title: Inspection of the engineered FhuA ΔC/Δ4L protein nanopore by polymer exclusion.
abstract: Extensive engineering of protein nanopores for biotechnological applications using native scaffolds requires further inspection of their internal geometry and size. Recently, we redesigned ferric hydroxamate uptake component A (FhuA), a 22-β-stranded protein containing an N-terminal 160-residue cork domain (C). The cork domain and four large extracellular loops (4L) were deleted to obtain an unusually stiff engineered FhuA ΔC/Δ4L nanopore. We employed water-soluble poly(ethylene glycols) and dextran polymers to examine the interior of FhuA ΔC/Δ4L. When this nanopore was reconstituted into a synthetic planar lipid bilayer, addition of poly(ethylene glycols) produced modifications in the single-channel conductance, allowing for the evaluation of the nanopore diameter. Here, we report that FhuA ΔC/Δ4L features an approximate conical internal geometry with the cis entrance smaller than the trans entrance, in accord with the asymmetric nature of the crystal structure of the wild-type FhuA protein. Further experiments with impermeable dextran polymers indicated an average internal diameter of ~2.4 nm, a conclusion we arrived at based upon the polymer-induced alteration of the access resistance contribution to the nanopore's total resistance. Molecular insights inferred from this work represent a platform for future protein engineering of FhuA that will be employed for specific tasks in biotechnological applications.
authors.affiliation: Department of Physics, Syracuse University, Syracuse, NY, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Niedzwiecki
authors.forename: Mohammad M
authors.initials: MM
authors.lastname: Mohammad
authors.forename: Liviu
authors.initials: L
authors.lastname: Movileanu
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D055592:Biophysical Phenomena; D003911:Dextrans / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry*; D015640:Ion Channel Gating; D008958:Models, Molecular; D008970:Molecular Weight; D058608:Nanopores; D011092:Polyethylene Glycols / Q000378:metabolism; D011108:Polymers / Q000378:metabolism*; D015202:Protein Engineering; D017384:Sequence Deletion; D012995:Solubility; D014867:Water / Q000737:chemistry
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003911:Dextrans; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D011108:Polymers; D014867:Water; D011092:Polyethylene Glycols