Figure 4.

pH dependence of optical spectra of mIrisGFP in the dark-adapted state (cis chromophore) (A–C) and immediately after 473-nm illumination (predominantly trans chromophore) (D–F). (A) Absorption, excitation, and emission spectra (solid, dotted, and dashed lines, respectively). Excitation and emission spectra were measured with emission at 540 nm and excitation at 390 nm. (B) CD spectra. (C) pH dependence of the (normalized) peak amplitudes of the A_C (open circles) and B_C (solid circles) absorption bands and of the emission intensity (triangles). (D and E) Absorption spectra and CD spectra, respectively. Arrows indicate increasing pH. (F) Normalized peak amplitudes of the A (open circles) and B (solid circles) bands and normalized peak shift of the A band in D (solid diamonds) and E (open diamonds) and of mutant Glu²¹²Gln (stars).