Current Blockades of Proteins inside Nanopores for Real-Time Metabolome Analysis

Abstract

Biological nanopores are emerging as powerful and low-cost sensors for real-time analysis of biological samples. Proteins can be incorporated inside the nanopore, and ligand binding to the protein adaptor yields changes in nanopore conductance. In order to understand the origin of these conductance changes and develop sensors for detecting metabolites, we tested the signal originating from 13 different protein adaptors. We found that the quality of the protein signal depended on both the size and charge of the protein. The engineering of a dipole within the surface of the adaptor reduced the current noise by slowing the protein dynamics within the nanopore. Further, the charge of the ligand and the induced conformational changes of the adaptor defined the conductance changes upon metabolite binding, suggesting that the protein resides in an electrokinetic minimum within the nanopore, the position of which is altered by the ligand. These results represent an important step toward understanding the dynamics of the electrophoretic trapping of proteins inside nanopores and will allow developing next-generation sensors for metabolome analysis.

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

“…This process may be repeated as the MSA-DNA complex sticks and slides, effectively tumbling down the length of the pore before finally completing translocation. Although tumbling in nanopores has previously been reported for other proteins,(49, 129) we did not observe deep and prolonged multilevel events for streptavidin alone in the same pore (see Figure 2), emphasizing the role of the attached DNA.…”

Single-sulfur atom discrimination of polysulfides with a protein nanopore for improved batteries

“…These nanopore-based sensors are now employed for the detection of subtle chemical modifications 29,30 or to read the sequence of biomolecules 31,32 . Nanopores are also used to study protein conformational changes and transitions 33,34 , epigenetics by detection of DNA methylations 35,36 or for metabolome analysis 37 . Further pushing this technique to its limits researchers have recently identified the twenty amino acids 38 , paving the way to protein sequencing.…”

Strategies for enzymological studies and measurements of biological molecules with the cytolysin A nanopore

“…Once a protein is inside the nanopore, the signal can be similar to the open pore current (low-noise signal) or display a variety of different current levels (Zernia, van der Heide, Galenkamp, Gouridis, & Maglia, 2020). Although the protein signal can be improved for example by introducing a charge dipole within the protein (Van Meervelt et al, 2017) or N-or C-terminal tag (Willems et al, 2019), the origin of the noise of proteins inside the nanopore is not understood.…”

“…When testing a new protein, the binding of the ligands might or might not bring a change in the I B . Thus far, 1/3 of the protein adaptors we tested showed no ligand-induced current changes (Zernia et al, 2020). Hence, when we test a ligand for the first time, we add it into the cis chamber at a final concentration near the bulk dissociation constant (K d ).…”