Redesign of a plugged beta-barrel membrane protein

Area of Science:

• Biophysics
• Protein Engineering
• Nanotechnology

Background:

• Bacterial outer membrane proteins and toxins offer robust β-barrel structures ideal for biosensing.
• Ferric hydroxamate uptake component A (FhuA) is a monomeric β-barrel protein from Escherichia coli.

Purpose of the Study:

• To engineer and characterize a functional nanopore from FhuA for enhanced biosensing applications.
• To investigate the impact of deleting extracellular loops and the cork domain on FhuA structure and function.

Main Methods:

• Membrane protein engineering techniques were employed to modify FhuA.
• Single-channel electrical recordings were used to analyze pore properties in planar lipid bilayers.

Main Results:

• Engineered FhuA variant (FhuAΔC/Δ4L) with extensive deletions formed a stable, open pore.
• The FhuAΔC/Δ4L nanopore exhibited a high unitary conductance of approximately 4.8 nanosiemens.
• This conductance is significantly higher than previously reported engineered FhuA channels.

Conclusions:

• Extensive engineering of FhuA can yield functional nanopores with superior conductance.
• Engineered FhuA holds promise for fundamental studies in protein folding and ion transport.
• Applications include stochastic single-molecule sensing of proteins and nucleic acids.