Protein-Bound Water Molecules in Primate Red- and Green-Sensitive Visual Pigments

Abstract

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.

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

“…Wat2a involves three hydrogen bonds to Ser186, Cys187, and Glu181, and Wat2b is located near Glu113 and stabilizes the protonated Schiff base of the retinal chromophore 34 . Alternatively, a previous analysis of the water signals for MG and MR indicated that the negative dipole moment of the oxygen side of the water molecule, which was possibly localized at the β-ionone ring side of retinal for MR, would associate with a spectral red-shift in absorbance 15 . The identification of MB water signals using site-directed mutational studies provides several insights into the mechanism of the spectral blue-shift.…”

“…This vector was expressed in the HEK293T cell line and regenerated with 11- cis -retinal 14, 15 . The regenerated sample was solubilized with a buffer that contained 2% (w/v) n-dodecyl-β-D-maltoside (DDM), 50 mM HEPES, 140 mM NaCl, and 3 mM MgCl 2 (pH 6.5) and purified by adsorption on an antibody-conjugated column.…”

Color Vision: “OH-Site” Rule for Seeing Red and Green

“…This could be one of the reasons for the presence of several internal water molecules in M/LWS pigments. 26 Residue W265 in helix VI is highly conserved in all the three pigments and continues to remain in van der Waals contact with the β-ionone ring of retinal. 13 …”

Optogenetics

“…By using the highly accurate Fourier transform infrared (FTIR) spectra, proteinbound water molecules in the red and green cone pigments have been identifi ed (Katayama et al 2012 ). The water signals were found to differ not only from those of rhodopsin but also between the red and green cone pigments ( Fig.…”

“…Light-minus-dark difference Fourier transform infrared (FTIR) spectra of monkey red ( a ), monkey green ( b ), bovine Rh ( c )(Katayama et al 2012 ), and squid Rh ( d )(Ota et al 2006 ), with 11cis -retinyl, in the 2,750-1,800 cm −1 region measured at 77 K. Red and blue lines represent the spectra in D 2 O and D 2 18 O, respectively, and green labeled frequencies correspond to those identifi ed as water-stretching vibrations. Light-minus-dark difference Fourier transform infrared (FTIR) spectra of monkey red ( a ), monkey green ( b ), bovine Rh ( c )(Katayama et al 2012 ), and squid Rh ( d )(Ota et al 2006 ), with 11cis -retinyl, in the 2,750-1,800 cm −1 region measured at 77 K. Red and blue lines represent the spectra in D 2 O and D 2 18 O, respectively, and green labeled frequencies correspond to those identifi ed as water-stretching vibrations.…”