Single-Molecule\nObservation of Competitive Protein–Protein\nInteractions Utilizing a Nanopore

Two or more protein ligands may compete against each\nother to interact\ntransiently with a protein receptor. While this is a ubiquitous phenomenon\nin cell signaling, existing technologies cannot identify its kinetic\ncomplexity because specific subpopulations of binding events of different\nligands are hidden in the averaging process in an ensemble. In addition,\nthe limited time resolution of prevailing methods makes detecting\nand discriminating binding events among diverse interacting partners\nchallenging. Here, we utilize a genetically encoded nanopore sensor\nto disentangle competitive protein–protein interactions (PPIs)\nin a one-on-one and label-free fashion. Our measurements involve binary\nmixtures of protein ligands of varying binding affinity against the\nsame receptor, which was externally immobilized on the nanopore tip.\nWe use the resistive-pulse technique to monitor the kinetics and dynamics\nof reversible PPIs without the nanopore confinement, with a high-time\nbandwidth, and at titratable ligand concentrations. In this way, we\nsystematically evaluate how individual protein ligands take their\nturn to reside on the receptor’s binding site. Further, our\nsingle-molecule determinations of these interactions are quantitatively\ncompared with data generated by a two-ligand, one-receptor queuing\nmodel. The outcomes of this work provide a fundamental basis for future\ndevelopments aimed at a better mechanistic understanding of competitive\nPPIs. Moreover, they may also form a platform in drug development\npipelines targeting high-complexity PPIs mediated by protein hubs.