Single-molecule studies of protein folding hold keys to unveiling protein folding pathways and elusive intermediate folding states-attractive pharmaceutical targets. Although conventional single-molecule approaches can detect folding intermediates, they presently lack throughput and require elaborate labeling. Here, we theoretically show that measurements of ionic current through a nanopore containing a protein can report on the protein's folding state. Our all-atom molecular dynamics (MD) simulations show that the unfolding of a protein lowers the nanopore ionic current, an effect that originates from the reduction of ion mobility in proximity to a protein. Using a theoretical model, we show that the average change in ionic current produced by a folding-unfolding transition is detectable despite the orientational and conformational heterogeneity of the folded and unfolded states. By analyzing millisecond-long all-atom MD simulations of multiple protein transitions, we show that a nanopore ionic current recording can detect folding-unfolding transitions in real time and report on the structure of folding intermediates.
Keywords: folding intermediates; ionic current; misfolding; molecular dynamics; nanopore; protein folding.