Shedding light on the dark and weakly fluorescent states of green fluorescent proteins

Recent experiments on various similar green fluorescent protein (GFP) mutan ts at the single-molecule level and in solution provide evidence of previou sly unknown short- and long-lived "dark" states and of related excited-stat e decay channels. Here, we present quantum chemical calculations on cis-tra ns photoisomerization paths of neutral, anionic, and zwitterionic GFP chrom ophores in their ground and first singlet excited states that explain the o bserved behaviors from a common perspective. The results suggest that favor able radiationless decay channels can exist for the different protonation s tates along these isomerizations, which apparently proceed via conical inte rsections. These channels are suggested to rationalize the observed dramati c reduction of fluorescence in solution. The observed single-molecule fast blinking is attributed to conversions between the fluorescent anionic and t he dark zwitterionic forms whereas slow switching is attributed to conversi ons between the anionic and the neutral forms. The predicted nonadiabatic c rossings are seen to rationalize the origins of a variety of experimental o bservations on a common basis and may have broad implications for photobiop hysical mechanisms in GFP.