Kota Katayama

An FTIR study of monkey green- and red-sensitive visual pigments.

Humans have two kinds of vision: twilight vision mediated by rhodopsin in rod photoreceptor cells and color vision achieved by multiple color pigments in cone photoreceptor cells. Humans have three color pigments: red-, green-, and blue-sensitive proteins maximally absorbing at 560, 530, and 425 nm, respectively, and specific perception of light by the red, green, blue (RGB) sensors is the origin of color vision. Rhodopsin and color-pigments both contain a common chromophore molecule, 11-cis retinal, whereas different chromophore–protein interactions allow preferential absorption of different colors. On the molecular level, studying rhodopsin is highly advantageous because large amounts of protein can be obtained from vertebrate and invertebrate native cells. Consequently, X-ray structures of bovine and squid rhodopsins were determined. In the case of bovine rhodopsin, the structures have been further determined for photointermediates and for the active-state complexed with the C-terminus peptide of the a subunit of G-protein. These structures provided insights into the mechanism of the chromophore–protein interaction and activation. On the other hand, structural studies of color pigments lag far behind those of rhodopsin. In fact, none of color pigments was crystallized. Catarrhini, including Old World monkeys and Hominoids, acquired green and red pigments, both of which belong to the L (long-wavelength absorbing) group, by gene duplication. They exhibit an approximately 30 nm difference in the lmax value and have 15 amino acid sequence differences. [2]

Protein-bound water molecules in primate red- and green-sensitive visual pigments.

Protein-bound water molecules play crucial roles in the structure and function of proteins. The functional role of water molecules has been discussed for rhodopsin, the light sensor for twilight vision, on the basis of X-ray crystallography, Fourier transform infrared (FTIR) spectroscopy, and a radiolytic labeling method, but nothing is known about the protein-bound waters in our color visual pigments. Here we apply low-temperature FTIR spectroscopy to monkey red (MR)- and green (MG)-sensitive color pigments at 77 K and successfully identify water vibrations using D(2)O and D(2)(18)O in the whole midinfrared region. The observed water vibrations are 6-8 for MR and MG, indicating that several water molecules are present near the retinal chromophore and change their hydrogen bonds upon retinal photoisomerization. In this sense, color visual pigments possess protein-bound water molecules essentially similar to those of rhodopsin. The absence of strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) is common between rhodopsin and color pigments, which greatly contrasts with the case of proton-pumping microbial rhodopsins. On the other hand, two important differences are observed in water signal between rhodopsin and color pigments. First, the water vibrations are identical between the 11-cis and 9-cis forms of rhodopsin, but different vibrational bands are observed at >2550 cm(-1) for both MR and MG. Second, strongly hydrogen-bonded water molecules (2303 cm(-1) for MR and 2308 cm(-1) for MG) are observed for the all-trans form after retinal photoisomerization, which is not the case for rhodopsin. These specific features of MR and MG can be explained by the presence of water molecules in the Cl(-)-biding site, which are located near positions C11 and C9 of the retinal chromophore. The averaged frequencies of the observed water O-D stretching vibrations for MR and MG are lower as the λ(max) is red-shifted, suggesting that water molecules are involved in the color tuning of our vision.

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

Significance Vertebrate rhodopsin (Rh) has been a model system for many G protein-coupled receptors for over a decade. However, due to its thus-far limited repertoire of active ligands, its use in assisting the development of new therapeutic modalities and drugs has been limited. This study elucidates a photocyclic G protein activation by Rh bound with a six-carbon ring retinal (Rh6mr), and thus broadens the diversity of such Rh signaling modulators. Rh6mr does not release its chromophore after light activation, but instead the resulting photoproduct is thermally reisomerized back to its inactive state, abrogating the necessity for a complex retinoid cycle to renew its chromophore. This photocyclic behavior of Rh6mr opens up several avenues for using optogenetic tools based on vertebrate Rhs. 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 one-way reaction that activates transducin (Gt) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C11=C12 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 Gt 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 Gt 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.

Color Tuning in Retinylidene Proteins

Retinylidene proteins (also called rhodopsins) are membrane-embedded photoreceptors that contain a vitamin A aldehyde linked to a lysine residue by a Schiff base as their light-sensing chromophore. The chromophore is surrounded by seven-transmembrane α-helices and absorbs light at different wavelengths due to differences in the electronic energy gap between its ground and excited states. The variation in the wavelength of maximal absorption (λmax: 360–620 nm) of rhodopsins arises due to interaction between the apoprotein (opsin) and the retinyl chromophore, the ‘opsin shift’. This chapter reviews the color tuning mechanisms in type-1 microbial and type-2 animal rhodopsins as revealed mainly by our experimental and theoretical studies.

FTIR study of the photoreaction of bovine rhodopsin in the presence of hydroxylamine.

In bovine rhodopsin, 11-cis-retinal forms a Schiff base linkage with Lys296. The Schiff base is not reactive to hydroxylamine in the dark, which is consistent with the well-protected retinal binding site. In contrast, under illumination it easily forms all-trans retinal oxime, resulting in the loss of color. This suggests that activation of rhodopsin creates a specific reaction channel for hydroxylamine or loosens the chromophore binding pocket. In the present study, to extract structural information on the Schiff base vicinity and to understand the changes upon activation of rhodopsin, we compared light-induced FTIR difference spectra of bovine rhodopsin in the presence and absence of hydroxylamine under physiological pH (approximately 7). Although the previous FTIR study did not observe the complex formation between rhodopsin and G-protein transducin in hydrated films, the present study clearly shows that hydrated films can be used for studies of the interaction between rhodopsin and hydroxylamine. Hydroxylamine does not react with the Schiff base of Meta-I intermediate trapped at 240 K, possibly because of decreased conformational motions under the frozen environment, while FTIR spectroscopy showed that hydroxylamine affects the hydrogen bonds of the Schiff base and water molecules in Meta-I. In contrast, formation of the retinal oxime was clearly observed at 280 K, the characteristic temperature of Meta-II accumulation in the absence of hydroxylamine, and time-dependent formation of retinal oxime was observed from Meta-II at 265 K as well. The obtained difference FTIR spectra of retinal oxime and opsin are different from that of Meta-II. It is likely that the antiparallel beta-sheet constituting a part of the retinal binding pocket at the extracellular surface is structurally disrupted in the presence of hydroxylamine, which allows the hydrolysis of the Schiff base into retinal oxime.

