Fig. 2: Monitoring real-time conversion of rho to opsin in detergent micelles and in native membranes, probing the effects of pulses of light and confirming the formation of N-ret-PE.

a, b, Changes in the population of rho and opsin in LMNG (blue and green, respectively; a) and in native membranes (red and yellow, respectively; b). Individual spectra are shown as zero-charge plots with illumination times stated. c, Plot of the relative abundance of rho during illumination, in either LMNG detergent micelles (blue) or native membranes (red) monitored as a change in mass as a function of time. Rate constants for hydrolysis are shown for the reaction in detergent micelles or in membranes (Extended Data Fig. 3). d, Schematic of the dark-adapted state undergoing light-activated conversion of cis-retinal to all-trans-retinal, hydrolysis of the Schiff base and dissociation from rho to form opsin. e, Monitoring the decay of cis-retinal rho in membranes pretreated with hydroxylamine (red), the generation of opsin in native membranes (grey) and the change of rho* calculated over the illumination period (orange). The inset, which is an expansion of the illumination period from 0–18 s, shows a less than 18-s increase in rho abundance at the expense of opsins. The schematic depicts a possible regeneration mechanism through isomerization of trans-retinal. f, Monitoring the conversion of rho to opsin and the regenerative reaction of opsin to rho following pulses of light at 0.51 min, 7 min and 35 min for time periods of 3 s, 12 s and 32 s, respectively. Zero-charge mass spectra are shown at time points during this conversion reaction, before illumination (dark) and at the peak of the three light pulses. g, Spectrum (orange) of N-ret-PE (18:0/18:1 representative structure shown) extracted from native membranes; the bar graph shows changes in the equilibrium between conjugated and free PE in response to illumination time. Data are presented as mean ± s.d. (n = 3).