Nanopores and pore-forming proteins

Big insights from small molecular structures.

Types of Nanopores

Nanopores, i.e. apertures with diameters in the nanometer range in an otherwise nonconductive surface, can be exploited for the detection and investigation of molecules on a single particle level as well as for applications like the sequencing of DNA, RNA, and even the primary structure of proteins. While solid-state nanopores are artificially engineered in materials like silicone nitride or aluminum oxide, lipid bilayers can provide a stable nonconductive matrix for the insertion of biological nanopores like e.g. alpha-Hemolysin or Aerolysin.

Native vs. engineered

channels, pores and toxins

DNA nanopores

DNA provides a powerful tool to design nanopores of freely definable size and shape for the transport of certain molecules or their usage in sequencing applications. Lipid bilayers provide the means to investigate and verify the properties of such tailor-made DNA pores on a single molecule level.

Figure representing tests whether DNA nanopores permit label-free, direct, and specific detection of IgG antibodies at the single-molecule level. Specific and label-free IgG sensing using DNA nanopores Sqr-10-Biot using bilayer Orbit mini / Orbit 16 TC.

Xing Y., et al. Nature Nanotechnology (2022)

Pore-forming toxins

Pore-forming toxins (PFTs) are an important area of study in toxin research. Unlike toxins that target ion channels, PFT create nano-scale pores in cell membranes themselves, disrupting cellular ion balance and affecting cell’s normal function. Understanding how these toxins form pores and interact with membranes provides valuable insights into cellular mechanisms and pathogen-host interactions.

Although PFTs represent the largest class of bacterial toxins, they are not exclusive to bacteria; these proteins are found across all kingdoms of life, including mammals, invertebrates, plants, and fungi. PFTs demonstrate a conserved molecular strategy across diverse life forms, highlighting their fundamental importance in cellular interactions, defense mechanisms, and survival tactics.

PFTs are classified into two main categories based on their pore-forming structures:

  1. α-PFTs: Form pores using α-helices (e.g., Cytolysin A of E. coli)
  2. β-PFTs: Create pores using β-barrels (e.g., α-hemolysin)

The creation of pores by PFTs leads to several detrimental effects on target cells:

  • Osmotic imbalance
  • Loss of membrane potential
  • Efflux of ions and nutrients
  • Dissipation of the protonmotive force (PMF)
  • Cellular death through necrosis or apoptosis

Applications:

Antimicrobial AgentsSome pore-forming peptides (e.g., melittin, Smp24) are studied as potential antibiotics for drug-resistant bacteria.
Biosensors and SequencingEngineered pore-forming toxins (e.g., α-Hemolysin) are used in biosensors and nanopore sequencing.
Cancer TherapySelective pore-forming toxins target tumor cells by exploiting differences in membrane composition.

 

In the recommended publication below, Orbit mini was used for the investigation of the pore-forming protein Bryoprin with artificial lipid bilayers.

Antimicrobial peptides

Antimicrobial peptides (AMPs), including pore-forming peptides, are considered promising candidates for combating multidrug-resistant pathogens. AMPs can directly kill bacteria, including multidrug-resistant strains, by rapidly permeabilizing bacterial membranes. Bacteria develop resistance to AMPs more slowly compared to conventional antibiotics, making these peptides potential next-generation antibiotics.

The general concept of using pore-forming peptides as antibiotics is supported. AMPs are being investigated for their ability to:

  1. Directly kill bacteria through membrane permeabilization
  2. Enhance the effectiveness of conventional antibiotics against resistant pathogens
  3. Prevent biofilm formation

Research is ongoing to design and optimize AMPs for therapeutic use, with a focus on their ability to selectively target pathogenic bacteria while minimizing toxicity to human cells.

Towards sequencing through nanopores

Nanopores, i.e. apertures with diameters in the nanometer range in an otherwise nonconductive surface, can be exploited for the detection and investigation of molecules on a single particle level and ultimately for applications like the sequencing of DNA, RNA and even the primary structure of proteins. While solid-state nanopores are artificially engineered in materials like silicone nitride or aluminum oxide, lipid bilayers can provide a stable nonconductive matrix for the insertion of biological nanopores like e.g. alpha-Hemolysin or Aerolysin. Ensslen et al, JACS 2022

Electrophysiology
in bilayers

Single ion channel recordings in lipid bilayers

Whole-cell patch clamping allows for the straightforward and high-throughput compatible investigation of ion channels in living cells. Detailed information about open probabilities, unitary currents, and the precise kinetic behavior of an ion channel can, however, only be obtained on a single channel level. The introduction of ion channels to lipid bilayers provides a convenient and reliable alternative to the tedious and difficult technique of cell-attached recordings on native membranes. Currents evoked by ion channels introduced into lipid bilayers can conveniently be recorded on the Orbit mini.

Yelshanskaya M.V., et al. Nature 2022

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