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. 2017 Aug 4;7:7349. doi: 10.1038/s41598-017-07461-9

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Molecular properties of purified Drosophila Rh7. (A–D) Absorption spectra of Rh7-Cap purified after it was regenerated with 11-cis-retinal (A,B) or 11-cis-3-hydroxyretinal (C,D). (A,C) Spectra were measured in the dark (black curve), after UV light irradiation (red curve), after subsequent yellow light (>500 nm) irradiation (green curve), after UV light re-irradiation (blue curve), and after yellow light re-irradiation (violet curve). (B,D) Difference spectra calculated based on the spectra in (A) or (C). The red curve was calculated by subtracting the spectrum before from that after UV light irradiation. The green curve was calculated by subtracting the spectrum after UV light irradiation from that after subsequent yellow light irradiation. The blue curve was calculated by subtracting the spectrum after yellow light irradiation from that after UV light re-irradiation. The violet curve was calculated by subtracting the spectrum after UV light re-irradiation from that after yellow light re-irradiation. The inset of (D) shows a comparison of the spectral properties of Rh7-Cap reconstituted with 11-cis-retinal (black curve) and 11-cis-3-hydroxyretinal (red curve). Difference spectra caused by UV light irradiation shown by red curve in (B) and (D) were normalized to be ~1.0 at the positive maxima. (E) Light-dependent retinal configuration changes of 11-cis-retinal bound Rh7-Cap. Left-hand panel, the retinal configurations were analyzed with HPLC after extraction of the chromophore as retinal oximes (syn and anti forms of 11-cis-, 13-cis-, and all-trans-retinal oximes). Right-hand panel, isomeric compositions of retinal before and after light irradiation. (F) Absorption spectra of Rh7-Cap purified after it was regenerated with all-trans-retinal. Spectra were measured in the dark (black curve), after yellow light (>500 nm) irradiation (red curve), and after subsequent UV light irradiation (blue curve). Inset, Difference spectra calculated by subtracting the spectrum before from that after yellow light irradiation (red curve) and the spectrum after yellow light irradiation from that after subsequent UV light irradiation (blue curve). (G) Time courses of G protein activation ability of Rh7-Cap reconstituted with 11-cis-retinal. Gq activation abilities were measured in the dark (black line), after UV light irradiation (red line), and after subsequent yellow light irradiation (green line). Data are presented as the means ± S.E. of three independent experiments.