed mutants, strategically designed by phylogenetic comparison. This site-directed mutant approach led us to identify many key features in the evolution of the photoreceptor molecules. In particular, we will discuss the evolution of the counterion, the reduction of agonist binding to the receptor, and the molecular properties that characterize rod opsins apart from cone opsins. We will show how the advances in molecular biology and biophysics have given us insights into how evolution works at the molecular level.

Covalent bond between ligand and receptor required for efficient activation in rhodopsin

Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.

Origin of the low thermal isomerization rate of rhodopsin chromophore

Low dark noise is a prerequisite for rod cells, which mediate our dim-light vision. The low dark noise is achieved by the extremely stable character of the rod visual pigment, rhodopsin, which evolved from less stable cone visual pigments. We have developed a biochemical method to quickly evaluate the thermal activation rate of visual pigments. Using an isomerization locked chromophore, we confirmed that thermal isomerization of the chromophore is the sole cause of thermal activation. Interestingly, we revealed an unexpected correlation between the thermal stability of the dark state and that of the active intermediate MetaII. Furthermore, we assessed key residues in rhodopsin and cone visual pigments by mutation analysis and identified two critical residues (E122 and I189) in the retinal binding pocket which account for the extremely low thermal activation rate of rhodopsin.

Red-Tuning of the Channelrhodopsin Spectrum Using Long Conjugated Retinal Analogues

As optogenetic studies become more popular, the demand for red-shifted channelrhodopsin is increasing, because blue-green light is highly scattered or absorbed by animal tissues. In this study, we developed a red-shifted channelrhodopsin by elongating the conjugated double-bond system of the native chromophore, all -trans-retinal (ATR1). Analogues of ATR1 and ATR2 (3,4-didehydro-retinal) in which an extra C═C bond is inserted at different positions (C6-C7, C10-C11, and C14-C15) were synthesized and introduced into a widely used channelrhodopsin variant, C1C2 (a chimeric protein of channelrhodopsin-1 and channelrhodopsin-2 from Chlamydomonas reinhardtii). C1C2 bearing these retinal analogues as chromophores showed broadened absorption spectra toward the long-wavelength side and photocycle intermediates similar to the conducting state of channelrhodopsin. However, the position of methyl groups on the retinal polyene chain influenced the yield of the pigment, absorption maximum, and photocycle pattern to a variable degree. The lack of a methyl group at position C9 of the analogues considerably decreased the yield of the pigment, whereas a methyl group at position C15 exhibited a large red-shift in the absorption spectra of the C1C2 analogue. Expansion of the chromophore binding pocket by mutation of aromatic residue Phe265 to Ala improved the yield of the pigment bearing elongated ATR1 analogues without a great alteration of the photocycle kinetics of C1C2. Our results show that elongation of the conjugated double-bond system of retinal is a promising strategy for improving the ability of channelrhodopsin to absorb long-wavelength light passing through the biological optical window.

Alternative Formation of Red-Shifted Channelrhodopsins: Noncovalent Incorporation with Retinal-Based Enamine-Type Schiff Bases and Mutated Channelopsin

Red-shifted channelrhodopsins (ChRs) are attractive for optogenetic tools. We developed a new type of red-shifted ChRs that utilized noncovalent incorporation of retinal and 3,4-dehydroretinal-based enamine-type Schiff bases and mutated channelopsin, C1C2-K296G. These ChRs exhibited absorption maxima that were shifted 10-30 nm toward longer wavelengths than that of C1C2-ChR regenerated with all-trans-retinal.

The C-Terminus of the G Protein α Subunit Controls the Affinity of Nucleotides

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.