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

Meishan Lin, Ge Zhang, Monifa Fahie, Leslie K. Morgan, Min Chen, Timothy A. Keiderling, Linda Kenney, Jie Liang

Research output: Contribution to journalArticle

Abstract

β-Barrelmembrane proteins (βMPs) form barrel-shaped pores in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Because of the robustness of their barrel structures, βMPs have great potential as nanosensors for single-molecule detection. However, natural βMPs 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 βMPs with desired properties. Here we report a method for engineering novel βMPs 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 β1–β6 strands of the protein OmpF that lack these motifs with β1–β6 strands of OmpG enriched with these motifs and computational verification of increased stability of its transmembrane section, we engineered a novel βMP 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 βMP can be successfully engineered through strand replacement based on sequence motif analysis and stability calculation.

Original languageEnglish (US)
Pages (from-to)1180-1189
Number of pages10
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1859
Issue number7
DOIs
StatePublished - Jul 1 2017
Externally publishedYes

Keywords

  • Membrane proteins
  • Nanopores
  • Porins
  • Protein engineering
  • Sequence motif
  • β-Barrel protein

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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