Uncovering hidden protein conformations with high bandwidth nanopore measurements

Advanced nanopore measurements allow structural probing of molecules with high spatial and temporal resolution. We report high signal-to-noise, 1-10 MHz bandwidth, translocation measurements of the multi-state folding of heme protein cytochrome c in KCl solution through optimally designed silicon nitride pores of 2.3-3.3 nm diameter and 3.6-3.8 nm effective thickness, and an optimal concentration of a denaturant (Gdm-Cl). The pore diameter is slightly smaller than the protein size, forcing the protein to squeeze through the pore. The sufficiently large pore thickness allows enough time for protein probing at an applied field of approximately 250 kV/cm. Through Bayesian Information Criterion score analysis, current blockades reveal six distinct levels, attributed to specific protein states. We calculate the transition probabilities between the states and the conditional probabilities of the protein leaving the pore from each state. We validate the model by simulating events and comparing them to experimental data.