Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states

Green fluorescent protein (GFP) from jellyfish Aequorea victoria, the power ful genetically encoded tag presently available in a variety of mutants fea turing blue to yellow emission, has found a red-emitting counterpart. The r ecently cloned red fluorescent protein DsRed, isolated from Discosoma coral s (Matz et al., 1999), with its emission maximum at 583 nm, appears to be t he long awaited tool for multi-color applications in fluorescence-based bio logical research. Studying the emission dynamics of DsRed by fluorescence c orrelation spectroscopy (FCS), it can be verified that this protein exhibit s strong light-dependent flickering similar to what is observed in several yellow-shifted mutants of GFP. FCS data recorded at different intensities a nd excitation wavelengths suggest that DsRed appears under equilibrated con ditions in at minimum three interconvertible states, apparently fluorescent with different excitation and emission properties. Light absorption induce s transitions and/or cycling between these states on time scales of several tens to several hundreds of microseconds, dependent on excitation intensit y. With increasing intensity, the emission maximum of the static fluorescen ce continuously shifts to the red, implying that at least one state emittin g at longer wavelength is preferably populated at higher light levels. In c lose resemblance to GFP, this light-induced dynamic behavior implies that t he chromophore is subject to conformational rearrangements upon population of the excited state.