Insights into the Size and Geometry of a Robust Engineered Membrane Protein Nanopore

The maximum utility of protein nanopores in biotechnological applications is generally dictated by the size and internal geometry of the protein. The knowledge of these two parameters is imperative, particularly when extensive protein redesign is employed to produce task-specific proteins.Recently, we used ferric hydroxamate uptake component A (FhuA), a β-barrel bacterial outermembrane protein, as a protein-engineering template to produce an unusually-rigid, open nanopore (FhuA ΔC/Δ4L). This new nanopore maintains its stability under harsh experimental conditions. It was essential to couple comprehensive protein redesign with protein refolding protocols to obtain such a nanopore. With the radical revamp of the FhuA template, it was necessary to shed light on the overall structure of the new FhuA ΔC/Δ4L protein. Thus, we employed water-soluble, flexible poly(ethylene glycols) and dextran polymers to inspect the interior of FhuA ΔC/Δ4L during single-channel recordings.The addition of poly(ethylene glycols) to solution produced alterations in the single-channel conductance, allowing for the calculation of the nanopore diameter. We report that FhuA ΔC/Δ4L features an approximate conical internal geometry with the cis entrance (extracellular) smaller than the trans entrance (periplasmic). This finding is in accord with the asymmetric nature of the crystal structure of the wild-type FhuA protein. Additionally, experiments with impermeable dextran indicated an average internal diameter of ∼2.4 nm, an estimate based on the polymer-induced alteration of the access resistance contribution to the total resistance of the nanopore. The insights into size and geometry of FhuA ΔC/Δ4L deduced from these polymers experiments will aid in future protein engineering of FhuA, opening new engineering avenues for specific tasks in biotechnological applications.This work is funded in part by grants from the US National Science Foundation (DMR-1006332, L.M.) and the National Institutes of Health (R01 GM088403, L.M.).