Inspection of the Engineered FhuA Δc/Δ4L Protein Nanopore by Polymer Exclusion

David J. Niedzwiecki, Mohammad M. Mohammad, Liviu Movileanu

Research output: Contribution to journalArticle

19 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)2115-2124
Number of pages10
JournalBiophysical Journal
Volume103
Issue number10
DOIs
StatePublished - Nov 21 2012

ASJC Scopus subject areas

  • Biophysics

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