Red-shifted mutants of green fluorescent protein: reversible photoconversions studied by hole-burning and high-resolution spectroscopy Original Research Article

Mutants of green fluorescent protein (GFP) are usually designed to absorb and emit light as “one color” systems, i.e. with a single, photostable conformation of the chromophore. We have studied two red-shifted GFP-mutants (S65T and EYFP) by means of hole-burning and high-resolution optical spectroscopy at low temperature, and compare the data to those previously reported for RS-GFP. We prove that these GFP-mutants are not “one color” systems because they can be reversibly phototransformed from one conformation into another. The results are rationalized in terms of energy-level schemes that are similar to that previously derived by us for wild-type GFP. In these schemes, each mutant can be interconverted by light among at least three conformations that are associated with the protonation-state of the chromophore. The results have relevance for the study of protein–protein interactions by fluorescence resonance energy transfer (FRET), where GFP-mutants of different colors are used as labels in donor–acceptor pairs. Furthermore, we present a detailed mechanism that explains the “on–off” and “blinking” behavior of single GFP-molecules with the proposed energy-level diagrams.