Figure 5
Spectral analyses of Rh7 K90E mutant. (A) Absorption spectra of Rh7-Cap K90E mutant. Absorption spectra of K90E mutant purified after it was regenerated with 11-cis-retinal were measured in the dark (black curve), after blue light (450 nm) irradiation (red curve), after subsequent orange light (>550 nm) irradiation (green curve), after blue light re-irradiation (blue curve), and after orange light re-irradiation (violet curve). (B) Difference spectra of Rh7-Cap K90E mutant calculated based on the spectra in (A). The red curve was calculated by subtracting the spectrum before irradiation from that after blue light irradiation. The green curve was calculated by subtracting the spectrum after blue light irradiation from that after subsequent orange light irradiation. The blue curve was calculated by subtracting the spectrum after orange light irradiation from that after blue light re-irradiation. The violet curve was calculated by subtracting the spectrum after blue light re-irradiation from that after orange light re-irradiation. (C) The calculated absorption spectra of Rh7-Cap K90E mutant. Difference spectrum of Rh7-Cap K90E mutant (green curve of (B)) was fitted with a template spectrum of squid retinochrome modeled by the Lamb and Govardovskii method to calculate the absorption spectrum of its all-trans-retinal bound form. Spectrum of 11-cis-retinal bound forms was determined by subtracting the difference spectrum in (B) from the calculated spectrum of all-trans-retinal bound form. Spectra of the dark (curve 2) and irradiated (curve 4) states of Rh7-Cap K90E mutant were compared with those of Rh7-Cap (curves 1 and 3).
