Abstract
We report a new method to quantify the affinity of small molecules for proteins. This method is based on Förster resonance energy transfer (FRET) between endogenous tryptophan (Trp) residues and the coumarin-derived fluorophore Pacific Blue (PB). Tryptophan residues are frequently found in proteins near ligand-binding sites, making this approach potentially applicable to a wide range of systems. To improve access to PB, we developed a scalable multigram synthesis of this fluorophore, starting with inexpensive 2,3,4,5-tetrafluorobenzoic acid. This route was used to synthesize fluorescent derivatives of biotin, as well as lower affinity thiobiotin, iminobiotin, and imidazolidinethione analogues that bind the protein streptavidin. Compared with previously published FRET acceptors for tryptophan, PB proved to be superior in both sensitivity and efficiency. These unique properties of PB enabled direct quantification of dissociation constants (Kd) as well as competitive inhibition constants (Ki) in the micromolar to nanomolar range. In comparison to analogous binding studies using fluorescence polarization, fluorescence quenching, or fluorescence enhancement, affinities determined using Trp-FRET were more precise and accurate as validated using independent isothermal titration calorimetry studies. FRET between tryptophan and PB represents a new tool for the characterization of protein-ligand complexes.