Engineering a Novel Porin OmpGF Via Strand Replacement from Computational Analysis of Sequence Motif.
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.
Department of Bioengineering, University of Illinois at Chicago, Chicago, IL 60612, United States.
Meishan
M
Lin
Department of Bioengineering, University of Illinois at Chicago, Chicago, IL 60612, United States; Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, United States.
Ge
G
Zhang
Department of Chemistry, University of Massachusetts Amherst, Amherst, MA 01003, United States.
Monifa
M
Fahie
Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612, United States.
Leslie K
LK
Morgan
Department of Chemistry, University of Massachusetts Amherst, Amherst, MA 01003, United States.
Min
M
Chen
Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, United States.
Timothy A
TA
Keiderling
Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612, United States; Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore. Electronic address: kenneyl@uic.edu.
Linda J
LJ
Kenney
Department of Bioengineering, University of Illinois at Chicago, Chicago, IL 60612, United States. Electronic address: jliang@uic.edu.
Jie
J
Liang
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D008051:Lipid Bilayers / Q000737:chemistry; D018272:Porins / Q000737:chemistry*; D015202:Protein Engineering; D017433:Protein Structure, Secondary
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D018272:Porins
Membrane proteins; Nanopores; Porins; Protein engineering; Sequence motif; β-Barrel protein