| The green-fluorescent protein (GFP) of Aequorea victoria is the ultimate light emitting protein in Aequorea bioluminescence and functions through energy transfer from the photoprotein aequorin. We have found that GFP forms reversible dimers by hydrophobic interactions at elevated protein and salt concentrations and have used this phenomenon to purify GFP in two gel filtration columns. GFP is chromatographed as a monomer (27 kDa) in the first column, but elutes at an apparent molecular weight of 21 kDa due to hydrophobic interactions with the gel matrix. In the second gel filtration step, GFP is chromatographed as a dimer and elutes at an apparent molecular weight of 44 kDa, a shift in molecular weight that allows it to become separated from the low molecular weight contaminants with which it was associated during the first column. Cloned GFP expressed in E. coli was purified by this method to greater than 85% purity and the first 60 amino terminal amino acids were sequenced. Other than the addition of an alanine at the first position in the recombinant and the presence of an acetylated serine in the first position in native GFP, the two have identical sequences. At least nine isoforms of native GFP have been isolated and, through sequencing and mass spectral analysis, the areas of heterogeneity have been elucidated. Protease experiments indicate that the isoforms may arise not only from multiple genes but through proteolysis at the C-terminal. GFP dimerization results in significant absorption spectral changes that have implications in the role of GFP as an energy transfer protein. Circular dichroism data indicate that dimerization of GFP in the presence of salt does not affect secondary structure and that the large spectral perturbations of the chromophore are due to the direct interaction of the salt ions or to the indirect interaction of water molecules as they solvate the protein surface. This is the first report of non-secondary spectral perturbations in the GFP chromophore. We have found that when GFP dimerizes, it generates a 4-fold suppression of the adsorption peak at 470 nm where energy transfer between aequorin and GFP is greatest. Thus, under dimer conditions, which we feel are present in vivo, less than 1% of the emitted light from aequorin would be reabsorbed by GFP in a radiative transfer system. We therefore propose that GFP and aequorin form a complex and transfer energy radiationlessly. Evidence using the Hummel and Dreyer chromatographic technique support this hypothesis as an 87,000 Da GFP/aequorin species has been isolated, suggesting a heterotetramer. |
