Unraveling Complex Protein Environments In Green Fluorescent Protein Using The Unnatural Amino Acid 4-Cyano-L-Phenylalanine

Spectroscopic reporter unnatural amino acids (UAAs) have the potential to serve as effective, site-specific probes of local protein structure and dynamics. Specifically, the vibrational reporter UAA 4-cyano-L-phenylalanine (pCNF) is an effective probe of local protein environments due to the position of the nitrile symmetric stretch, the sensitivity of this stretch, and its small size. Here, pCNF was genetically incorporated individually into three distinct sites of superfolder green fluorescent protein (sfGFP) via the amber codon suppression methodology. Temperature-dependent infrared (IR) spectroscopy was utilized to assess the local solvation environments of each of these sites. One site was found to be fully solvated while a second site was partially solvated. The third site resulted in an IR spectrum whose nitrile IR absorbance band consisted of three subcomponents unlike the other two sites whose nitrile IR absorbance band consisted of a single component. Furthermore, each of the three subcomponents had a unique temperature dependence of the nitrile stretching frequency. Molecular dynamics (MD) simulations, in conjunction with X-ray crystallography, were utilized to identify specific protein-protein or protein-solvent interactions potentially responsible for the three components. Based upon this analysis, mutagenesis was performed surrounding the incorporated UAA. Subsequent temperature-dependent IR characterization of these constructs aided in the determination of the possible molecular origin of the multiple components present in the nitrile IR absorbance band. Data resulting from these constructs will be presented.