Facilitated translocation of polypeptides through a single nanopore

Abstract

The transport of polypeptides through nanopores is a key process in biology and medical biotechnology. Despite its critical importance, the underlying kinetics of polypeptide translocation through protein nanopores is not yet comprehensively understood. Here, we present a simple two-barrier, one-well kinetic model for the translocation of short positively charged polypeptides through a single transmembrane protein nanopore that is equiped with negatively charged rings, simply called traps. We demonstrate that the presence of these traps within the interior of the nanopore dramatically alters the free energy landscape for the partitioning of the polypeptide into the nanopore interior, as revealed by significant modifications in the activation free energies required for the transitions of the polypeptide from one state to other. Our kinetic model permits the calculation of the relative and absolute exit frequencies of the short cationic polypeptides through either opening of the nanopore. Moreover, this approach enabled quantitative assessment of the kinetics of translocation of the polypeptides through a protein nanopore, which is strongly dependent on several factors, including the nature of the translocating polypeptide, the position of the traps, the strength of the polypeptide-attractive trap interactions and the applied transmembrane voltage.

OccK Channels fromPseudomonas aeruginosaExhibit Diverse Single-Channel Electrical Signatures but Conserved Anion Selectivity

“…In this case, the attractive sites are aromatic residues, thus reducing the activation free energies for sugar translocation from one side of the membrane to the other (37). In the past, we also demonstrated that attractive sites present within the channel lumen have major implications even for larger molecules, such as polypeptides traversing the channel (29;38), which is in accord with the theoretical predictions (39–42). …”

Interference-Free Detection of Genetic Biomarkers Using Synthetic Dipole-Facilitated Nanopore Dielectrophoresis

“…1a right). The K131D 7 pore was previously used by the Movileanu group to trap proteins containing positively charged tags 37, 38 . The nanocarrier used for driving the target nucleic acid into the nanopore contained a polycationic peptide tag (charge +8 e , Fig.…”

Global redesign of a native β-barrel scaffold

“…S17). For example, in a range of transmembrane potentials between +20 and +80 mV, the dwell time of Syn B2 within the pore interior of FhuA ΔC/Δ5L was between 0.08 and 0.18 ms. A biphasic voltage dependence of the dwell time or rate constant indirectly suggests that the relatively short Syn B2 polypeptide rapidly navigates across the pore interior of FhuA ΔC/Δ5L at potentials greater than a critical value [36–38]. The conspicuous lack of the 33% blockades might likely be determined by the rapid Syn B2 translocation.…”