pH dependence of the on-switching kinetics (trans-cis isomerization). (A) Thermally activated recovery of the anionic cis chromophore with rate coefficient, kth, measured at T = 290 K (open circles) and T = 310 K (solid circles). Stars, mIrisGFP Glu212Gln at T = 310 K; solid line, two-site protonation with pKa1 = 7.8 and pKa2 = 9.5; dotted line, one-site protonation with pK = 7.8; dashed line, one-site protonation with pK = 10.3. (B) Photoactivated trans-cis isomerization: kon(405) for P405 = 13 mW cm−2. Solid line shows one-site protonation with pK = 7.8.