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

Abstract

Structural studies of color visual pigments lag far behind those of rhodopsin for scotopic vision. Using difference FTIR spectroscopy at 77 K, we report the first structural data of three primate color visual pig­ments, monkey red (MR), green (MG), and blue (MB), where the batho-intermediate (Batho) exhibits photo­equilibrium with the unphotolyzed state. This photo­chromic property is highly advantageous for limited samples since the signal-to-noise ratio is improved, but may not be applicable to late intermediates, because of large structural changes to proteins. Here we report the photochromic property of MB at 163 K, where the BL intermediate, formed by the relaxation of Batho, is in photoequilibrium with the initial MB state. A comparison of the difference FTIR spectra at 77 and 163 K provided information on what happens in the process of transition from Batho to BL in MB. The coupled C 11 =C 12 HOOP vibration in the planer structure in MB is decoupled by distortion in Batho after retinal photoisomerization, but returns to the coupled C 11 =C 12 HOOP vibration in the all- trans chromophore in BL. The Batho formation accompanies helical structural perturbation, which is relaxed in BL. Protein-bound water molecules that form an extended water cluster near the retinal chromophore change hydrogen bonds differently for Batho and BL, being stronger in the latter than in the initial state. In addition to structural dynamics, the present FTIR spectra show no signals of protonated carboxylic acids at 77 and 163 K, suggesting that E181 is deprotonated in MB, Batho and BL.

Early Proton Transfer Reaction in a Primate Blue-Sensitive Visual Pigment

“…The two positive and one negative absorbances at 344, 469, and 406 nm correspond to photointermediates and the ground state of MB, respectively. Previously, light illumination at 163 K has been shown to form the BL intermediate with a red-shifted absorbance from the ground state . In fact, at 223 K, a red-shifted photointermediate with a peak at 466 nm was observed upon illumination by 380 nm light (Figure b, black solid curve).…”

“…However, considering that the counterion switches from Glu113 to Glu181 during Meta-I formation in bovine rhodopsin (counterion switch model) , and that MB shows photochromism like bistable invertebrate rhodopsin with photochromism, the proton acceptor in Lumi might be Glu181. In fact, we infer that Glu181 is deprotonated because none of the C=O stretching vibration of the protonated carboxylate was observed in the >1700 cm –1 region for both 77 and 163 K spectra in MB . Thus, based on these results, both Glu113 and Glu181 are potential candidates for being the proton acceptor that leads to the origin of the positive 1714 cm –1 band in the Lumi intermediate.…”

“…In fact, we infer that Glu181 is deprotonated because none of the C=O stretching vibration of the protonated carboxylate was observed in the >1700 cm −1 region for both 77 and 163 K spectra in MB. 32 Thus, based on these results, both Glu113 and Glu181 are potential candidates for being the proton acceptor that leads to the origin of the positive 1714 cm −1 band in the Lumi intermediate.…”

“…Previously, light illumination at 163 K has been shown to form the BL intermediate with a red-shifted absorbance from the ground state. 32 In fact, at 223 K, a redshifted photointermediate with a peak at 466 nm was observed upon illumination by 380 nm light (Figure 1b S2). Unlike other early photointermediates such as Batho (77 K) and BL (163 K), another photointermediate formed by 420 nm light illumination in Figure 1a was blue-shifted to 344 nm.…”

“…We recently reported that MB exhibits a photochromic property at 77 K as well as 163 K where the BL intermediate is in photoequilibrium with the ground state of MB. 32 FTIR spectra of BL state clearly showed that the distorted retinal structure in Batho is relaxed during the transition to BL, which is accompanied by local structural relaxation. Thus, the structural features of both the retinal and local protein environment can be attributed to the photochromic property of MB at 163 K.…”