Probing Local Environments with the Infrared Probe: L-4-Nitrophenylalanine

The genetic incorporation of unnatural amino acids (UAAs) with high efficiency and fidelity is a powerful tool for the study of protein structure and dynamics with site-specificity in a relatively nonintrusive manner. Here, we illustrate the ability of L-4-nitrophenylalanine to serve as a sensitive IR probe of local protein environments in the 247 residue superfolder green fluorescent protein (sfGFP). Specifically, the nitro symmetric stretching frequency of L-4-nitrophenylalanine was shown to be sensitive to both solvents that mimic different protein environments and N-15 isotopic labeling of the three-atom nitro group of this UAA. (NO2)-N-14 and (NO2)-N-15 variants of this UAA were incorporated utilizing an engineered orthogonal aminoacyl tRNA synthetase/tRNA pair into a solvent exposed and a partially buried position in sfGFP with high efficiency and fidelity. The combination of isotopic labeling and difference FTIR spectroscopy permitted the nitro symmetric stretching frequency of L-4-nitrophenylalanine to be experimentally measured at either site in sfGFP. The (NO2)-N-14 symmetric stretching frequency red-shifted 7.7 cm(-1) between the solvent exposed and partially buried position, thus illustrating the ability of this UAA to serve as an effective IR probe of local protein environments.