Engineering Enhanced Pore Sizes Using FhuA Δ1-160 from E. coli Outer Membrane as Template

Biological membranes are the perfect example of a molecular filter using membrane channels to control the permeability of small water-soluble molecules. To allow filtering of larger hydrophilic molecules we started from the known mutant channel FhuA Delta 1-160 in which the cork domain closing the channel had been removed. Here we further expand the pore diameter by copying the amino acid sequence of two beta-strands in a stepwise manner increasing the total number of beta-strands from 22 to 34. The pore size of the respective expanded channel protein was characterized by single-channel conductance. Insertion of additional beta-strands increased the pore conductance but also induced more ion current flickering on the millisecond scale. Further, polymer exclusion measurements were performed by analyzing single-channel conductance in the presence of differently sized polyethylene glycol of known polymer random coil radii. The conclusion from channel conductance of small channel penetrating polymers versus larger excluded ones suggested an increase in pore radii from 1.6 nm for FhuA Delta 1-160 up to a maximum of about 2.7 nm for +8 beta insertion. Integration of more beta-strand caused instability of the channel and exclusion of smaller sized polymer. FhuA Delta 1-160 + 10 beta and FhuA Delta 1-160 + 12 beta effective radius decreased to 1.4 and 1.3 nm, respectively, showing the limitations of this approach.