h l D

chromophore is formed via the mTagBFP-like blue the DsRed chromophore can be formed from a blue protonated chromophore intermediate with an absorbance maximum atanionic conjugate of imidazolone with p-hydroxybenzylidene (Ormo ̈ et al., 1996). Chromophore protonation results in an absorbance shift to 400 nm (Brejc et al., 1997). Blue and Fluorescent Timers, which change fluorescence color from blue to red over time, have been developed on the basis of mCherry (Subach et al., 2009), and their crystal structures havecyan color variants of GFP contain the substitutions of tyrosine in the chromophore with histidine and tryptophan (Heim and Tsien, 1996; Heim et al., 1994). In red tyrosine-based chromo-been determined (Pletnev et al., 2010). On the basis of all thesedata,we hypothesized that a formation pathway for a red chromophore via a blue intermediate should beintermediate