The Nature of Transient Dark States in a Photoactivatable Fluorescent Protein

Fluorescent proteins (FPs) of the green fluorescent protein family blink and bleach like all fluorophores. However, contrary to organic dyes, the mechanisms by which transient losses of fluorescence occur in FPs have received little attention. Here, we focus on the photoactivatable IrisFP, for which a transient non-fluorescent chromophoric state with distorted geometry was recently reported (Adam, V.; et al. J. Am. Chem. Soc. 2009, 131, 18063). We investigated the chemical nature of this blinked state by employing quantum chemical/molecular mechanical calculations. Our findings suggest two previously unidentified dark states that display similar distorted chromophores with a transiently ruptured pi-electron system. Both are protonated at atom C-alpha of the chromophore methylene bridge. Transient protonation may occur via proton transfer from the nearby Arg66 either in the triplet state T-1 after intersystem crossing or in an anionic radical (doublet) ground state. As Arg66 is conserved in green-to-red photoconvertible FPs, these dark states are predicted to be common to all these proteins. We also suggest that C-alpha protonated dark states may accelerate photobleaching by favoring decarboxylation of the fully conserved Glu212.