title: Global redesign of a native β-barrel scaffold.
abstract: One persistent challenge in membrane protein design is accomplishing extensive modifications of proteins without impairing their functionality. A truncation derivative of the ferric hydroxamate uptake component A (FhuA), which featured the deletion of the 160-residue cork domain and five large extracellular loops, produced the conversion of a non-conductive, monomeric, 22-stranded β-barrel protein into a large-conductance protein pore. Here, we show that this redesigned β-barrel protein tolerates an extensive alteration in the internal surface charge, encompassing 25 negative charge neutralizations. By using single-molecule electrophysiology, we noted that a commonality of various truncation FhuA protein pores was the occurrence of 33% blockades of the unitary current at very high transmembrane potentials. We determined that these current transitions were stimulated by their interaction with an external cationic polypeptide, which occurred in a fashion dependent on the surface charge of the pore interior as well as the polypeptide characteristics. This study shows promise for extensive engineering of a large monomeric β-barrel protein pore in molecular biomedical diagnosis, therapeutics, and biosensor technology.
authors.affiliation: Department of Physics, Syracuse University, 201 Physics Building, Syracuse, NY 13244-1130, USA; Structural Biology, Biochemistry, and Biophysics Program, Syracuse University, 111 College Place, Syracuse, NY 13244-4100, USA.
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Wolfe
authors.affiliation: Department of Physics, Syracuse University, 201 Physics Building, Syracuse, NY 13244-1130, USA.
authors.forename: Mohammad M
authors.initials: MM
authors.lastname: Mohammad
authors.affiliation: Department of Physics, Syracuse University, 201 Physics Building, Syracuse, NY 13244-1130, USA; Structural Biology, Biochemistry, and Biophysics Program, Syracuse University, 111 College Place, Syracuse, NY 13244-4100, USA.
authors.forename: Avinash K
authors.initials: AK
authors.lastname: Thakur
authors.affiliation: Department of Physics, Syracuse University, 201 Physics Building, Syracuse, NY 13244-1130, USA; Structural Biology, Biochemistry, and Biophysics Program, Syracuse University, 111 College Place, Syracuse, NY 13244-4100, USA; The Syracuse Biomaterials Institute, Syracuse University, 121 Link Hall, Syracuse, NY 13244, USA. Electronic address: lmovilea@syr.edu.
authors.forename: Liviu
authors.initials: L
authors.lastname: Movileanu
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015640:Ion Channel Gating; D008564:Membrane Potentials / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011991:Receptors, Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D055672:Static Electricity; D013329:Structure-Activity Relationship
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D011991:Receptors, Virus; D011994:Recombinant Proteins
keywords: FhuA; Ion channel; Membrane protein engineering; Single-molecule electrophysiology; Spontaneous gating