Background: Variants of the green fluorescent protein (GFP) with different colors would be very useful for simultaneous comparisons of multiple protein fates, developmental lineages and gene expression levels. The simplest way to shift the emission color of GFP is to substitute histidine or tryptophan for the tyrosine in the chromophore, but such blue-shifted point mutants are only dimly fluorescent. The longest wavelengths previously reported for the excitation and emission peaks of GFP mutants are 488 and 511 nm, respectively.
Results: Additional substitutions, mainly in residues 145-163, have improved the brightness of the blue-shifted GFP mutants with histidine and tryptophan in place of tyrosine 66. Separate mutations have pushed the excitation and emission peaks of the most red-shifted mutant to 504 and 514 nm, respectively. At least three different colors of GFP mutants can now be cleanly distinguished from each other under the microscope, using appropriate filter sets. A fusion protein consisting of linked blue- and green-fluorescent proteins exhibits fluorescence resonance energy transfer, which is disrupted by proteolytic cleavage of the linker between the two domains.
Conclusions: Our results demonstrate that the production of more and better GFP variants is possible and worthwhile. The production of such variants facilitates multicolor imaging of differential gene expression, protein localization or cell fate. Fusions between mutants of different colors may be useful substrates for the continuous in situ assay of proteases. Demonstration of energy transfer between GFP variants is an important step towards a general method for monitoring the mutual association of fusion proteins.