Production of a Light-Gated Proton Channel by Replacing the Retinal Chromophore with Its Synthetic Vinylene Derivative

Abstract

Rhodopsin is widely distributed in organisms as a membrane-embedded photoreceptor protein, consisting of the apoprotein opsin and vitamin-A aldehyde retinal, A1-retinal and A2-retinal being the natural chromophores. Modifications of opsin (e.g., by mutations) have provided insight into the molecular mechanism of the light-induced functions of rhodopsins as well as providing tools in chemical biology to control cellular activity by light. Instead of the apoprotein opsin, in this study, we focused on the retinal chromophore and synthesized three vinylene derivatives of A2-retinal. One of them, C(14)-vinylene A2-retinal (14V-A2), was successfully incorporated into the opsin of a light-driven proton pump archaerhodopsin-3 (AR3). Electrophysiological experiments revealed that the opsin of AR3 (archaeopsin3, AO3) with 14V-A2 functions as a light-gated proton channel. The engineered proton channel showed characteristic photochemical properties, which are significantly different from those of AR3. Thus, we successfully produced a proton channel by replacing the chromophore of AR3.

Vectorial Proton Transport Mechanism of RxR, a Phylogenetically Distinct and Thermally Stable Microbial Rhodopsin

“…In order to quantitatively estimate proton pumping activities, we obtained the initial slope amplitudes of extracellular pH changes from 0 to 10 sec after illumination in the absence of CCCP 21,22 . We used 15 mW/cm 2 as a light www.nature.com/scientificreports www.nature.com/scientificreports/ intensity because the relationship between light intensities and initial slope amplitudes of pH changes showed a linear regression at light intensities below 104 mW/cm 2 (see the text of Experimental procedures for details) (Fig.…”

“…Consequently, these genes were inserted into pET21a (for wild-type RxR) or pET22b (for all RxR mutants) plasmid vectors (Novagen, USA) with NdeI and XhoI restriction enzyme sites. An expression plasmid of histidine-tagged Archaerhodopsin-3 (AR3) was constructed as previously described 22 . E. coli cells harboring the plasmids were grown at 37 °C in LB medium (1% Bacto tryptone, 0.5% yeast extract and 0.5% NaCl) containing 50 μg/mL ampicillin and protein expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG, final conc.…”

Synthesis of One Double Bond-Inserted Retinal Analogs and Their Binding Experiments with Opsins: Preparation of Novel Red-Shifted Channelrhodopsin Variants

“…Spectral data were shown in the literature. 20) Preparation of ChR Analogs and Spectroscopy In this study, two ChR variants, ReaCh (NCBI accession number, KF448069) 11) and ChrimsonR (Chrimson (KF992060) K176R mutant) 19) were used. These ChR variants were expressed by HEK293 system as reported previously.…”

“…We anticipated that it might be possible to produce the even more red-shifted new ChRs by changing opsins for binding. As other opsins, we chose ReaChR 11) and ChrimsonR 19) because their absorption maxima were 61 nm and 110 nm red-shifted, respectively, compared to that of C1C2. Although the data of A2 analogs (3b, 4b and 5b) were already reported in the literature, 20) we describe here a full ac-count of the synthesis of six kinds of chromophores and their binding experiments with other opsins.…”

Redshifted and Near‐infrared Active Analog Pigments Based upon Archaerhodopsin‐3

“…In view of its application as a fluorescent voltage sensor, several variants of AR3 have been engineered to achieve an up to eight‐fold increase in brightness, using retinal analogs , site‐saturation mutagenesis and a combination of both . In another approach, incorporation of conjugated‐chain extended retinals into AR3 induced a switch of function from light‐driven proton pump to light‐driven proton channel .…”