Recent advances in our understanding of the mechanism of chromophore formation in green fluorescent protein (GFP) are presented. GFP is the best-studied member of the family of GFP-like proteins, proteins that exhibit bright coloration spanning most of the visible spectrum. GFPs undergo a post-translational self-modification process that yields an intrinsic fluorophore constructed from an internal main-chain cross-link that is susceptible to air oxidation. A combination of protein X-ray crystallographic and kinetic experiments has led to the development of a mechanistic model that entails conformational pre-organization, electrophilic and base catalysis, and production of hydrogen peroxide upon protein oxidation. The process is concluded by a slow proton abstraction step from a tyrosine-derived carbon acid.
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