Direct Observation of Electric Field-Driven Protein Translocation through the Single-Walled Carbon Nanotube Embedded in a Lipid Bilayer Membrane Revealed a High Dipole Moment of the Transition State

The translocation of molecules through nanoscale pores is ubiquitous in biology and widely used in biotechnology for nanopore sequencing and characterizing single biological molecules. Numerous artificial channels mimicking biological pores have been developed, and one of those is a single-walled carbon nanotube porin (CNTP) embedded in a lipid bilayer membrane. Although transport of ssDNA through CNTP has been demonstrated experimentally, protein translocation through CNTP has rarely been achieved. Here, we report the experimental observations of protein (cytochrome c ) translocation through a single-walled carbon nanotube of an inner diameter of ∼1.3 nm embedded in a lipid bilayer membrane without using any chemical denaturant, enzyme motor, or tagging with an oligonucleotide. The electric field was found to sufficiently unfold the cytochrome c protein to facilitate the translocation, as confirmed by a decrease in the average residence time of the protein at the CNTP upon increasing the transmembrane potential. A simple free energy model suggests that the relatively fast (microseconds) dynamic interconversions of protein conformational states appear to dictate the slow (∼100 ms) translocation and account for the observed voltage-dependent rate constants from the exponential residence time distributions. Our report on the experimental evidence of protein translocation through CNTP may open the opportunity to explore the applications of CNTP in targeted protein delivery, in nanopore proteomics, and probing protein conformational transitions at the single-molecule level.