Alpha-hemolysin nanopore allows discrimination of the microcystins variants.

Microcystins (MCs) are a class of cyclic heptapeptides with more than 100 variants produced by cyanobacteria present in surface waters. MCs are potent hepatotoxic agents responsible for fatal poisoning in animals and humans. Several techniques are employed in the detection of MCs, however, there is a shortage of methods capable of discriminating variants of MCs. In this work we demonstrate that the α-hemolysin (αHL) nanopore can detect and discriminate the variants (LR, YR and RR) of MCs in aqueous solution. The discrimination process is based on the analysis of the residence times of each variant of MCs within the unitary nanopore, as well as, on the amplitudes of the blockages in the ionic current flowing through it. Simulations of molecular dynamics and calculation of the electrostatic potential revealed that the variants of MCs present different charge distribution and correlated with the three patterns on the amplitudes of the blockages in the ionic current. Additionally, molecular docking analysis indicates different patterns of interaction of the variants of MCs with two specific regions of the nanopore. We conclude that αHL nanopore can discriminate variants of microcystins by a mechanism based mainly on electrostatic interaction. Finally, we propose the use of nanopore-based technology as a promising method for analyzing microcystins in aqueous solutions.

Inhibition of the hemolytic activity caused by Staphylococcus aureus alpha-hemolysin through isatin-Schiff copper(II) complexes.

A great number of pathogens secrete pore-forming proteins during infection. Such molecules, from either bacterial or viral origin, are considered important virulence factors, which makes them attractive targets in the study of new therapeutic agents. Thus, the inhibitory activity of isatin-Schiff base copper(II) complexes was evaluated against membrane damage activity of Staphylococcus aureus α-hemolysin (α-HL). For this purpose, a standard hemolysis assay with rabbit erythrocytes and micromolar concentrations of the compounds was employed. Additionally, planar artificial lipid membranes with a single α-HL ion channel and molecular docking studies were used to elucidate the molecular mechanism of the complexes. Accordingly, the compounds were observed to possess a significant anti-hemolytic activity, capable of interacting with the constriction region of α-HL channel and blocking it in a potential dependent manner. Based on these results, it is expected that such isatin-Schiff base Copper(II) complexes may be employed as cotherapeutic agents for the treatment of staphylococcal infections.

Hofmeister effect in confined spaces: halogen ions and single molecule detection.

Despite extensive research in the nanopore-sensing field, there is a paucity of experimental studies that investigate specific ion effects in confined spaces, such as in nanopores. Here, the effect of halogen anions on a simple bimolecular complexation reaction between monodisperse poly(ethylene glycol) (PEG) and α-hemolysin nanoscale pores have been investigated at the single-molecule level. The anions track the Hofmeister ranking according to their influence upon the on-rate constant. An inverse relationship was demonstrated for the off-rate and the solubility of PEG. The difference among anions spans several hundredfold. Halogen anions play a very significant role in the interaction of PEG with nanopores although, unlike K(+), they do not bind to PEG. The specific effect appears dominated by a hydration-dehydration process where ions and PEG compete for water. Our findings provide what we believe to be novel insights into physicochemical mechanisms involved in single-molecule interactions with nanopores and are clearly relevant to more complicated chemical and biological processes involving a transient association of two or more molecules (e.g., reception, signal transduction, enzyme catalysis). It is anticipated that these findings will advance the development of devices with nanopore-based sensors for chemical and biological applications.

Mechanism of KCl enhancement in detection of nonionic polymers by nanopore sensors.

The mechanisms of KCl-induced enhancement in identification of individual molecules of poly(ethylene glycol) using solitary alpha-hemolysin nanoscale pores are described. The interaction of single molecules with the nanopore causes changes in the ionic current flowing through the pore. We show that the on-rate constant of the process is several hundred times larger and that the off-rate is several hundred times smaller in 4 M KCl than in 1 M KCl. These shifts dramatically improve detection and make single molecule identification feasible. KCl also changes the solubility of poly(ethylene glycol) by the same order of magnitude as it changes the rate constants. In addition, the polymer-nanopore interaction is determined to be a strong non-monotonic function of voltage, indicating that the flexible, nonionic poly(ethylene glycol) acts as a charged molecule. Therefore, salting-out and Coulombic interactions are responsible for the KCl-induced enhancement. These results will advance the development of devices with sensor elements based on single nanopores.

Single-molecule mass spectrometry in solution using a solitary nanopore.

We introduce a two-dimensional method for mass spectrometry in solution that is based on the interaction between a nanometer-scale pore and analytes. As an example, poly(ethylene glycol) molecules that enter a single alpha-hemolysin pore cause distinct mass-dependent conductance states with characteristic mean residence times. The conductance-based mass spectrum clearly resolves the repeat unit of ethylene glycol, and the mean residence time increases monotonically with the poly(ethylene glycol) mass. This technique could prove useful for the real-time characterization of molecules in solution.

Single polymer molecules in a protein nanopore in the limit of a strong polymer-pore attraction.

The capture and release of single poly(ethylene glycol) molecules by the alpha-Hemolysin pore are observed as time-resolved reversible steps in ion conductance. The capture on rate, inferred from the step frequency, decreases monotonically with polymer size. However, the polymer residence time shows a crossover behavior, first increasing and then decreasing with molecular weight. Our interpretation is that, in the case of polymers which are too large to be accommodated within the pore, the out-of-the-pore part of the molecule pulls on the trapped part, thus acting as an entropic spring.

Field-dependent effect of crown ether (18-crown-6) on ionic conductance of alpha-hemolysin channels.

Closing linear poly(ethylene glycol) (PEG) into a circular "crown" dramatically changes its dynamics in the alpha-hemolysin channel. In the electrically neutral crown ether (C2H4O)6, six ethylene oxide monomers are linked into a circle that gives the molecule ion-complexing capacity and increases its rigidity. As with linear PEG, addition of the crown to the membrane-bathing solution decreases the ionic conductance of the channel and generates additional conductance noise. However, in contrast to linear PEG, both the conductance reduction (reporting on crown partitioning into the channel pore) and the noise (reporting on crown dynamics in the pore) now depend on voltage strongly and nonmonotonically. Within the whole frequency range accessible in channel reconstitution experiments, the noise power spectrum is "white", showing that crown exchange between the channel and the bulk solution is fast. Analyzing these data in the framework of a Markovian two-state model, we are able to characterize the process quantitatively. We show that the lifetime of the crown in the channel reaches its maximum (a few microseconds) at about the same voltage (approximately 100 mV, negative from the side of protein addition) where the crown's reduction of the channel conductance is most pronounced. Our interpretation is that, because of its rigidity, the crown feels an effective steric barrier in the narrowest part of the channel pore. This barrier together with crown-ion complexing and resultant interaction with the applied field leads to behavior usually associated with voltage-dependent binding in the channel pore.