Cell-to-cell signaling, or quorum sensing (QS), in many Gram-negative bacteria is governed by small molecule signals (N-acyl-l-homoserine lactones, AHLs) and their cognate 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. Reports of quantitative direct-binding experiments on LuxR-type proteins are scarce, and robust and generalizable methods that provide such data are largely nonexistent. We report herein a Förster resonance energy transfer (FRET) assay that leverages (1) conserved tryptophans located in the LuxR-type protein ligand-binding site and synthetic fluorophore–AHL conjugates, and (2) isolation 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 proteins. These methods will advance fundamental investigations of LuxR-type protein mechanism and the development of small molecule QS modulators.

许多革兰氏阴性细菌中的细胞间信号传导或群体感应 (QS) 由小分子信号（ N-酰基-L-高丝氨酸内酯，AHL）及其同源受体（LuxR 型蛋白）控制。由于分离和操作大多数 LuxR 型蛋白的挑战，这些细菌中 QS 的机制基础受到严重限制。 LuxR 型蛋白定量直接结合实验的报告很少，而且提供此类数据的稳健且可推广的方法基本上不存在。我们在此报告了福斯特共振能量转移（FRET）测定，该测定利用（1）位于LuxR型蛋白质配体结合位点的保守色氨酸和合成荧光团-AHL缀合物，以及（2）分离与弱激动剂结合的蛋白质。 FRET 测定可以直接测量配体与受体的结合亲和力（无论是在体外还是在细胞中），并被证明与六种 LuxR 型蛋白兼容。这些方法将推进 LuxR 型蛋白质机制的基础研究和小分子 QS 调节剂的开发。