Color‐Discriminating Retinal Configurations of Sensory Rhodopsin I by Photo‐Irradiation Solid‐State NMR Spectroscopy

Abstract

SRI (sensory rhodopsin I) can discriminate multiple colors for the attractant and repellent phototaxis. Studies aimed at revealing the color-dependent mechanism show that SRI is a challenging system not only in photobiology but also in photochemistry. During the photoreaction of SRI, an Mintermediate (attractant) transforms into a P-intermediate (repellent) by absorbing blue light. Consequently, SRI then cycles back to the G-state. The photoreactions were monitored with the 13 C NMR signals of [20-13 C]retnal-SrSRI using in situ photo-irradiation solid-state NMR spectroscopy. The M-intermediate was trapped at À40 8C by illumination at 520 nm. It was transformed into the P-intermediate by subsequent illumination at 365 nm. These results reveal that the G-state could be directly transformed to the P-intermediate by illumination at 365 nm. Thus, the stationary trapped M-and P-intermediates are responsible for positive and negative phototaxis, respectively.

Solid-state NMR analysis of the sodium pump Krokinobacter rhodopsin 2 and its H30A mutant

“…In combination with illumination protocols, detection of the retinal within certain photointermediate states became accessible. Since such experiments caused significant technical challenges in terms of sensitivity, examples were restricted to bacteriorhodopsin, sensory and visual rhodopsin (Concistre et al, 2008;Etzkorn et al, 2010;Hu et al, 1998;Kimata et al, 2016;Yomoda et al, 2014). However, with the advent of dynamic nuclear polarization (DNP), new opportunities arose.…”

Recent Solid-State NMR Studies of Membrane-Bound Peptides and Proteins

“…The individual intermediates of the photocycle of SrSRI have been observed using in situ photo-irradiation solid-state NMR spectroscopy [204]. Using this technique, 13 C CP-MAS NMR signals from the [20-13 C]-retinal of the long-lived M-and P-intermediates and pathways from the G-state to the photointermediates were observed, thereby providing insights into the mechanism of the photocycle of SrSRI.…”

“…In the system used in most analysis described in this section, the sample is illuminated from inside the rotor tube through a glass rod inserted into the rotor [203,204]. This allows for illumination of the sample with extremely high efficiency and opens the door to studies of the photoreaction processes of photoreceptor membrane proteins [205].…”

“…An optical fiber is guided from the bottom to the top of the probe head and accurately aligned along the top of the MAS rotor. The top part of zirconia rotor is capped by a glass rod in which the tip part is ground in order to illuminate the light perpendicular to the spinner axis such that the strong light irradiates the sample from inside the sample tube [204]. M-intermediate can be eliminated by illuminating the sample with 365 nm LED light, as the M-intermediate has a maximum absorbance frequency of 390 nm, which differs from that of the other intermediates.…”

“…We developed an in situ photo-irradiation system for solid-state MAS NMR measurements under MAS analyses [203][204][205]. In situ continuous photo-irradiation was carried out using an optical fiber passed from outside the magnet through a tightly sealed cap made of a glass rod glued to a zirconia rotor (Fig.…”