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

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 pig-ments, monkey red (MR), green (MG), and blue (MB), where the batho-intermediate (Batho) exhibits photo-equilibrium with the unphotolyzed state. This photo-chromic 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 C₁₁=C₁₂ HOOP vibration in the planer structure in MB is decoupled by distortion in Batho after retinal photoisomerization, but returns to the coupled C₁₁=C₁₂ 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.