ABSTRACT
The green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria (avGFP) has revolutionized microscopy and cell biology. After the cloning of the gene (Prasher et al. 1992) and the demonstration that no specific jellyfish cofactor is needed for its production in other species (Chalfie et al. 1994), GFP soon became the most popular fluorescent tag in life sciences research. Starting in 1994, many research groups have worked on mutagenizing the GFP gene to isolate enhanced or spectroscopically altered variants of GFP. Besides random mutagenesis approaches, the crystal structure published in 1996 (Ormö et al. 1996) also allowed rationalized mutagenesis approaches. The crystal structure demonstrated that GFP consists of an outer β-barrel that is built up by 11 β-sheets. A short α-helix, to which the chromophore is attached, is positioned inside the β-barrel. The avGFP chromophore is formed via an autocatalytic reaction involving three consecutive amino acids: Ser65, Tyr66, and Gly67. Cyclization (imidazolinone formation) of the chromophore occurs by a nucleophilic attack of the amide of Gly67 (which is the best nucleophile because of the minimal steric hindrance) on the carbonyl of residue 65, followed by a dehydration step. The imidazolinone-conjugated system is then connected to the aromatic group by the oxidation of the α−β bond of residue 66 (Tsien 1998). Because the chromophore is buried inside the β-barrel, it is well protected from the extracellular environment, and GFP therefore shows remarkably similar spectroscopic properties in cells, in buffer solutions after purification, or even in protein crystals after crystallization (Chudakov et al. 2010).
