Peptide properties predict multistate translocation kinetics via protective antigen nanopores.

Single-molecule analysis of guest-host peptide translocations via anthrax toxin protective antigen (PA) nanopores reveals a multistate kinetic mechanism. K-means clustering identified four distinct conductance states for all peptides tested, including a fully blocked state (state 0), two intermediates (states 1 and 2), and a fully open pore (state 3). Multiexponential kinetic analysis of state-to-state transitions was performed, and the resulting lifetimes and amplitudes were correlated with molecular properties of the guest residue. These correlations revealed which physical properties govern the overall mechanism. The fully blocked state 0 acts as a "hydrophobic trap," with the lifetime of entry transitions (e.g., 1→0) strongly predicted by side-chain hydrophobicity. Conversely, escaping this trap is a steric process governed by molecular size, though the probability of a fast escape is uniquely facilitated by aromaticity, suggesting a specific ungating interaction with the pore's ϕ clamp, which is consistent with clamp site dilation. Rearrangements between partially blocked states are also dominated by hydrophobicity, reflecting solvation/desolvation of guest residues and clamp site during conformational rearrangements. Final dissociation to open nanopore is a multipathway process where the dominant physical force depends on the starting state: escape from deeper states is an energetic battle against hydrophobicity and aromaticity, whereas escape from shallower states presents a final steric hurdle. Overall, this work dissects the peptide translocation process, demonstrating how distinct physical forces-hydrophobicity, sterics, and aromaticity-govern specific, sequential steps of intrapore dynamics and release, providing a detailed energy landscape for peptide-nanopore interactions.