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 outcomes are in accord with a lower diffusion constant of the polypeptide and higher depletion-attraction forces between the polypeptide and transmembrane protein pore under crowding and confinement conditions.

WD40 repeat proteins are frequently involved in processing cell signaling and scaffolding large multi-subunit machineries. Despite their significance in physiological and disease-like conditions, their reversible interactions with other proteins remain modestly examined. Here, we show the development and validation of a protein nanopore for the detection and quantification of WD40 repeat protein 5 (WDR5), a chromatin-associated hub involved in epigenetic regulation of histone methylation. Our nanopore sensor is equipped with a 14-residue Win motif of mixed lineage leukemia 4 methyltransferase (MLL4Win), a WDR5 ligand. Our approach reveals a broad dynamic range of MLL4Win-WDR5 interactions and three distant subpopulations of binding events, representing three modes of protein recognition. The three binding events are confirmed as specific interactions using a weakly binding WDR5 derivative and various environmental contexts. These outcomes demonstrate the substantial sensitivity of our nanopore sensor, which can be utilized in protein analytics.

There have been only a few studies reporting on the impact of polyhistidine affinity tags on the structure, function, and dynamics of proteins. Because of their relatively short size, they are often assumed to have little or no effect on the conformation and activity of a protein. Here, using membrane protein design and single-molecule electrophysiology, we determined that the presence of a hexahistidine arm at the N-terminus of a truncated FhuA-based protein nanopore, leaving the C-terminus untagged, produces an unusual increase in the unitary conductance up to ~8 nS in 1 M KCl. To our knowledge, this is the largest single-channel conductance ever recorded with a monomeric β-barrel outer membrane protein. The hexahistidine arm was captured by an anti-polyhistidine tag monoclonal antibody added to the side of the channel-forming protein addition, but not to the opposite side, documenting that this truncated FhuA-based protein nanopore inserts into a planar lipid bilayer with a preferred orientation. This finding is in agreement with the protein insertion in vivo, in which the large loops face the extracellular side of the membrane. The aberrantly large single-channel conductance, likely induced by a greater cross-sectional area of the pore lumen, along with the vectorial insertion into a lipid membrane, will have profound implications for further developments of engineered protein nanopores.