Spectral Tuning Mechanism of Primate Blue-sensitive Visual Pigment Elucidated by FTIR Spectroscopy

Protein-bound water molecules are essential for the structure and function of many membrane proteins, including G-protein-coupled receptors (GPCRs). Our prior work focused on studying the primate green- (MG) and red- (MR) sensitive visual pigments using low-temperature Fourier transform infrared (FTIR) spectroscopy, which revealed protein-bound waters in both visual pigments. Although the internal waters are located in the vicinity of both the retinal Schiff base and retinal β-ionone ring, only the latter showed differences between MG and MR, which suggests their role in color tuning. Here, we report FTIR spectra of primate blue-sensitive pigment (MB) in the entire mid-IR region, which reveal the presence of internal waters that possess unique water vibrational signals that are reminiscent of a water cluster. These vibrational signals of the waters are influenced by mutations at position Glu113 and Trp265 in Rh, which suggest that these waters are situated between these two residues. Because Tyr265 is the key residue for achieving the spectral blue-shift in λmax of MB, we propose that these waters are responsible for the increase in polarity toward the retinal Schiff base, which leads to the localization of the positive charge in the Schiff base and consequently causes the blue-shift of λmax.

Complex binding pathways determine the regeneration of mammalian green cone opsin with a locked retinal analogue

Phototransduction is initiated when the absorption of light converts the 11-cis-retinal chromophore to its all-trans configuration in both rod and cone vertebrate photoreceptors. To sustain vision, 11-cis-retinal is continuously regenerated from its all-trans conformation through a series of enzymatic steps comprising the “visual or retinoid” cycle. Abnormalities in this cycle can compromise vision because of the diminished supply of 11-cis-retinal and the accumulation of toxic, constitutively active opsin. As shown previously for rod cells, attenuation of constitutively active opsin can be achieved with the unbleachable analogue, 11-cis-6-membered ring (11-cis-6mr)-retinal, which has therapeutic effects against certain degenerative retinal diseases. However, to discern the molecular mechanisms responsible for this action, pigment regeneration with this locked retinal analogue requires delineation also in cone cells. Here, we compared the regenerative properties of rod and green cone opsins with 11-cis-6mr-retinal and demonstrated that this retinal analogue could regenerate rod pigment but not green cone pigment. Based on structural modeling suggesting that Pro-205 in green cone opsin could prevent entry and binding of 11-cis-6mr-retinal, we initially mutated this residue to Ile, the corresponding residue in rhodopsin. However, this substitution did not enable green cone opsin to regenerate with 11-cis-6mr-retinal. Interestingly, deletion of 16 N-terminal amino acids in green cone opsin partially restored the binding of 11-cis-6mr-retinal. These results and our structural modeling indicate that a more complex binding pathway determines the regeneration of mammalian green cone opsin with chromophore analogues such as 11-cis-6mr-retinal.

Identical Hydrogen-Bonding Strength of the Retinal Schiff Base between Primate Green- and Red-Sensitive Pigments: New Insight into Color Tuning Mechanism.

Three aspects are generally considered in the color-tuning mechanism of vision: (I) chromophore distortion, (II) electrostatic interaction between the protonated Schiff base and counterion, and (III) polarity around the β-ionone ring and polyene chain. Primate green- and red-sensitive proteins are highly homologous but display maximum absorption at 530 and 560 nm, respectively. In the present study, the N-D stretching frequency of monkey green-sensitive protein was identified by using C15-D retinal. The hydrogen-bonding strength between monkey green and red was identical. Together with a previous resonance Raman study, we conclude that the 30 nm difference originates exclusively from the polarity around the β-ionone ring and polyene chain. Three amino acids (Ala, Phe, and Ala in monkey green and Ser, Tyr, and Thr in monkey red, respectively) may be responsible for color tuning together with protein-bound water molecules around the β-ionone ring and polyene chain but not at the Schiff base region.

FTIR study of primate color visual pigments.

How do we distinguish colors? Humans possess three color pigments; red-, green-, and blue-sensitive proteins, which have maximum absorbance (λmax) at 560, 530, and 420 nm, respectively, and contribute to normal human trichromatic vision (RGB). Each color pigments consists of a different opsin protein bound to a common chromophore molecule, 11-cis-retinal, whereas different chromophore-protein interactions allow preferential absorption of different colors. However, detailed experimental structural data to explain the molecular basis of spectral tuning of color pigments are lacking, mainly because of the difficulty in sample preparation. We thus started structural studies of primate color visual pigments using low-temperature Fourier-transform infrared (FTIR) spectroscopy, which needs only 0.3 mg protein for a single measurement. Here we report the first structural data of monkey red- (MR) and green- (MG) sensitive pigments, in which the information about the protein, retinal chromophore, and internal water molecules is contained. Molecular mechanism of color discrimination between red and green pigments will be discussed based on the structural data by FTIR spectroscopy.