Autoinducer-Fluorophore Conjugates Enable FRET in LuxR Proteins in Vitro and in Cells

Cell-to-cell signaling, or quorum sensing (QS), in Gram-negative bacteria is governed by small molecule signals (N-acyl L-homoserine lactones, AHLs) and their cognate intracellular receptors (LuxR-type proteins). The mechanistic underpinnings of QS in these bacteria are severely limited due to the challenges of isolating and manipulating most LuxR-type proteins. Quantitative assays to characterize the direct binding of ligands to these receptors are largely non-existent. We report herein a Förster Resonance Energy Transfer (FRET) assay that leverages (i) conserved endogenous tryptophans located in the LuxR-type protein ligand-binding site and synthetic fluorophore-AHL conjugates, and (ii) isolation/stabilization of the proteins bound to weak agonists. The FRET assay permits straightforward measurement of ligand-binding affinities with receptor—either in vitro or in cells—and was shown to be compatible with six LuxR-type receptors. These methods will advance fundamental investigations of the mechanisms of LuxR-type proteins and the development of small molecule modulators of QS.