GFP Maturation Visualized♦

♦ See referenced article, J. Biol. Chem. 2010, 285, 15978–15984

Although green fluorescent protein (GFP) and its relatives have become extremely valuable experimental tools, recently a group of related, but colorless, GFP-like proteins have also been identified. These colorless relatives, like the jellyfish Aequorea coerulescens GFPL, can be made fluorescent by mutating conserved residues and thus can serve as models for understanding the maturation of GFP side chains into functional chromophores. In this Paper of the Week, Nadya Pletneva and colleagues have solved high resolution crystal structures of three different acGFPL mutant species: the green fluorescent aceGFP (five mutations to acGFPL), the colorless aceGFP-G222E (which contains an immature chromophore), and UV-stimulated aceGFP-G222E (which photoconverts the chromophore back to a green fluorescent state). The structures showed that aceGFP-G222E chromophore was trapped in an intermediate state as the important Tyr-66 had not yet oxidized. Pletneva and colleagues hypothesized that the nearby presence of Tyr-220 pushed Glu-222 away from Tyr-66 to prevent the required co-planar arrangement, and indeed mutating Tyr-220 to leucine resulted in a mature chromophore and GFP-like spectral properties without the need of UV photoconversion. Interestingly, the authors also noted that in regular aceGFP (containing Gly-222) the chromophore maturation may occur through an alternate reaction using Tyr-220, suggesting that the environment of the chromophore may not only influence fluorescence properties but also the maturation pathway.

Illustration of the chromophore in aceGFP-G222E (yellow) and avGFP (green), highlighting the extensive shift of the Glu-222 side chain in aceGFP-G222E relative to its conventional position.