Abstract
The redesign of biological nanopores is focused on bacterial outer membrane proteins and pore-forming toxins, because their robust β-barrel structure makes them the best choice for developing stochastic biosensing elements. Using membrane protein engineering and single-channel electrical recordings, we explored the ferric hydroxamate uptake component A (FhuA), a monomeric 22-stranded β-barrel protein from the outer membrane of Escherichia coli. FhuA has a luminal cross-section of 3.1 × 4.4 nm and is filled by a globular N-terminal cork domain. Various redesigned FhuA proteins were investigated, including single, double, and multiple deletions of the large extracellular loops and the cork domain. We identified four large extracellular loops that partially occlude the lumen when the cork domain is removed. The newly engineered protein, FhuAΔC/Δ4L, was the result of a removal of almost one-third of the total number of amino acids of the wild-type FhuA (WT-FhuA) protein. This extensive protein engineering encompassed the entire cork domain and four extracellular loops. Remarkably, FhuAΔC/Δ4L forms a functional open pore in planar lipid bilayers, with a measured unitary conductance of ∼4.8 nanosiemens, which is much greater than the values recorded previously with other engineered FhuA protein channels. There are numerous advantages and prospects of using such an engineered outer membrane protein not only in fundamental studies of membrane protein folding and design, and the mechanisms of ion conductance and gating, but also in more applicative areas of stochastic single-molecule sensing of proteins and nucleic acids.
Highlights
Extraordinary stiffness to the core of the protein,  barrels are open to remodeling in various ways, including direct genetic engineering and covalent modifications [1, 6, 7]
We explored ferric hydroxamate uptake component A (FhuA), a monomeric 22-stranded -barrel outer membrane protein of E. coli
Single-channel electrical recordings performed with the wild-type FhuA (WT-FhuA) protein revealed that the native channel is not fully closed (ϳ300 pS in 1 M KCl)
Summary
Plasmid Constructs—pPR-IAB1 plasmids that contained wt fhua and fhua⌬1–160, with an internal 6ϫHisϩ cloned into the coding region for the surfaced-exposed loop L5, were gifted by Professor Ulrich Schwaneberg (Jacobs University Bremen, Bremen, Germany). To construct fhua⌬322–355, inverse PCR was performed on the wt fhua-containing plasmid with the following two phosphorylated primers: p-322, 5Ј-GTG ATC GAA GCT GTA GCC GAC-3Ј, and p-355, 5Ј-AAT GCT TAC AGC AAA CAG TGT-3Ј. To construct fhua⌬1–160/⌬322–355 and fhua⌬1–160/⌬335–355, we applied the above overall strategy except that we performed inverse PCR on the fhua⌬1–160containing plasmid. The fhua gene, which lacked the regions coding for the cork domain and loops 3–5 and 11, named fhua⌬C/⌬4L, was constructed by de novo synthesis (GENEART, Regensburg, Germany) in the pMK-RQ plasmid flanked by EcoRI and XhoI restriction sites. In this construct, the deleted loops were replaced with the polypeptide NSEG(S).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
