The microscopic details of how peptides translocate one at a time through nanopores are crucial determinants for transport through membrane pores and important in developing nano-technologies. To date, the translocation process has been too fast relative to the resolution of the single molecule techniques that sought to detect its milestones. Using pH-tuned single-molecule electrophysiology and molecular dynamics simulations, we demonstrate how peptide passage through the α-hemolysin protein can be sufficiently slowed down to observe intermediate single-peptide sub-states associated to distinct structural milestones along the pore, and how to control residence time, direction and the sequence of spatio-temporal state-to-state dynamics of a single peptide. Molecular dynamics simulations of peptide translocation reveal the time- dependent ordering of intermediate structures of the translocating peptide inside the pore at atomic resolution. Calculations of the expected current ratios of the different pore-blocking microstates and their time sequencing are in accord with the recorded current traces.

Protein and solid-state nanometer-scale pores are being developed for the detection, analysis and manipulation of single molecules. In the simplest embodiment, the entry of a molecule into a nanopore causes a reduction in the latter’s ionic conductance. The ionic current blockade depth and residence time have been shown to provide detailed information on the size, adsorbed charge and other properties of molecules. Here we describe the use of the nanopore formed by Staphylococcus aureus α-hemolysin and polypeptides with oppositely charged segments at the N- and C-termini to increase both the polypeptide capture rate and mean residence time of them in the pore, regardless of the polarity of the applied electrostatic potential. The technique provides the means to improve the signal to noise of single molecule nanopore-based measurements.