Single-molecule sensing inside stereo- and regio-defined hetero-nanopores

Area of Science:

• Biochemistry
• Nanotechnology
• Molecular Biology

Background:

• Heteromeric pore-forming proteins are crucial for molecular recognition but challenging to engineer for specific applications.
• Producing position isomers and purifying regio-defined heteromeric proteins for single-molecule sensing remains a significant hurdle.

Purpose of the Study:

• To develop an in situ strategy for creating stereo- and regio-specific heteromeric nanopores.
• To overcome challenges in preparing heteromeric proteins for precise single-molecule sensing applications.

Main Methods:

• Utilized single-molecule chemical modification of a heptameric pore-forming protein (K238C aerolysin).
• Employed real-time ionic current recording for controlled voltage manipulation and stoichiometric modification.
• Applied mass spectrometry and single-particle cryogenic electron microscopy for characterization.
• Conducted all-atom molecular dynamics simulations to analyze pore structure.

Main Results:

• Successfully constructed a stereo- and regio-specific heteromeric nanopore with a 3:4 subunit ratio.
• Demonstrated asymmetric stereo- and regio-defined residue structure through simulations and experiments.
• Achieved 95% accuracy in label-free discrimination of four peptide stereoisomers with subtle structural differences.

Conclusions:

• The developed in situ strategy enables the creation of customized heteromeric nanopores.
• This approach overcomes preparation obstacles for heteromeric protein engineering.
• The engineered hetero-nanopore shows significant potential for advancing single-molecule sensing technologies.