Unraveling Complex Local Protein Environments with 4-Cyano-l-phenylalanine.

We present a multifaceted approach to effectively probe complex local protein environments utilizing the vibrational reporter unnatural amino acid (UAA) 4-cyano-l-phenylalanine (pCNPhe) in the model system superfolder green fluorescent protein (sfGFP). This approach combines temperature-dependent infrared (IR) spectroscopy, X-ray crystallography, and molecular dynamics (MD) simulations to provide a molecular interpretation of the local environment of the nitrile group in the protein. Specifically, a two-step enantioselective synthesis was developed that provided an 87% overall yield of pCNPhe in high purity without the need for chromatography.