Red fluorescent proteins (RFPs) are indispensable tools for deep tissue imaging, fluorescence resonance energy transfer (FRET), and super-resolution microscopy. Recent time-resolved spectroscopy studies revealed a new intermediate formed on sub-�s timescale and decaying on �s-ms timescale. This intermediate is characterized by red-shifted absorption at 1.68-1.72 eV (?max=720-740 nm). The intermediate was found to be common for at least three members of RFP family (DsRed, mRFP, and KillerRed). Based on the results of electronic structure calculations we assign the new specie to an unusal open-shell dianionic chromophore (dianion-radical) formed via photoreduction. Doubly charged state that is not stable for the isolated (gas-phase) chromophore is stabilized by the electrostatic field of the protein. Mechanistic implications for photobleaching, blinking, and phototoxicity are discussed.

Chromophore photoreduction in red fluorescent proteins and its role in bleaching and phototoxicity

247th National Meeting (2014)

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