Controlled Translocation of Proteins through a Biological Nanopore for Single-Protein Fingerprint Identification.

After the successful sequencing of nucleic acids, nanopore technology has now been applied to proteins. Recently, it has been demonstrated that an electro-osmotic flow can be used to induce the transport of unraveled polypeptides across nanopores. Polypeptide translocation, however, is too fast for accurate reading its amino acid compositions.

Blobs form during the single-file transport of proteins across nanopores.

The transport of biopolymers across nanopores is an important biological process currently under investigation for the rapid analysis of DNA and proteins. While the transport of DNA is generally understood, methods to induce unfolded protein translocation have only recently been discovered (Yu et al., 2023, Sauciuc et al.

Controlling Electroosmosis in Nanopores Without Altering the Nanopore Sensing Region.

Nanopores are powerful tools for single-molecule sensing of biomolecules and nanoparticles. The signal coming from the molecule to be analyzed strongly depends on its interaction with the narrower section of the nanopore (constriction) that may be tailored to increase sensing accuracy. Modifications of nanopore constriction have also been commonly used to induce electroosmosis, that favors the capture of molecules in the nanopore under a voltage bias and independently of their charge.

Unravelled proteins form blobs during translocation across nanopores.

The electroosmotic-driven transport of unravelled proteins across nanopores is an important biological process that is now under investigation for the rapid analysis and sequencing of proteins. For this approach to work, however, it is crucial that the polymer is threaded in single file. Here we found that, contrary to the electrophoretic transport of charged polymers such as DNA, during polypeptide translocation blob-like structures typically form inside nanopores.

Translocation of linearized full-length proteins through an engineered nanopore under opposing electrophoretic force.

Nanopores have recently been used to identify and fingerprint proteins. However, because proteins, unlike DNA, do not have a uniform charge, the electrophoretic force cannot in general be used to translocate or linearize them. Here we show that the introduction of sets of charges in the lumen of the CytK nanopore spaced by ~1 nm creates an electroosmotic flow that induces the unidirectional transport of unstructured natural polypeptides against a strong electrophoretic force.

Mechanisms of the neuromuscular blocking activity of the aminoglycoside antibiotic streptomycin.

While increasing the spontaneous quantal transmitter release in frog neuromuscular junctions, streptomycin blocked the evoked transmitter release by decreasing the quantal content (m). The quantal parameter of release n (number of release sites) was significantly decreased, while p (probability of release through transmitter activation) increased. Streptomycin competes with calcium ions for the membrane release sites while the intracellular mechanisms of release remain unaffected.