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

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

Research output: Research - peer-reviewArticle

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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.

LanguageEnglish (US)
Pages2115-2124
Number of pages10
JournalBiophysical Journal
Volume103
Issue number10
DOIs
StatePublished - Nov 21 2012

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Nanopores
Polymers
Proteins
iron (III) hydroxamate
Ethylene Glycols
Protein Engineering
Dextrans
Lipid Bilayers
Water

ASJC Scopus subject areas

  • Biophysics

Cite this

Inspection of the Engineered FhuA Δc/Δ4L Protein Nanopore by Polymer Exclusion. / Niedzwiecki, David J.; Mohammad, Mohammad M.; Movileanu, Liviu.

In: Biophysical Journal, Vol. 103, No. 10, 21.11.2012, p. 2115-2124.

Research output: Research - peer-reviewArticle

Niedzwiecki, David J. ; Mohammad, Mohammad M. ; Movileanu, Liviu. / Inspection of the Engineered FhuA Δc/Δ4L Protein Nanopore by Polymer Exclusion. In: Biophysical Journal. 2012 ; Vol. 103, No. 10. pp. 2115-2124
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