Discovery of excited state proton transfer in flavin-based fluorescent protein with large Stokes shift

Nikolaev, A.; Markeeva, E.; Maksimov, E. G.; Yudenko, A.; Tropina, E. V.; Boldyrev, K. N.; Nikolaeva, Y. S.; Cherepanov, D. A.; Kovalev, K.; Yang, Y.; Borshchevskiy, V. I.; Kuznetsova, E.; Kuzmin, A.; Semenov, O.; Remeeva, A.; Gushchin, I.

doi:10.1101/2025.06.07.658458

bioRxiv · 10.1101/2025.06.07.658458

Discovery of excited state proton transfer in flavin-based fluorescent protein with large Stokes shift

Nikolaev, A.·Markeeva, E.·Maksimov, E. G.·Yudenko, A.·Tropina, E. V.·Boldyrev, K. N.·Nikolaeva, Y. S.·Cherepanov, D. A.·Kovalev, K.·Yang, Y.·Borshchevskiy, V. I.·Kuznetsova, E.·Kuzmin, A.·Semenov, O.·Remeeva, A.·Gushchin, I.

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Abstract

Flavin-binding proteins (flavoproteins) are widespread in nature, revealing versatile oxidation-reduction reactions and photochemistry. Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics, as well as fluorescent markers and photogenerators of reactive oxygen species. Despite extensiev efforts, all currently used LOV-derived proteins have similar absorption spectra with maxima around 275, 35-0375, and 450-485 nm. Here, we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein, LSSFbFP, which can be obtainedin vivo and in vitro, with absorption maxima at 340-350 and 395-405 nm. Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ~500 nm. We sho that the protein binds lumichrome as the chromophore and use low temperature and time-resolved spectroscopy, X-ray crystallography and modeling to prove that the apparent Stokes shift of LSSFbFP occurs due to excited state proton phenomena observed in flavoproteni s and pave the way for engineering of new flavin-based molecular instruments.

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Nikolaev, A., Markeeva, E., Maksimov, E. G., Yudenko, A., Tropina, E. V., Boldyrev, K. N., Nikolaeva, Y. S., Cherepanov, D. A., Kovalev, K., Yang, Y., Borshchevskiy, V. I., Kuznetsova, E., Kuzmin, A., Semenov, O., Remeeva, A., Gushchin, I.. 2025-06-08. Discovery of excited state proton transfer in flavin-based fluorescent protein with large Stokes shift. https://doi.org/10.1101/2025.06.07.658458

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