Despite decades of green fluorescent protein (GFP) research, a recent review states “there remains substantial room for improvement” of fluorescent proteins. In that spirit, we have undertaken a project to introduce site-directed mutations in stave 11 of GFP, at residues expected to have a strong influence on the chromophore. Specifically, we have targeted L220, E222 and V224 of the cycle 3 version of GFP (GFPuv), which are located 6.9, 3.5 and 6.6 Angstrom units, respectively, from the chromophore. It is well known that changing E222 affects both the maturation rate and the degree of protonation of the chromophore, with E222Q being the most common mutation. In our hands, all non-acidic mutations at E222 alone give maturation rates which are not useful, as judged by consistently non-fluorescent phenotypes. As a result, we endeavored to increase the acidity of the chromophore environment (and presumably the maturation rate) by introducing either E or D at positions 220 and 224, with no change at E222. We find that only V224E gives fluorescent colonies, and, in contrast to expectations, the isolated protein continues to mature long after it is purified. Thus, an additional acidic amino acid near the chromophore in this case slows down maturation, and gives us the opportunity to study the maturation conveniently by isolating the protein anaerobically, followed by monitoring fluorescence growth after addition of a variety of oxidants (O2, H2O2, I2, etc.). Isolation of a relatively non-fluorescent GFP which then “turns on” in the presence of oxidants has clear analytical applications. The E222D mutation gives fluorescent colonies, but in this case the proportion of the phenolic form of the chromophore is increased, suggesting an increase in the acidity of the chromophore environment, despite their being only a single CH2 group difference between E and D. Here again, however, the maturation rate is slowed down, showing there is a subtle interplay between maturation rate vs. the degree of protonation of the chromophore as a function of acidity. Experiments with stave 11 mutations in addition to V224E are presented, and the analytical and mechanistic implications of these new findings are discussed.

Acidic GFP stave 11 mutations slow down chromophore maturation

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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