Global redesign of a native β-barrel scaffold

Area of Science:

• Biochemistry
• Biophysics
• Protein Engineering

Background:

• Membrane protein design faces challenges in extensive modification without functional impairment.
• A truncated ferric hydroxamate uptake component A (FhuA) was engineered into a large-conductance protein pore.

Purpose of the Study:

• To investigate the tolerance of the engineered FhuA protein pore to extensive internal surface charge alterations.
• To characterize the behavior of these modified pores using single-molecule electrophysiology.

Main Methods:

• Protein engineering of FhuA by deleting the cork domain and extracellular loops.
• Single-molecule electrophysiology to measure ionic current.
• Analysis of current blockades and their dependence on surface charge and polypeptide characteristics.

Main Results:

• The redesigned beta-barrel protein pore tolerated 25 negative charge neutralizations on its internal surface.
• A consistent 33% blockade of unitary current was observed at high transmembrane potentials.
• Current transitions were dependent on interactions with external cationic polypeptides and pore interior surface charge.

Conclusions:

• Extensive engineering of the monomeric beta-barrel protein pore is feasible.
• The modified FhuA protein pore demonstrates potential for applications in molecular biomedical diagnosis, therapeutics, and biosensor technology.