Investigations of combinations of mutations in the jellyfish green fluorescent protein (GFP) that afford brighter fluorescence, and use of a version (VisGreen) in plant, bacterial, and animal cells.

Among the GFPs used for imaging green fluorescence, the Emerald version has been considered the best GFP to use but there is no formal report on its construction or the relevance of the amino acid (aa) substitutions in it relative to the commonly used GFPs. Here, we have shown that a version of Emerald makes Escherichia coli host cells visibly green even under dim room light conditions. Exploiting this feature, we have determined for the first time whether the changes in the structure of Emerald protein brought about by the aa substitutions are all indeed essential for brightness. F64L and S72A accompanying the classical S65T substitution on the chromophore-bearing helix are essential. Two amino acid changes, one on the surface (N149K) of the beta barrel that encases the helix and the other (I167T) near the chromophore enhance the visible green colour individually and additively when present together. The other two substitutions, M153T (on the surface) and H231L (on the surface), do not contribute to the visible green phenotype, even though in earlier studies M153T has been reported to enhance GFP fluorescence. The GFP version with F64L-S65T-S72A-N149K-I167T is referred to as VisGreen. We found VisGreen and Emerald to be indistinguishable in their quantum yield, molar extinction coefficient, folding efficiency, or photosensitivity. VisGreen rendered bacterial, plant, and animal cells highly fluorescent. Interestingly, N149K in the above combination was not essential to render bacterial cells highly fluorescent.