Green-fluorescent protein mutants with altered fluorescence excitation spectra

Using random mutagenesis and visual selection of fluorescent clones, we have isolated a T203I and a E222G mutant of the Aequorea green-fluorescent protein. Each mutant has one of the two fluorescence excitation bands of the wild type deleted and retains the other without a wavelength shift. This finding is consistent with each excitation band corresponding to a distinct spectroscopic state of the chromophore. Both mutations are single amino acid exchanges which in the linear sequence are located remotely from the chromophore but in the folded protein may be situated in its vicinity. We conclude that the mutations influence the fluorescence properties by changing the interactions between the chromophore and its protein environment.