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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.

Eyes gather information and color forms an extremely important component of the information, more so in the case of animals to forage and navigate within their immediate environment. By using the ONIOM (QM/MM) method, we report a comprehensive theoretical analysis of the structure and molecular mechanism of spectral tuning of monkey-red and green-sensitive visual pigments. We show that, interaction of retinal with three hydroxyl-bearing amino acids near the β-ionone ring part of the retinal in opsin, A164S, F261Y and A269T, increases the electron delocalization, decreases the BLA of the retinal and leads to variation in the wavelength of maximal absorbance in the red- and green-sensitive visual pigments. Based on the analysis, we propose the “OH-site” rule for seeing red and green. This rule is also shown to account for the spectral shifts obtained from hydroxyl-bearing amino acids near the Schiff base in different visual pigments: at site 292 (A292S, A292Y, and A292T) in bovine and at site 111 (Y111) in squid opsins. Therefore, the OH-site rule is shown to be site-specific and not pigment-specific and thus can be used for tracking spectral shifts in any visual pigment.

Light energy is first captured in animal and microbial rhodopsins by ultrafast photoisomerization, whose relaxation accompanies protein structural changes for each function. Here, we report a microbial rhodopsin, marine bacterial TAT rhodopsin, that displays no formation of photointermediates at >10–5 s. Low-temperature ultraviolet–visible and Fourier transform infrared spectroscopy revealed that TAT rhodopsin features all-trans to 13-cis photoisomerization like other microbial rhodopsins, but a planar 13-cis chromophore in the primary K intermediate seems to favor thermal back isomerization to the original state without photocycle completion. The molecular mechanism of the early photoreaction in TAT rhodopsin will be discussed.

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.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.

Rot sehen (und grün): Einen ersten Einblick in Strukturen, die für die Farbabstimmung der Sehpigmente wichtig sind, lieferte die lichtinduzierte FT‐IR‐Spektroskopie der grünen und roten Affen‐Retinalpigmente in D2O. Ihre FT‐IR‐Spektren sind im niederfrequenten Bereich demjenigen von Rhodopsin ähnlich, unterscheiden sich aber in den X‐H‐ und X‐D‐Streckschwingungsbereichen erheblich, was Informationen über Chromophor‐Protein‐Wechselwirkungen in Farbpigmenten liefert.

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