Figure 1.

Spectral, biochemical and photochemical properties of the purified PAmCherry variants. (a) Absorbance, excitation and emission spectra of PAmCherry1 before (pre) and after (post) photoactivation with the 399 nm laser line. (b) Maturation kinetics for the indicated proteins at 37 °C. (c) Equilibrium pH dependence for the red fluorescence of the indicated proteins. (d) Rate and efficiency of PAmCherry1 photoactivation at different physiological pH values, normalized to the fluorescence intensities at the respective pH (data shown in c). (e,f) Photoactivation (e) and photobleaching (f) kinetics for the indicated proteins using arc lamp illumination through ×60 oil objective with 390/40 nm (e) and 570/30 nm (f) filters. (g–i) Cycling illumination of PAmCherry1, rsCherry and rsCherryRev using an arc lamp, ×100 oil objective, 570/30 filter for 12 s and one of the following filters: 390/40 nm for 0.6 s (g), 436/20 for 0.5 s (h) and 480/40 for 0.4 s (i). The maxima of the fluorescence signals shown in g–i were normalized per the relative brightness for each protein indicated in Table 1 to achieve 100%. The ground states of the proteins are indicated with the respective symbols.