Site-specific vibrational spectroscopy with non-canonical amino acids containing frequency-resolved labels

Unnatural protein side-chains with frequencies within the “transparent window” spectral region (∼1800 - 2700 cm−1), where no native protein vibrations are found, can single out specific residues in vibrational spectra. These moieties contain most often triple bonds, like nitriles (R-C≡N), which can be conveniently attached to proteins in the form of genetically encoded non-canonical amino acids (ncAA) e.g. 4-cyanophenylalanine (CNF). However, the full potential of ncAA as vibrational reporters has not been exploited yet, particularly for Raman spectroscopy. Here we show how CNF residues introduced at multiple positions of the bacterial transcription factor EL222 provide a detailed picture of local changes along the photocycle. Time-resolved infrared spectra in the CNF absorption region disclose two additional kinetic events not sensed by the native probes, one occurring before flavin chromophore relaxation and the other after protein backbone relaxation. In addition, we incorporate an ncAA carrying a diacetylene (R-C≡C-C≡CH) via genetic code expansion technology into EL222 variants. The conjugated diyne features an intense Raman signal which is sensitive to the microenvironment around the triple bonds. We then measure light-induced conformational changes in the flavoprotein EL222 with single-residue precision. Our results suggest that vibrational spectroscopy assisted by ncAA can reveal protein structural dynamics residue-by-residue.