Electrical unfolding of cytochrome during translocation through a nanopore constriction.

Prabhat Tripathi, Abdelkrim Benabbas, Behzad Mehrafrooz, Hirohito Yamazaki, Aleksei Aksimentiev, Paul M. Champion, and Meni Wanunu
Proceedings of the National Academy of Sciences 118(17) e2016262118 (2021)
DOI:10.1073/pnas.2016262118  PMID:33883276  BibTex

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Can localized electric fields drive the complete unfolding of a protein molecule? Protein unfolding prior to its translocation through a nanopore constriction is an important step in protein transport across biological membranes and also an important step in nanopore-based protein sequencing. We studied here the electric-field–driven translocation behavior of a model protein (cyt c) through nanopores of diameters ranging from 1.5 to 5.5 nm. These single-molecule measurements show that electric fields at the nanopore constriction can select both partially and fully unfolded protein conformations. Zero-field free energy gaps between these conformations, found using a simple thermodynamic model, are in remarkable agreement with previously reported studies of cyt c unfolding energetics.

Abstract

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome (cyt ) through ultrathin silicon nitride (SiN) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of ∼30 to 100 MV/m, cyt is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field-induced deformability.

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MD simulations of cyt c passage through nanopores: The movie illustrates MD trajectories where a single cyt c protein (shown as a cartoon enclosed by a semitransparent surface) was forced to pass through a 2.0 nm nanopore (gray) using the G-SMD protocol under a 3 V effective bias.

MD simulations of cyt c passage through nanopores: The movie illustrates MD trajectories where a single cyt c protein (shown as a cartoon enclosed by a semitransparent surface) was forced to pass through a 2.5 nm nanopore (gray) using the G-SMD protocol under a 3 V effective bias.

MD simulations of cyt c passage through nanopores: The movie illustrates MD trajectories where a single cyt c protein (shown as a cartoon enclosed by a semitransparent surface) was forced to pass through a 5.5 nm nanopore (gray) using the G-SMD protocol under a 1 V effective bias.