Interactions of a Polypeptide with a Protein Nanopore Under Crowding Conditions

Molecular crowding, a ubiquitous feature of the cellular environment, has significant implications in the kinetics and equilibrium of biopolymer interactions. In this study, a single charged polypeptide is exposed to competing forces that drive it into a transmembrane protein pore versus forces that pull it outside. Using single-molecule electrophysiology, we provide compelling experimental evidence that the kinetic details of the polypeptide–pore interactions are substantially affected by high concentrations of less-penetrating polyethylene glycols (PEGs). At a polymer concentration above a critical value, the presence of these neutral macromolecular crowders increases the rate constant of association but decreases the rate constant of dissociation, resulting in a stronger polypeptide–pore interaction. Moreover, a larger-molecular weight PEG exhibits a lower rate constant of association but a higher rate constant of dissociation than those values corresponding to a smaller-molecular weight PEG. These out...