A chemically reactive and Raman-active non-canonical amino acid reveals photocycle complexity in a blue-light receptor
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Photosensory protein function spans multiple time and length scales, demanding integrative approaches. We introduce 4-diacetylenyl-phenylalanine (DAF), a dual-purpose non-canonical amino acid (ncAA) that enables both chemical control and spectroscopic readout of photoreceptor dynamics. Genetically encoded in E. coli , DAF combines a reactive diyne for bioorthogonal ligations (thiols, azides, tetrazines) with a strong, solvatochromic Raman signal in the cell-silent region. Applied to the light-oxygen-voltage (LOV) transcription factor EL222, DAF enables multifaceted interrogation of its photocycle. We engineer a covalently cross-linked variant that suppresses light-driven conformational changes and DNA binding, and generate a donor–acceptor construct for Förster resonance energy transfer (FRET) tracking of photoinduced structural dynamics. Time-resolved stimulated Raman spectroscopy following flavin mononucleotide (FMN) excitation reveals additional processes -from vibrational energy transfer to local unfolding-spanning femtoseconds to milliseconds. DAF thus constitutes a versatile tool to resolve protein dynamics with high spatiotemporal resolution.
