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

Abstract

The proton transfer reaction belongs to one of the key triggers for the functional expression of membrane proteins. Rod and cone opsins are light-sensitive G-protein-coupled receptors (GPCRs) that undergo the cis–trans isomerization of the retinal chromophore in response to light. The isomerization event initiates a conformational change in the opsin protein moiety, which propagates the downstream effector signaling. The final step of receptor activation is the deprotonation of the retinal Schiff base, a proton transfer reaction which has been believed to be identical among the cone opsins. Here, we report an unexpected proton transfer reaction occurring in the early photoreaction process of primate blue-sensitive pigment (MB). By using low-temperature UV–visible spectroscopy, we found that the Lumi intermediate of MB formed in transition from the BL intermediate shows an absorption maximum in the UV region, indicating the deprotonation of the retinal Schiff base. Comparison of the light-induced difference FTIR spectra of Batho, BL, and Lumi showed significant α-helical backbone C=O stretching and protonated carboxylate C=O stretching vibrations only in the Lumi intermediate. The transition from BL to Lumi thus involves dramatic changes in protein environment with a proton transfer reaction between the Schiff base and the counterion resulting in an absorption maximum in the UV region.

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“…17,18 Conversely, our recent findings indicate that during the formation of the lumi intermediate in MB, a significant perturbation of the α-helix, most likely in TM3, takes place, concomitant with the deprotonation of the Schiff base. 32 Additionally, using the O−H stretching band of Thr117 3.33 as a probe, we inferred that TM3 is primarily perturbed upon the formation of the lumi intermediate. The physiological significance of deprotonation during the early conformational change process in MB remains unclear.…”

“…32 Furthermore, FTIR spectroscopy unraveled the relaxation of retinal distortion, accompanied by a substantial conformational change in the α-helices�akin to the changes observed in rhodopsin and MB. 32 Additionally, as seen in rhodopsin, we detected shifts in the hydrogen bonding involving protonated carboxylic amino acids. Nevertheless, this protonation signal's source differed from that of rhodopsin, as inferred from sitedirected mutagenesis experiments.…”

“…Recently, we conducted spectroscopic measurements of lumi intermediates of MB, revealing a fascinating phenomenon: the maximum absorption wavelength (λ max ) shifts into the ultraviolet (UV) region upon the deprotonation of the retinal Schiff base . Additionally, we observed that the lumi intermediate is photoreversible and capable of reverting to its resting state when exposed to UV light.…”

“…In this study, we investigated the structural changes associated with the formation of the lumi intermediate of MG, employing low-temperature UV-visible and FTIR spectroscopic techniques . Our analysis unveiled photoabsorption changes reminiscent of those observed in rhodopsin, featuring a blue-shift of 50 nm, while no shift was observed in the UV region attributable to the Schiff base’s deprotonation reaction, a phenomenon observed in MB . Furthermore, FTIR spectroscopy unraveled the relaxation of retinal distortion, accompanied by a substantial conformational change in the α-helicesakin to the changes observed in rhodopsin and MB .…”

“…Our analysis unveiled photoabsorption changes reminiscent of those observed in rhodopsin, featuring a blue-shift of 50 nm, while no shift was observed in the UV region attributable to the Schiff base’s deprotonation reaction, a phenomenon observed in MB . Furthermore, FTIR spectroscopy unraveled the relaxation of retinal distortion, accompanied by a substantial conformational change in the α-helicesakin to the changes observed in rhodopsin and MB . Additionally, as seen in rhodopsin, we detected shifts in the hydrogen bonding involving protonated carboxylic amino acids.…”