Superfolder green fluorescent protein (sfGFP) is a 244 residue, ß-barrel protein that has been used in a wide range of applications including as a biosensor for subcellular imaging of organelles and protein complexes. The photophysical properties of sfGFP are primarily the result of a cyclized tripeptide chromophore inside the ß-barrel, consisting of residues T65, Y66, and G67. The protonation state of the central tyrosine 66 residue (Y66) is directly related to the photophysical properties of the proteins - specifically the electronic absorption and emission spectral properties. Here we have replaced this tyrosine residue with several unnatural amino acids (UAAs) that were selected to vary the pKa of the side-chain hydroxyl group to alter the photophysical properties of the protein. The UAAs were site-specifically incorporated with high efficiency and fidelity using the Amber codon suppression methodology. The successful incorporation of these UAAs was verified through time-of-flight mass spectrometry. The altered photophysical properties of these sfGFP constructs in addition to the experimentally determined pKa of the tyrosine hydroxyl group in the protein will be presented in addition to initial efforts to determine the X-ray crystal structures of these constructs.
