Direct Observation of Single-Protein Transition State Passage by Nanopore Ionic Current Jumps

Conformational transitions of proteins are governed by chemical kinetics, often toggled by passage through an activated state separating two conformational ensembles. The passage time of a protein through the activated state can be too fast to be detected by single-molecule experiments without the aid of viscogenic agents. Here, we use high-bandwidth nanopore measurements to resolve microsecond-duration transitions that occur between conformational states of individual protein molecules partly blocking pore current. We measure the transition state passage time between folded and unfolded states of a two-state lambda(6-85) mutant and between metastable intermediates and the unfolded state of the multistate folder cytochrome c. Consistent with the principle of microscopic reversibility, the transition state passage time is the same for the forward and backward reactions. A passage time distribution whose tail is broader than a single exponential observed in cytochrome c suggests a multidimensional energy landscape for this protein.

Automated summary

Protein conformational transitions are controlled by chemical kinetics, with passage through an activated state separating different conformational ensembles. The passage time between different states of protein molecules was measured using high-bandwidth nanopore measurements, showing a multidimensional energy landscape for cytochrome c.