Engineering a Novel Porin OmpGF Via Strand Replacement from Computational Analysis of Sequence Motif

beta-Barrelmembrane proteins (beta MPs) form barrel-shaped pores in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Because of the robustness of their barrel structures, beta MPs have great potential as nanosensors for single-molecule detection. However, natural beta MPs currently employed have inflexible biophysical properties and are limited in their pore geometry, hindering their applications in sensing molecules of different sizes and properties. Computational engineering has the promise to generate beta MPs with desired properties. Here we report a method for engineering novel beta MPs based on the discovery of sequence motifs that predominantly interact with the cell membrane and appear in more than 75% of transmembrane strands. By replacing beta 1-beta 6 strands of the protein OmpF that lack these motifs with beta 1-beta 6 strands of OmpG enriched with these motifs and computational verification of increased stability of its transmembrane section, we engineered a novel beta MP called OmpGF. OmpGF is predicted to form a monomer with a stable transmembrane region. Experimental validations showed that OmpGF could refold in vitro with a predominant beta-sheet structure, as confirmed by circular dichroism. Evidence of OmpGF membrane insertion was provided by intrinsic tryptophan fluorescence spectroscopy, and its pore-forming property was determined by a dye-leakage assay. Furthermore, single-channel conductance measurements confirmed that OmpGF function as a monomer and exhibits increased conductance than OmpG and OmpF. These results demonstrated that a novel and functional beta MP can be successfully engineered through strand replacement based on sequence motif analysis and stability calculation. (C) 2017 Elsevier B.V. All rights reserved.