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

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 obtained in 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.