Fig. 4: Comparison of single-flash and multi-flash experiments of GtACR1 WT.

a GtACR1 amplitude spectra of the reaction after the first flash (black) and the second flash (red). Shown are the reactions from L₂ to M (1st flash T₄ = 44 ms, 2nd flash T₄ = 88 ms), from M to N/O (1st flash T₅ = 150 ms, 2nd flash T₅ = 1438 ms) and from N/O to ground state (1st flash T₆ = 16 s, 2nd flash T₆ = 93 s). Due to the low amplitude of the T₆ spectra and the spectra after the second flash, they have been amplified as indicated. b, c Time course of GtACR1 marker absorption bands after one flash (b) and after the second flash (c). The time-resolved data were obtained from rapid-scan FTIR difference spectroscopic measurements. The band assignment is based on the work by Dreier et al.⁶⁶ assigning the 1644 cm⁻¹ band (light blue) to conformational changes during channel opening and closing in the amide I region, the 1691 cm⁻¹ band (magenta) to channel opening and therefore the conducting state, the 1184 cm⁻¹ band (black) to protonated 13-cis retinal and the 1529 cm⁻¹ band (blue) to the retinal C=C vibrations in the ground state. d GtACR1 amplitude spectra at T₆ of the reaction after one flash (black, t_1/2 = 16 s), two flashes (gray, t_1/2 = 93 s), and five flashes (red, t_1/2 = 69 s). Spectra were scaled to the 1531 cm⁻¹ marker band for all-trans retinal. A reduction in the amplitude of the band at 1708 cm⁻¹ and an amplification in the amplitude at 1691 cm⁻¹ can be observed. e GtACR1 amplitude spectra at T₆ of the reaction after one flash (black, t_1/2 = 16 s) and 30 s of continuous illumination (red, t_1/2 = 68 s). Spectra were scaled to the 1531 cm⁻¹ marker band for all-trans retinal. A reduction in the amplitude of the bands at 1708 cm⁻¹ and 1691 cm⁻¹ can be observed. f GtACR1 amplitude spectra at T₆ of the reaction after one flash (black, t_1/2 = 16 s), two flashes (gray, t_1/2 = 93 s), and five flashes (red, t_1/2 = 69 s). Spectra were scaled to the 1184 cm⁻¹ marker band for protonated 13-cis retinal. A shift towards 1176 cm⁻¹ can be observed with a growing number of flashes. g GtACR1 amplitude spectra at T₆ of the reaction after one flash (black, t_1/2 = 16 s) and 30 s of continuous illumination (red, t_1/2 = 68 s). Spectra were scaled to the 1184 cm⁻¹ marker band for protonated 13-cis retinal. A shift towards 1179 cm⁻¹ can be observed.