Spectral versatility of single reef coral fluorescent proteins detected by spectrally-resolved single molecule spectroscopy

We use spectrally-resolved room temperature single molecule spectroscopy to yield insights into the occurrence and dynamics of spectral forms of single tetramers of DsRed and its variants DsRed2, Fluorescent Timer, DsRed_N42H and AG4. The red-emitting chromophore in DsRed and all studied variants readily converts into a high quantum efficiency super-red emitting form. We propose the existence of two super-red forms of different quantum efficiencies. The observed emission from the green-emitting chromophore is consistent with bulk spectroscopy. We further observe distinct new spectral forms from each variant, which we attribute to a photoinduced chemical reaction leading to a truncated form of the red-emitting chromophore analogous to the chromophore in the visible fluorescent protein zFP538. Our results have implications for the accurate interpretation of biological and biochemical processes illuminated by fluorescent proteins as well as for choosing appropriate experimental configurations.