Quiet Outer Membrane Protein G (OmpG) Nanopore for Biosensing

Area of Science:

• Biotechnology
• Nanotechnology
• Biophysics

Background:

• Nanopore technology offers unique applications in biosensing and sequencing.
• The E. coli outer membrane protein OmpG is a promising protein nanopore candidate.
• Flexibility in OmpG's extracellular loops limits its biosensing capabilities.

Purpose of the Study:

• To engineer a more stable and reliable OmpG protein nanopore.
• To reduce the flexibility of OmpG's extracellular loops, particularly loop 6.
• To enhance OmpG's suitability for advanced biosensing applications.

Main Methods:

• Genetic modification of the OmpG protein by deleting residues in loop 6.
• Nuclear Magnetic Resonance (NMR) spectroscopy to determine the dynamic structure of the modified OmpG.
• Electrophysiological measurements to assess pore stability and flickering.
• Modification of the pore lumen with a copper chelating moiety.

Main Results:

• The engineered OmpG construct exhibited reduced flexibility in loops 1, 2, 6, and 7.
• The modified nanopore demonstrated significantly reduced flickering between open and closed states over a wide pH range.
• The copper chelating moiety facilitated small molecule detection.
• Concurrent single-molecule biosensing of glutamate and adenosine triphosphate was successfully demonstrated.

Conclusions:

• Engineering OmpG by reducing loop flexibility enhances nanopore stability and performance.
• The modified OmpG nanopore is a robust platform for precise single-molecule biosensing.
• This improved nanopore design opens new avenues for detecting various small molecules with high sensitivity.