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

Abstract

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.

Light-induced difference FTIR spectroscopy of primate blue-sensitive visual pigment at 163 K

“…The band shape of the coupled C11=C12 HOOP mode is sharper in MB (at 960 cm -1 ) than in MG and MR [27], implying that C11=C12 retains its geometric planar structure compared to other pigments in their dark states. Upon retinal photoisomerization, the strongly coupled double bond at C11 and C12 of the retinal polyene chain are decoupled, leading to C11H and C12H wagging modes at 928 and 863 cm -1 , respectively, in Batho.…”

“…MB cDNA was tagged with the Rho1D4 epitope sequence and introduced into the pFastBac HT expression vector. This construct was expressed in the Sf9 cell line and regenerated with 11-cis-retinal [25,27]. The regenerated sample was solubilized with a buffer containing 2% (w/v) n-dodecyl--D-maltoside (DDM) (final concentration was 1% (w/v)), 50 mM HEPES, 140 mM NaCl, and 3 mM MgCl2 (pH 7.0) and purified by adsorption on an antibody-conjugated column and eluted with a buffer containing 0.10 mg/mL 1D4 peptide, 0.02% DDM, 50 mM HEPES, 140 mM NaCl, and 3 mM MgCl2 (pH 7.0).…”

“…spectroscopy was applied to the films hydrated with H2O at 77 K as described previously [29,30]. Low-temperature FTIR spectroscopy was applied to the films hydrated with H2O, D2O, or D2 18 O at 163 K, as described previously [20,21,27]. For the formation of the Batho intermediate of MB, samples were irradiated with 400 nm light (by using an interference filter) for 5 min.…”

“…In bovine rhodopsin, the O-H group of T118, whose stretching frequency is located at 3463 cm -1 [45], is only deuterated at high temperatures [46], which was used to investigate protein fluctuation as the origin of dark noise [47][48][49][50]. A previous study suggested that the bands at 3520 (+)/3472 (-) cm -1 are candidates of the O-H stretch of T118 in MB [27], where a high frequency shift indicates a weaker hydrogen bond upon formation of Batho. Interestingly, the frequency shift (3504 (-)/3483 (+) cm -1 ) was opposite at 163 K (Figure 5b), where the hydrogen bond is stronger in BL than in MB.…”

“…Consequently, we have reported structural information for MR and MG since 2010 [20][21][22][23][24][25][26]. Although a lower expression level was reported for bluesensitive pigments, we successfully obtained light-induced difference FTIR spectroscopy of MB from marmoset [27].…”