title: Engineering a rigid protein tunnel for biomolecular detection.
abstract: One intimidating challenge in protein nanopore-based technologies is designing robust protein scaffolds that remain functionally intact under a broad spectrum of detection conditions. Here, we show that an extensively engineered bacterial ferric hydroxamate uptake component A (FhuA), a β-barrel membrane protein, functions as a robust protein tunnel for the sampling of biomolecular events. The key implementation in this work was the coupling of direct genetic engineering with a refolding approach to produce an unusually stable protein nanopore. More importantly, this nanostructure maintained its stability under many experimental circumstances, some of which, including low ion concentration and highly acidic aqueous phase, are normally employed to gate, destabilize, or unfold β-barrel membrane proteins. To demonstrate these advantageous traits, we show that the engineered FhuA-based protein nanopore functioned as a sensing element for examining the proteolytic activity of an enzyme at highly acidic pH and for determining the kinetics of protein-DNA aptamer interactions at physiological salt concentration.
authors.affiliation: Department of Physics, Syracuse University, New York 13244-1130, United States.
authors.forename: Mohammad M
authors.initials: MM
authors.lastname: Mohammad
authors.forename: Raghuvaran
authors.initials: R
authors.lastname: Iyer
authors.forename: Khalil R
authors.initials: KR
authors.lastname: Howard
authors.forename: Mark P
authors.initials: MP
authors.lastname: McPike
authors.forename: Philip N
authors.initials: PN
authors.lastname: Borer
authors.forename: Liviu
authors.initials: L
authors.lastname: Movileanu
mesh_terms: D052157:Aptamers, Nucleotide / Q000032:analysis*; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering; D008958:Models, Molecular; D017510:Protein Folding
chemical_list: D052157:Aptamers, Nucleotide; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli