A fundamental question in peptide folding/unfolding is how the peptide fleets through a set of transition states which dominate the dynamics of biomolecular folding path. Owing to their rapid duration and sub-nm structure difference, however, they have always been oversimplified because of limited instrumental resolution.^1-3 Moreover, the most experiments indicate a single fold pathway while the simulations suggest peptides owns the preference in multiple pathways.

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... ing the electrochemical confined effect of a solid-state nanopore, we measured the multiple transit paths of peptide inside nanopores. </a>Combining with Markov chain modelling, this new single-molecule technique is applied to clarify the 5 transition paths of the <a>β-hairpin </a>peptide which shows 4 nonequilibrium fluctuating stages. These results enable experimental access to previously obscured peptide dynamics which are essential to understand the misfolding in peptides. The statistical analysis of each peptide from high throughput shows that 78.5% of the peptide adopts the Pathways I during their folding/unfolding in a nanopore while 21.5% of the peptide undergoes the hidden folding/unfolding of transit Pathways II-IV. The frequency of the ionic fluctuation reveals a harmonic structure difference of the <a>metastable</a> peptide. Our results suggest the folding/unfolding of β-hairpin undergo four major structure vibrations which agree well with the theoretical expectation. These measurements provide a first look at the critical experiment picture of the mechanical folding/unfolding of a peptide, opening exciting avenues for the high <a></a><a>throughput</a> investigation of transition paths.