Electrical Unfolding of Cytochrome C During Translocation through a Nanopore Constriction

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 of transport, should be critical factors that govern whether protein translocation proceeds via squeezing, unfolding/threading, or both. To directly probe this, we studied the electric-field-driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiNx) 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 ∼30-100 MV/m cyt c is able to squeeze into the pore. Further, as electric fields inside the pore further increase the unfolded state of cyt c is stabilized, facilitating translocation. In contrast, for 1.5 nm and 2 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 transition energies between the metastable, intermediate, and unfolded protein states are extracted by exploiting differences in the state-dependent electric dipole interactions with the applied electric field. These experiments also map the various modes of protein translocation through a constriction, which opens new avenues for exploring protein folding structures, internal contacts, and electric field-induced deformability.