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

Abstract β -Barrelmembrane proteins ( β MPs) form barrel-shaped pores in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Because of the robustness of their barrel structures, β MPs have great potential as nanosensors for single-molecule detection. However, natural β 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 β MPs with desired properties. Here we report a method for engineering novel β 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 β 1– β 6 strands of the protein OmpF that lack these motifs with β 1– β 6 strands of OmpG enriched with these motifs and computational verification of increased stability of its transmembrane section, we engineered a novel β 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 β -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 β MP can be successfully engineered through strand replacement based on sequence motif analysis and stability calculation.