Sensitive, Site-Specific, and Stable Vibrational Probe of Local Protein Environments: 4‑Azidomethyl‑l‑Phenylalanine

We have synthesized the unnatural amino acid (UAA), 4-azidomethyl-l-phenylalanine (pN3CH2Phe), to serve as an effective vibrational reporter of local protein environments. The position, extinction coefficient, and sensitivity to local environment of the azide asymmetric stretch vibration of pN3CH2Phe are compared to the vibrational reporters: 4-cyano-l-phenylalanine (pCNPhe) and 4-azido-l-phenylalanine (pN3Phe). This UAA was genetically incorporated in a site-specific manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase in response to an amber codon with high efficiency and fidelity into two distinct sites in superfolder green fluorescent protein (sfGFP). This allowed for the dependence of the azide asymmetric stretch vibration of pN3CH2Phe to different protein environments to be measured. The photostability of pN3CH2Phe was also measured relative to the photoreactive UAA, pN3Phe.