Microbe response inspires tools to study proteins

NCCR Bio-Inspired Materials researchers at the Adolphe Merkle Institute have developed tunable nanopores that may help decipher the structure and function of proteins. Their approach is modelled on how immune system recognizes pathogens.

 Proteins are the foundation for all biological processes in living organisms, and working out the shape of a protein enables scientists to understand how that protein functions. Building on their previous work, the scientists at the University of Fribourg have designed tunable nanosized tubes, called nanopores, that can help to reveal the shape of a protein — and even its orientation in the 3D space. The approach could find applications not only in detecting and characterizing proteins, but also as a tool to deliver drugs inside cells — or even as a miniature weapon to kill cancer cells and bacteria. The inspiration for the nanopores came indeed from the way our body fights pathogens: when the immune system recognizes a harmful microbe, it activates a complex of proteins that kill the pathogen by punching tiny holes through its cell membrane. “If we could use that killing machine to our advantage, it would be very powerful,” says study senior author and NCCR Principal Investigator Prof. Michael Mayer.

To develop the nanopores, Mayer’s team took advantage of a peptide called CtxA, which a fly native to sub-Saharan Africa produces to protect its eggs from bacteria. A handful of CtxA peptides typically assemble to form molecular-scale tubes that can kill microbes — very much like our immune system does with pathogens. First, the researchers had the CtxA peptide synthesized in the lab; then, they attached a DNA strand to it. “We created a peptide-DNA hybrid,” Mayer explains. Because DNA is made of complementary sites that can bind to one another, individual molecules of the right sequence will self-assemble, forcing the structure to form a desired shape.

By creating structures with 4, 8 or 12 peptide-DNA hybrids, the researchers were able to tune the diameter of the nanopores, making the inner tube bigger. They also attached to the structure a fatty molecule, which increases the affinity of the nanopore to the lipid membrane of cells. The findings were published in the journal ACS Nano. This is not the first time that researchers have made nanopores: the tiny tubes are already used to sequence DNA molecules. When a nanopore inserts into a membrane that separates two liquid compartments — one with a positive charge and one with a negative charge — it opens a path for charged particles, or ions, to flow from one compartment to the other. When ions move through a nanopore, they create an electrical current. But when a DNA molecule enters the pore, it reduces the flow of ions across the membrane. By monitoring changes to the electrical current produced by this flow of ions, researchers can determine the sequence of a specific DNA strand.

Previous work by Mayer’s team showed that it’s possible to use nanopores to identify the fingerprint of specific proteins. “The blockade is not just proportional to the volume of a protein, but it also represents its orientation,” Mayer says. “We use that signal to make estimates about the shape of a protein, which is important to understand its function.” However, analyzing the shape of proteins with nanopores presents many challenges, Mayer says. For one, proteins are substantially bigger than DNA molecules, so larger pores would be helpful, but Mayer notes that bigger pores seem to be less stable than smaller ones. “Nature doesn’t normally make those bigger pores, except to kill microbes.”

For this reason, the team is now trying to build nanopores using one of the proteins that make up the immune system’s ‘killing machine.’ Preliminary results suggest that using this approach, it’s possible to make stable nanopores that are up to 12 nanometers in inner diameter. “They might actually work for what we want to do,” Mayer says. “At the end, we’re using exactly what nature uses — of course, we need some tricks to make this work in a test tube, but the results look very promising.”

Reference:  Fennouri, A.; List, J.; Ducrey, J.; Dupasquier, J.; Sukyte, V.; Mayer, S. F.; Vargas, R. D.; Pascual Fernandez, L.; Bertani, F.; Rodriguez Gonzalo, S.; Yang, J.; Mayer, M. Tuning the Diameter, Stability, and Membrane Affinity of Peptide Pores by DNA-Programmed Self-Assembly. ACS Nano 2021, 15 (7), 11263–11275. https://doi.org/10.1021/acsnano.0c10311

Author: Giorgia Guglielmi