Researchers staple proteins to engineer a dynamic bacterial sieve to detect molecules

Image: Stapled and Unstapled CymA Protein. Credit: Authors, https://doi.org/10.1021/acsnano.5c16470

In a boost for nanopore and diagnostic technology, researchers have engineered a smart bacterial pore that can change shape to detect different types of molecules. The study by researchers from BRIC-Rajiv Gandhi Centre for Biotechnology, Tata Institute of Fundamental Research, and VIT-AP University, focuses on a protein called CymA, found in the outer membrane of the bacteria Klebsiella oxytoca. By chemically stapling and unstapling a specific loop within this protein, the researchers have proven that the pore is not static and can act as a dynamic machine. The team have harnessed this movement to create a highly tunable sensor.

Nanopore technology involves sequencing nucleic acids (DNA/RNA) by passing them through a tiny hole, or nanopore, in a membrane. If an electric current is passed through this hole, the flow of ions creates a steady signal. However, when a molecule, like a sugar or a piece of DNA, passes through, it blocks the hole, causing a dip in the current. By analysing these dips, researchers can identify precisely what molecule just passed by. This technology is currently revolutionising how we sequence DNA and detect disease markers. However, most biological nanopores used today are rigid and cannot easily adapt to detect molecules of different sizes.

To address the rigidity, the researchers of the new study turned to the CymA protein. Unlike a simple open tube, CymA has a long N-terminal loop, which is a string of amino acids that hangs inside the pore, functioning like a gate. For years, researchers have debated the function of this loop. Some computer models suggested it moved in and out to allow food to enter the bacteria, but there was no direct experimental evidence. The research team devised a way to settle this debate and utilise the mechanism. They used site-directed mutagenesis to replace specific amino acids in the protein with cysteine. This allowed them to create a chemical staple, specifically, a disulfide bond, that locked the N-terminal loop to the inner wall of the barrel, effectively pinning the gate in a fixed position.

When the researchers tested this stapled CymA using electrical recordings, they found that the pore became constricted. The electrical current dropped significantly because the loop was blocking the flow of ions. In this locked state, the pore was too narrow for large sugar molecules, known as cyclodextrins, to pass through. However, it proved to be an excellent sensor for small peptides. The fixed loop acted like a precise filter, allowing the team to detect these small molecules with high resolution. This showed that when the loop is held still, the pore behaves like a narrow channel, suited to screening small targets.

The team then took the experiment a step further by adding a chemical called dithiothreitol (DTT), which acts as a key to unlock the staple. This chemical reaction broke the disulfide bond, releasing the loop from the barrel wall. Almost immediately, the electrical properties of the pore changed. The channel opened up, the current increased, and the pore regained its ability to transport bulky cyclic sugars. This transition from a stapled to an unstapled state provided the first direct experimental evidence that the N-terminal loop is naturally dynamic. It must move and flex to allow large molecules to enter the cell.

Building on this discovery, the researchers engineered an improved version of the CymA protein, dubbed the CymA R5C mutant. In the natural protein, the loop is often held loosely in place by electrostatic forces, like a weak magnet. This can cause gating or electrical noise that interferes with sensing. The R5C mutant was designed to remove this sticky interaction, allowing the loop to move freely without getting stuck. This advanced pore proved to be the most effective sensor of all, capable of detecting a wide variety of cyclic sugars at the single-molecule level without the background noise found in the natural protein.

In the past, researchers trying to use CymA as a sensor would often simply cut off the N-terminal loop entirely to make a clear hole. While this created an open pore, it also removed the protein's natural selectivity. Other studies relied on computer simulations to guess how the loop moved. This new research not only confirms the movement experimentally but also demonstrates that the loop is a feature, not a bug. By controlling the loop rather than removing it, the researchers created a dual-mode sensor that can be tuned for different tasks.

By engineering biological pores that can be tuned to detect specific molecules, we move closer to developing rapid, handheld diagnostic devices. These sensors could be used to detect biomarkers for diseases, analyse complex sugars involved in biological processes, or even sequence proteins. The ability to switch a sensor's mode from small-molecule to large-molecule detection suggests that future devices could perform multiple tests on a single chip, making medical diagnostics cheaper, faster, and more versatile.

This article was written with the help of generative AI and edited by an editor at Research Matters.