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Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution
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MspA nanopores from subunit dimers.

Mikhail Pavlenok1, Ian M Derrington, Jens H Gundlach

  • 1Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

Plos One
|June 22, 2012
PubMed
Summary

Researchers engineered Mycobacterium smegmatis porin A (MspA) into functional dimeric pores using peptide linkers. These engineered MspA dimers are suitable for DNA sequencing and offer better control over pore properties for nanotechnological applications.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Nanotechnology

Background:

  • Mycobacterium smegmatis porin A (MspA) forms octameric channels and is a key pore protein.
  • Controlling MspA subunit stoichiometry is crucial for nanotechnological applications, including nanopore sequencing.
  • MspA's unique pore geometry and robustness make it ideal for DNA sequencing applications.

Purpose of the Study:

  • To investigate the feasibility of creating functional dimeric MspA pores by linking monomers with peptide linkers.
  • To assess the suitability of these engineered MspA dimers for DNA translocation and sequencing.
  • To explore the potential for tailoring MspA pores with altered stoichiometries for enhanced control over their properties.

Main Methods:

  • Chemically linking MspA monomers with peptide linkers of varying lengths (17-62 amino acids).

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  • Purifying engineered MspA constructs from M. smegmatis.
  • Conducting lipid bilayer experiments to assess channel function, voltage gating, and DNA translocation.
  • Restoring porin function in M. smegmatis cells lacking native porins using dimeric MspA constructs.

    • Main Results:

    • Engineered MspA dimers formed functional channels in lipid bilayers, indicating correct folding and localization.
    • Expression levels of dimeric MspA were reduced tenfold compared to wild-type MspA.
    • Voltage gating properties of MspA monomers and dimers were identical, suggesting similar structural and dynamic characteristics.
    • Dimeric MspA pores successfully facilitated glucose uptake in M. smegmatis and showed identical ionic current blockades with DNA hairpins compared to monomeric pores.

    Conclusions:

    • Production of single-chain MspA pores in M. smegmatis is feasible, providing a proof of principle for generating MspA pores with controlled stoichiometries.
    • MspA subunit dimers offer enhanced control over the chemical and physical properties of the pore's constriction zone.
    • Engineered MspA dimers are suitable for DNA sequencing and hold promise for advancing mycobacterial transport studies and nanopore sequencing technology.