Time-resolved emission spectra of green fluorescent protein

The time-resolved emission spectra of wild-type green fluorescent protein (wtGFP) and the T203V GFP mutant have been recorded with picosecond time resolution, allowing the separate characterization of the two spectral components associated with the neutral and anionic forms of the GFP chromophore. Significantly, neither component shifts as a function of time. It is suggested that the absence of spectral shift is a result of highly restricted movement of the protein residues in the vicinity of the chromophore. The shapes of the separated spectra are discussed and their relative ratio analyzed in a steady-state analysis.