The green fluorescent protein: Roles of conserved residues in chromophore maturation

The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea Victoria has come to be a routinely used tool for the molecular and cellular biologist. However, much still remains unknown about the rare post-translational modification that results in the chromophore that gives GFP its characteristic green fluorescence. Here, a mutational investigation was set forth in order to gain insight into this unusual protein chemistry, and the R96K, R96M, E222Q, and G67A variants were made via site-directed mutagenesis in the enhanced green fluorescent protein (EGFP).; Limited proteolysis experiments among the different pools of the EGFP variants were carried out in order to investigate whether there was a correlation between overall protein stability and efficiency of generating the chromophore. Trypsin resistance among the EGFP, EGFP-R96K and EGFP-E222Q variants were all very similar, while the percentage of chromophore contained in each protein pool varied significantly. Therefore, the results show only a weak correlation between the efficiency of chromophore maturation and relative protein stability.; Spectral characterization of the EGFP variants show a blue shift in absorbance for the anionic form of the chromophore from 489 nm to 470 nm. This shift is consistent with a decrease in charge delocalization over the entire chromophore network. Additionally, the pKa of the chromophores were measured for the variants. Among the Arg variants, there is a rise in chromophore pK a, from that of EGFP (6.0), consistent with a weakened interaction with a stabilizing positive charge near the chromophore. The EGFP-E222Q variant showed a decrease in chromophore pKa (5.3), consistent with the elimination of a charge repulsion interaction with the chromophore anion.; The EGFP-R96M and EGFP-E222Q variants were used to study chromophore maturation in a pH-dependent manner, and base catalysis was observed in both proteins. The base catalyzed rate-enhancement in EGFP-E222Q was most pronounced, with a 600-fold increase over the pH range of 6--10, and that for the EGFP-R96M variant was 2.7 fold. This, along with MALDI mass spectrometry data for chromophore containing peptides support a role in which Glu222 and Arg96 act as catalysts in the cyclization step of chromophore formation.