Alpha-hemolysin nanopore allows discrimination of the microcystins variants

Abstract

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.

Fig. 1. Structure of microcystin-LR. Variants differ with respect to the identity of l-amino acids in position 2 and 4. Numbers represent the position of the amino acid residue.

Fig. 2. α-Hemolysin nanopore detects microcystins. (A) Structural representation of variants MC-RR, MC-LR and MC-YR with residues at position X2 highlighted in black, (arginine), blue (leucine) and red (tyrosine) circles, respectively. (B) The profile of the microcystin-induced ionic current blockages differs to the MCs variants. Solution: 4 M KCl, 5 mM Tris–HCl, (MC-RR, 1 μM; or MC-LR, 1 μM; or MC-YR, 1 μM), pH 7.5. Transmembrane potential, 40 mV. The all-point histograms are shown at the right of each record and can be used to give the mean value of blockage amplitudes.

Fig. 3. α-Hemolysin nanopore is capable of discriminating MCs variants. (A) The profile of the microcystin-induced ionic current blockages for the three MC variant simultaneously in solution: 4 M KCl, 5 mM Tris–HCl, (MC-RR, 1 μM; and MC-LR, 1 μM; and MC-YR, 1 μM). The coloured lines indicate the blockage pattern for to MC-LR (blue), MC-RR (green) and MC-YR (red). (B) Docked MCs variants in the region of constriction of the αHL nanopore. In the upper structures, the MC-nanopore complexes are represented as surface in the context of the entire protein. In the lower images, the MC-RR, MC-LR and MC-YR molecular structures are shown as they interact with Glu111 residues of the region of constriction of the αHL nanopore.

Fig. 4. Influence of the transmembrane potential in the dwell time (τ_off) of MCs variants within αHL nanopore. (A) MC-LR, (B) MC-RR, (C) MC-YR. Concentration of MCs variants is 1000 μM in solution (4 M KCl, Tris–HCl 5 mM, pH 7.5). The result of at least three independent experiments (mean ± SE) is presented in each case.

Fig. 5. Influence of the electrostatic potential in interaction of MC-αHL nanopore. (A) Residence time for MC variants in nanopore, solutions: KCl 4 M or LiCl 4 M. The microcystin was added on the same side (cis or trans of the nanopore) in which the potential of 100 mV was applied. (B) Electrostatic potential represented in VMD for the outer surface of the α-HL nanopore. (C) Electrostatic potential represented in VMD for the inner surface (lumen) of the α-HL nanopore. (D–F) Electrostatic potential represented in VMD for the MCs variants. Electrostatic potentials are shown at isosurfaces of 5 kTe (blue) to −5 kTe (red).

Fig. 6. (A) Influence of the transmembrane potential on the on-rate constant (k_on) of the MC-αHL nanopore. (B) Transition rate and MCs variants concentration. (Data points) Means of at least three separate experiments. Solution (4 M KCl, Tris–HCl 5 mM, pH 7.5). (Data points) Means of at least three separate experiments.