Figure 3.

pH variation of the optical absorption spectra of mIrisGFP in the dark-adapted state (cis chromophore). (A) Absorption spectra in the pH range 3.6–10.4. Arrows point in the direction of increasing pH. (B) Peak absorption of the A_C (open circles) and B_C (solid circles) bands. Solid lines show the fit of the model depicted in panel c in the pH range 3.6–8. An additional protonation step described by the Henderson-Hasselbach equation was added to fit the B_C band amplitudes at pH >8. Dotted line shows one-site protonation with pK = 5.3. Open triangles indicate pH dependence of the reaction rate coefficient, k_off(473), for off-switching of the fluorescence with 473-nm light. (C) Four-state model describing the protonation equilibrium of the chromophore, C, interacting with a titratable amino acid, X. (D) pH dependence of the peak positions of the A_C (open circles) and B_C (solid circles) absorption bands of mIrisGFP, as well as the peak position of B_C of the Glu²¹²Gln mutant (stars).