Researchers quickly discovered that, in some regards, the wild-type protein is not optimal with respect to the demands of typical biological fluorescence imaging applications6. For example, the wild-type protein had evolved to fold and undergo the chromophore-forming reaction most efficiently at the cool temperatures of the northern Pacific Ocean (the habitat of Aequorea victoria)2,6. Engineering of Aequorea green fluorescent protein to fold more efficiently at the physiological temperature of 37 degrees celsius was an early example of how protein engineering could provide substantial improvements in the protein's usefulness with respect to imaging applications6.
Another example of engineering Aequorea green fluorescent protein to be more useful for certain imaging applications involved the introduction of mutations that abolished the tendency of the protein to dimerize at high concentrations12. In fact, in these conditions, AFPs might interact with each other, which results in a false-positive interaction as determined by FRET5. This tendency to dimerize can be reduced greatly or eliminated by mutating the hydrophobic amino acids that are in the dimerization interface to positively charged residues. It would seem prudent to routinely use non-dimerizing mutants when testing protein–protein interactions, or when AFP fusion proteins seem to be causing mis-targeting or dysfunction5.
In addition, variants with improved brightness and photo-stability have been engineered, along with GFP molecules with varying excitation and emission spectra2,6,13. Examples of each of the main color classes include: a blue fluorescent protein known as BFP14,15; a cyan fluorescent variant known as CFP13; a yellow fluorescent variant known as YFP16; a violet-excitable green fluorescent variant known as Sapphire17; and a cyan-excitable green fluorescing variant known as enhanced green fluorescent protein or EGFP18.
As shown in A and B, the blue and cyan fluorescing variants were created through modification of the chromophore structure6. In contrast, the yellow variant retains the Aequorea green fluorescent protein chromophore structure but the fluorescence is red-shifted due to an amino acid substitution that creates a p-p stacking interaction with the chromophore. In the Sapphire and EGFP variants, the chromophore environment has been modified such that the ground state equilibrium is shifted either towards the neutral phenol or anionic phenolate form, respectively6.
Engineered fluorescent protein chromophores not known to occur in nature6: