Flavin-tag: A Facile Method for Site-Specific Labeling of Proteins with a Flavin Fluorophore

Site-specific protein labeling methods are highly valuable tools for research and applications. We present a new protein labeling method that allows covalent attachment of a chromo- and fluorogenic flavin (FMN) to any targeted protein using a short flavinylation peptide-tag. We show that this peptide can be as short as 7 residues and can be located at the N-terminus, C-terminus, or in internal regions of the target protein. Analogous to kinase-catalyzed phosphorylation, the flavin is covalently attached via a stable phosphothreonyl linkage. The site-specific covalent tethering of FMN is accomplished by using a bacterial flavin transferase. The covalent coupling of FMN was shown to work in Escherichia coli and Saccharomyces cerevisiae cells and could be performed in vitro , rendering the "Flavin-tag" method a powerful tool for the selective decoration of proteins with a biocompatible redox-active fluorescent chromophore.