Characterization of LOV domain-ribityllumazine complex with blue-shifted fluorescence.

Flavin-based fluorescent proteins (FbFPs) are small tags derived from natural LOV domains that can be used for anaerobic imaging. However, their applications are limited due to absence of strongly color-shifted variants. Here, we explore the possibility of reconstituting an FbFP with 6,7-dimethyl-8-ribityllumazine (LUM), an endogenous biosynthetic precursor of riboflavin (RF). We show that CagFbFP binds LUM with low-micromolar affinity (Kd = 9.5 ± 1.1 μM), forming a fluorescent complex with the excitation maximum at 419 nm and emission maximum at 462 nm. Under blue-light irradiation, CagFbFP-bound LUM is converted irreversibly to 6,7-dimethyllumazine, whereas the more traditional chromophore RF undergoes reversible reduction to hydroquinone form. Crystal structure of the CagFbFP-LUM complex determined at the anisotropic resolution reaching 1.27 Å confirms that the chromophore occupies the canonical flavin-binding pocket. The obtained results pave the way towards engineering of LUM-based FbFP variants with blue-shifted fluorescence emission.