Extended Data Fig. 7: Influence of lutein or fucoxanthin on the retinal photoisomerization in HeimdallR1 at 77 K.

UV-visible (a, b) and FTIR (c-h) spectra obtained for lipid-reconstituted HeimdallR1 with (red; H(F)) or without (black; H(-)) fucoxanthin (top) and HeimdallR1 with (red; H(L)) or without (black; H(-)) lutein (middle) are compared to those for lipid-reconstituted Kin4B8-xanthorhodopsin with (green; K(L)) or without (black; K(-)) lutein (bottom) previously published⁷. a, UV-visible absorption spectra of H(F) (red) or H(-) (black) (top), H(L) (red) or H(-) (middle), and K(L) (green) or K(-) (black) (bottom) at 77 K. One division of the y-axis corresponds to 1.0 absorbance units. b, Difference UV-visible spectra upon illumination of H(F) (red) or H(-) (black) (top), H(L) (red) or H(-) (black) (middle), and K(L) (green) or K(-) (black) (bottom) at 77 K. Hydrated films of lipid-reconstituted protein were illuminated at 540 nm light, which forms the red-shifted K intermediate. One division of the y-axis corresponds to 0.08 absorbance units. c, Light-minus-dark difference FTIR spectra upon illumination of H(F) (red) or H(-) (black) (top), H(L) (red) or H(-) (black) (middle), and K(L) (green) or K(-) (black) (bottom) at 77 K. Hydrated films of lipid-reconstituted protein with H₂O were first illuminated at 540 nm light (solid lines), which forms the K intermediate, and the K intermediate was then reverted by illumination at >590 nm light (dotted lines). Spectral acquisition was repeated to improve signal-to-noise ratio. Positive and negative signals originate from the K intermediate and unphotolyzed state, respectively. One division of the y-axis corresponds to 0.0032 absorbance units. d, Enlarged spectra of the C = C stretching frequency region of the retinal chromophore (1600-1450 cm⁻¹) from (c). Negative peaks are different between H(F) (1534 cm⁻¹) and H(-) (1532 cm⁻¹) (top) or between H(L) (1534 cm⁻¹) and H(-) (1532 cm⁻¹) (middle), but not for K(L) and K(-). One division of the y-axis corresponds to 0.003 absorbance units. e, Enlarged spectra of the C-C stretching frequency region of the retinal chromophore (1250-1150 cm⁻¹) from (c). Spectra are identical with and without xanthophylls. One division of the y-axis corresponds to 0.002 absorbance units. f, Enlarged spectra of the hydrogen out-of-plane (HOOP) vibrational region of the retinal chromophore (1020-945 cm⁻¹) from (c). Positive peaks are different between H(F) (986 cm⁻¹) and H(-) (985 cm⁻¹) (top) or between H(L) (986 cm⁻¹) and H(-) (985 cm⁻¹) (middle), but not for the 960-cm⁻¹ band (bottom). One division of the y-axis corresponds to 0.0015 absorbance units. g, Spectral comparison of the HOOP bands between H₂O (red or green) and D₂O (blue) hydrations. Down-shifts of the positive peaks at 986 cm⁻¹ (top, middle) and 960 cm⁻¹ (bottom) show that these HOOP bands originate from the Schiff base region. One division of the y-axis corresponds to 0.0015 absorbance units. h, Enlarged spectra of the amide-I region of the peptide backbone (1680-1575 cm⁻¹) from (c). Spectra are identical with and without xanthophylls. One division of the y-axis corresponds to 0.001 absorbance units.

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