Threading single proteins through pores to compare their energy landscapes

Prabhat Tripathi, Arash Firouzbakht, Martin Gruebele, Meni Wanunu

Research output: Contribution to journalArticlepeer-review


Translocation of proteins is correlated with structural fluctuations that access conformational states higher in free energy than the folded state. We use electric fields at the solid-state nanopore to control the relative free energy and occupancy of different protein conformational states at the single-molecule level. The change in occupancy of different protein conformations as a function of electric field gives rise to shifts in the measured distributions of ionic current blockades and residence times. We probe the statistics of the ionic current blockades and residence times for three mutants of the λ-repressor family in order to determine the number of accessible conformational states of each mutant and evaluate the ruggedness of their free energy landscapes. Translocation becomes faster at higher electric fields when additional flexible conformations are available for threading through the pore. At the same time, folding rates are not correlated with ease of translocation; a slow-folding mutant with a low-lying intermediate state translocates faster than a faster-folding two-state mutant. Such behavior allows us to distinguish among protein mutants by selecting for the degree of current blockade and residence time at the pore. Based on these findings, we present a simple free energy model that explains the complementary relationship between folding equilibrium constants and translocation rates.

Original languageEnglish (US)
Article numbere2202779119
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number39
StatePublished - Sep 27 2022


  • electric field unfolding
  • nanopore biophysics
  • protein folding dynamics
  • protein translocation
  • reciprocal relation

ASJC Scopus subject areas

  • General


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