Single-Molecule Observation of Competitive Protein–Protein Interactions Utilizing a Nanopore

Two or more protein ligands may compete against each other to interact transiently with a protein receptor. While this is a ubiquitous phenomenon in cell signaling, existing technologies cannot identify its kinetic complexity because specific subpopulations of binding events of different ligands are hidden in the averaging process in an ensemble. In addition, the limited time resolution of prevailing methods makes detecting and discriminating binding events among diverse interacting partners challenging. Here, we utilize a genetically encoded nanopore sensor to disentangle competitive protein-protein interactions (PPIs) in a one-on-one and label-free fashion. Our measurements involve binary mixtures of protein ligands of varying binding affinity against the same receptor, which was externally immobilized on the nanopore tip. We use the resistive-pulse technique to monitor the kinetics and dynamics of reversible PPIs without the nanopore confinement, with a high-time bandwidth, and at titratable ligand concentrations. In this way, we systematically evaluate how individual protein ligands take their turn to reside on the receptor's binding site. Further, our single-molecule determinations of these interactions are quantitatively compared with data generated by a two-ligand, one-receptor queuing model. The outcomes of this work provide a fundamental basis for future developments aimed at a better mechanistic understanding of competitive PPIs. Moreover, they may also form a platform in drug development pipelines targeting high-complexity PPIs mediated by protein hubs.