Nanopore Tweezer Design and Operating Principles for Resolving Native Protein Conformational Dynamics.

Single-molecule methods that track protein conformational dynamics in real time can reveal transient states hidden by ensemble averaging. Nanopore tweezers achieve this by confining natively folded proteins within engineered nanopore lumens and monitoring ionic current signatures that report discrete conformational states. However, nanopore architecture and recording conditions may affect both measurement quality and analyte behavior. Here, we systematically examine how luminal surface properties, pore size, and applied voltage influence capture, residence time, and conformational-state resolution for the West Nile virus NS2B/NS3 protease, a two-component flaviviral enzyme with functionally important open-closed transitions. We show that apparent protease dynamics are sensitive to single-residue changes within the lumen and to applied voltage, while signal resolution also depends on pore size. These results define an analyte-specific "Goldilocks" operating window that optimizes current separation and dwell times while minimizing perturbation of native dynamics, guiding rational nanopore design and recording-parameter selection.