Figure 2.

Regeneration rates of wild-type and mutant rhodopsins. (A) Regeneration of wild-type, E122Q, and E122I rhodopsins monitored by change of absorbance at 530 nm. 11-cis-Retinal solution (2.5 nmol) in ethanol (5 μl) was added to the respective opsin solution (220 μl) at 2°C, and increase of absorbance at 530 nm due to the regeneration of pigment was recorded. The maximal absorbance due to the full regeneration is normalized. Solid curves are the fitted single exponential curves with time constants of 26 (wild type), 1.3 (E122Q), and 0.94 (E122I) min, respectively. (B) Rate constants of pigment regeneration in wild-type and mutant rhodopsins and native chicken rhodopsin (Rh), chicken green (cG), and chicken red (cR) (Inset). Rate constants of wild-type and native rhodopsins were normalized to 1, and the rate constants of mutant rhodopsins and native cone pigments are represented relative to those of their respective rhodopsins. The standard deviations were estimated from three independent experiments using different preparations.