Kinetic control and entropic barrier modulation of protein unfolding during translocation through stepped graphene nanopores.

Controlling protein unfolding and translocation through solid-state nanopores remains a significant challenge due to the steep entropic barriers and the propensity for kinetic jamming. We propose a stepped graphene nanopore architecture designed to decouple the thermodynamic and kinetic requirements of the translocation process. Using all-atom molecular dynamics simulations, we demonstrate that the stepped geometry reconfigures the electrostatic potential, smoothing the field gradient and partitioning the entropic barrier into manageable stages. Our results reveal a non-monotonic dependence of translocation success on the average trans-pore electric field, identifying an intermediate-field kinetic regime around 0.048 V Å-1 in which electrophoretic driving and protein conformational relaxation are better balanced under the present accelerated-sampling conditions. At a lower field of 0.024 V Å-1, translocation is suppressed by entropic rejection as the driving force fails to overcome the entry barrier. Conversely, at an elevated field of 0.096 V Å-1, a critical rate mismatch occurs: the excessive drift velocity outpaces the unfolding rate, triggering lateral buckling and irreversible steric jamming at the final constriction. Compared with a conventional single-step nanopore, the stepped architecture facilitates high-fidelity observation of unfolding intermediate states at moderate driving forces, thereby avoiding excessive driving conditions that promote kinetic jamming and reduce structural resolution. By establishing a kinetic-competition framework based on native-contact loss, residue-passage timing, and productive axial linearization, this study provides a physical framework for designing blockage-resistant nanopore sensors for high-resolution protein structural analysis.