Figure 4
Mutational effects of Asp74 and Asp205 located around the retinal chromophore. (A) Comparison of absorption spectra of wild-type RxR and the D74N and D205N RxR mutants in detergent n-dodecyl-β-D-maltoside (DDM) micelles. Absorption spectra of wild-type RxR and D74N were measured in NaCl solution, whereas those of D205N were measured in NaCl, in Na2SO4 and in no salt solutions. All spectra were normalized at peak absorbance. (B) Time-resolved absorption changes of D75N in NaCl solution at 25 °C; the fitting curve is shown as gray. (C) (upper panel) Light-induced pH changes of suspensions of E. coli cells expressing wild-type RxR and D205N in the absence or presence of the protonophore, CCCP (red and blue, respectively) in Na2SO4 solution. The suspensions were illuminated with yellow light (>480 nm) for 3 min (white background). Images on the right show E. coli cells expressing wild-type RxR and D205N. (lower panel) Quantitative evaluation of the proton pumping activities of wild-type RxR and D205N in Na2SO4 solution. The initial slope amplitudes of the light-induced pH changes, which were obtained from the data in Fig. S4, were normalized against the total amounts of photoactive proteins. All error bars represent the SEM of three independent measurements (n = 3). An asterisk (*) indicates a significant difference from the wild-type RxR (P < 0.05; Dunnett’s test). (D,E) Time-resolved absorption changes of D205N in NaCl (D) and in Na2SO4 solution (E); the fitting curves are shown as gray.
