We report on the ability to control the dynamics of a single peptide capture and passage across a voltage-biased, α-hemolysin nanopore (α-HL), under conditions that the electroosmotic force exerted on the analyte dominates the electrophoretic transport. We demonstrate that by extending outside the nanopore, the electroosmotic force is able to capture a peptide at either the lumen or vestibule entry of the nanopore, and transiently traps it inside the nanopore, against the electrophoretic force. Statistical analysis of the resolvable dwell-times of a metastable trapped peptide, as it occupies either the β-barrel or vestibule domain of the α-HL nanopore, reveals rich kinetic details regarding the direction and rates of stochastic movement of a peptide inside the nanopore. The presented approach demonstrates the ability to shuttle and study molecules along the passage pathway inside the nanopore, allows to identify the mesoscopic trajectory of a peptide exiting the nanopore through either the vestibule or β-barrel moiety, thus providing convincing proof of a molecule translocating the pore. The kinetic analysis of a peptide fluctuating between various microstates inside the nanopore, enabled a detailed picture of the free energy description of its interaction with the α-HL nanopore. When studied at the limit of vanishingly low transmembrane potentials, this provided a thermodynamic description of peptide reversible binding to and within the α-HL nanopore, under equilibrium conditions devoid of electric and electroosmotic contributions.

Peptide nucleic acids (PNAs) are artificial, oligonucleotides analogues, where the sugar-phosphate backbone has been substituted with a peptide-like N-(2-aminoethyl)glycine backbone. Because of their inherent benefits, such as increased stability and enhanced binding affinity toward DNA or RNA substrates, PNAs are intensively studied and considered beneficial for the fields of materials and nanotechnology science. Herein, we designed cationic polypeptide-functionalized, 10-mer PNAs, and demonstrated the feasible detection of hybridization with short, complementary DNA substrates, following analytes interaction with the vestibule entry of an α-hemolysin (α-HL) nanopore. The opposite charged state at the polypeptide-functionalized PNA-DNA duplex extremities, facilitated unzipping of a captured duplex at the lumen entry of a voltage-biased nanopore, followed by monomers threading. These processes were resolvable and identifiable in real-time, from the temporal profile of the ionic current through a nanopore accompanying conformational changes of a single PNA-DNA duplex inside the α-HL nanopore. By employing a kinetic description within the discrete Markov chains theory, we proposed a minimalist kinetic model to successfully describe the electric force-induced strand separation in the duplex. The distinct interactions of the duplex at either end of the nanopore present powerful opportunities for introducing new generations of force-spectroscopy nanopore-based platforms, enabling from the same experiment duplex detection and assessment of interstrand base pairing energy.

The synergy of life sciences discoveries, biomolecular and protein engineering advances, and groundbreaking nanofabrication technologies, has introduced over the past years the wide use of the nanopore-based investigations of matter at the molecular level. This review focuses on the fundamental principles of α-hemolysin (α-HL) protein-based nanopores, as sensitive investigative tools that combine single-molecule detection with the ability to simultaneously manipulate single molecules, in otherwise complex samples. Herein, there are presented some of the efforts directed to control the capture dynamics and translocation speed of tailored polypeptides through the α-HL nanopore, by harnessing the electro-osmotic flow and nanopore-tweezing influence on individual molecules, which are engineered to resemble macrodipoles. The reported applications of this approach suggest a solution to enhance the temporal resolution of nanopore detection, prove the capability of the system in distinguishing between groups of distinct amino acids from the studied poly peptides, and propose a strategy to translate such single-molecule sensors in devices suitable for polypeptide sequencing at unimolecular level.

Metal ions binding exert a crucial influence upon the aggregation properties and stability of peptides, and the propensity of folding in various substates. Herein, we demonstrate the use of the α-HL protein as a powerful nanoscopic tool to probe Cu(2+)-triggered physicochemical changes of a 20 aminoacids long, antimicrobial-derived chimera peptide with a His residue as metal-binding site, and simultaneously dissect the kinetics of the free- and Cu(2+)-bound peptide interaction to the α-HL pore. Combining single-molecule electrophysiology on reconstituted lipid membranes and fluorescence spectroscopy, we show that the association rate constant between the α-HL pore and a Cu(2+)-free peptide is higher than that of a Cu(2+)-complexed peptide. We posit that mainly due to conformational changes induced by the bound Cu(2+) on the peptide, the resulting complex encounters a higher energy barrier toward its association with the protein pore, stemming most likely from an extra entropy cost needed to fit the Cu(2+)-complexed peptide within the α-HL lumen region. The lower dissociation rate constant of the Cu(2+)-complexed peptide from α-HL pore, as compared to that of Cu(2+)-free peptide, supports the existence of a deeper free energy well for the protein interaction with a Cu(2+)-complexed peptide, which may be indicative of specific Cu(2+)-mediated contributions to the binding of the Cu(2+)-complexed peptide within the pore lumen.

Recent evidence shows that metal coordination by amyloid beta peptides (Aβ) determines structural alterations of peptides, and His-13 from Aβ is crucial for Cu(2+) binding. This study used the truncated, more soluble Aβ1-16 isoforms derived from human and rat amyloid peptides to explore their interaction with Cu(2+) by employing the membrane-immobilized α-hemolysin (α-HL) protein as a nanoscopic probe in conjunction with single-molecule electrophysiology techniques. Unexpectedly, the experimental data suggest that unlike the case of the human Aβ1-16 peptide, Cu(2+) complexation by its rat counterpart leads to an augmented association and dissociation kinetics of the peptide reversible interaction with the protein pore, as compared to the Cu(2+)-free peptide. Single-molecule electrophysiology data reveal that both human and rat Cu(2+)-complexed Aβ peptides induce a higher degree of current flow obstruction through the α-HL pore, as compared to the Cu(2+)-free peptides. It is suggested that morphology changes brought by Cu(2+) binding to such amyloidic fragments depend crucially upon the presence of the His-13 residue on the primary sequence of such peptide fragments, and the α-HL protein-based approach provides unique opportunities and challenges to probing metal-induced folding of peptides.