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bioRxiv · 10.64898/2026.05.26.727966

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.
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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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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.. 2026-05-29. A chemically reactive and Raman-active non-canonical amino acid reveals photocycle complexity in a blue-light receptor. https://doi.org/10.64898/2026.05.26.727966

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