A chemically reactive and Raman-active non-canonical amino acid reveals photocycle complexity in a blue-light receptor
Chatterjee, A.; Pham, P. N.; Mukherjee, A.; Cubakova, P.; Kaziannis, S.; Dostal, J.; Kloz, M.; Chaudhari, A. S.; Finocchiaro, G.; Springer, T.; Homola, J.; Honc, O.; Kizovsky, M.; Safarik, M.; Stursa, J.; Werner, L.; Sebestik, J.; Fuertes, G.
Show abstract
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 Forster 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.
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