Protein engineering of FhuA Δ1-160 to enhance pore size

01/12/2017
 

Congratulations Zhanzhi Liu on his recent publication!

FhuA Δ1-160 Copyright: ACS Publications

Biological membranes are perfect examples for a molecular filter that uses membrane channels to control the permeability of small water-soluble molecules. We started from the known mutant channel FhuA Δ1-160 in which the cork domain closing the channel had been removed to allow filtering of larger hydrophilic molecules. Here, we further expanded the pore diameter by copying the amino acid sequence of two β-strands in a stepwise manner increasing the total number of β-strands from 22 to 34. The pore size of the respective expanded channel protein was characterized by single-channel conductance analysis. Furthermore, polymer exclusion measurements were performed by analyzing single-channel conductance in the presence of differently sized polyethylene glycols 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 Δ1-160 up to a maximum of about 2.7 nm for FhuA Δ1-160 + 8 β. Integration of more β-strand caused instability of the channel and exclusion of smaller sized polymer. FhuA Δ1-160 + 10 β and FhuA Δ1-160 + 12 β effective radius decreased to 1.4 and 1.3 nm, respectively, showing the limitations of this approach.

Access information of this article can be found under 10.1021/acssensors.7b00481 and

Liu, Z., Ghai, I., Winterhalter, M., Schwaneberg, U.; Engineering Enhanced Pore Sizes Using FhuA Δ1-160 from E. coli Outer Membrane as Template; ACS Sens., 2017, 2 (11), pp 1619–1626