Unique retinal binding pocket of primate blue-sensitive visual pigment

Abstract

The visual pigments of humans contain 11-cis retinal as the chromophore of light perception, and its photoisomerization to the all-trans form initiates visual excitation in our eyes. It is well known that three isomeric states of retinal (11-cis, all-trans, and 9-cis) are in photoequilibrium at very low temperatures such as 77 K. Here we report the lack of formation of the 9-cis form in monkey blue (MB) at 77 K, as revealed by light-induced difference FTIR spectroscopy. This indicates that the chromophore binding pocket of MB does not accommodate the 9-cis form, even though it accommodates the all-trans form by twisting the chromophore. Mutation of the blue-specific tyrosine at position 265 into tryptophan, which is highly conserved in other animal rhodopsins, led to formation of the 9-cis form in MB, suggesting that Y265 is one of the determinants of the unique photochemistry in blue pigments. We also found that 9-cis retinal does not bind to MB opsin, implying that the chromophore binding pocket does not accommodate the 9-cis form at physiological temperature. The unique property of MB is discussed based on the present results.3 Humans have two kinds of vision: twilight vision mediated by rhodopsin in rod photoreceptor cells and color vision achieved by multiple color pigments in cone photoreceptor cells. 1 Humans also possess three color pigments, red-, green-, and blue-sensitive proteins, that maximally absorb at 560, 530, and 425 nm, respectively. 2 Rhodopsin and color pigments both contain a common chromophore molecule, 11-cis retinal, and different chromophore-protein interactions allow preferential absorption of different colors. 3,4 Purified proteins are a prerequisite to understanding the mechanism of color tuning and the activation process by light. Studying rhodopsin is highly advantageous because large amounts of protein can be obtained from vertebrate and invertebrate native cells. Bovine rhodopsin is a standard protein, and its crystal structures have been reported for the unphotolyzed state, 5 opsin, 6 photobleaching intermediates, 7,8 active state, 9,10 and the active-state complexed with the C-terminus peptide of the  subunit of G-protein, 10-12 or engineered mini-Go, 13 and arrestin. 14 These structures have provided insight into the mechanism of the chromophore-protein interaction and activation.On the other hand, structural studies of color pigments lag far behind those of rhodopsin.No color visual pigments have yet been crystallized. Under such circumstances, we started structural studies of primate color pigments by using low-temperature difference FTIR spectroscopy. 15,16 To achieve this, monkey red-(MR), and monkey green-(MG)-sensitive color visual pigments were expressed in HEK cells, and light-induced difference FTIR spectra were measured at 77 K, where the photoproduct, batho-intermediate (Batho), was reverted to the initial state by light. 17-21 Consequently, photoconversions from the initial state to Batho, and Batho to the initial state were repeated. This photochromic property h...