Controlling Nanopore Dynamics via Loop Stapling and Unstapling for Tunable Substrate Transport

Biological nanopores with diverse geometries are versatile tools for nanopore biosensing. CymA, a membrane porin specialized for large cyclic sugar uptake, has potential sensing applications owing to its unique architecture. This architecture features an unusual, constricted N terminus in the pore center, proposed to regulate substrate transport, although its dynamic role remains poorly understood. Therefore, we investigate the CymA conformational dynamics using protein engineering, electrical recordings, and molecular dynamics simulations. We engineered a mutant, "stapled CymA" with the N terminus confined within the pore lumen by a disulfide bond, restricting large cyclic sugar translocation while facilitating passage of small peptides. The disruption of this disulfide bond enables the transition to "unstapled CymA," restoring N terminus dynamic flexibility, facilitating cyclic sugar translocation. This conformational control of the pore enables dual-mode sensing of small and large analytes. Based on these insights, we engineered a superior CymA featuring a dynamic, untethered N terminus for single-molecule detection of various cyclic sugars. In this design, the electrostatic interactions between the N terminus and the pore barrel are disrupted, thereby minimizing gating and facilitating effective sensing. These findings establish N terminus flexibility as a tunable structural element for regulating molecular transport and offer an advanced strategy for designing dynamic nanopores for sensing.