Inspection of the Engineered FhuA [Delta]C/[Delta]4L Protein Nanopore by Polymer Exclusion

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...

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Bibliographic Details
Published in:Biophysical journal 2012-11, Vol.103 (10), p.2115
Main Authors: Niedzwiecki, David J, Mohammad, Mohammad M, Movileanu, Liviu
Format: Article
Language:English
Subjects:
Biophysics
Molecular structure
Molecules
Nanotechnology
Polyethylene glycol
Proteins
Tissue engineering
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Summary: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 ...C/...4L nanopore. We employed water-soluble poly(ethylene glycols) and dextran polymers to examine the interior of FhuA ...C/...4L. 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 ...C/...4L 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. (ProQuest: ... denotes formulae/symbols omitted.)
ISSN:0006-3495
1542-0086