Smooth Voltage-Driven Translocation Of Full-Length Proteins Through Nanopores

Biological nanopores have become useful nanosensors that can read single DNA and RNA molecules with high resolution. The most common way to use these nanosensors is to insert a protein channel (e.g. α-hemolysin) into a phospholipid bilayer and apply voltage to drive ion current through the pore. When a DNA molecule traverses the pore, electromotive force on the DNA molecule drives it through the pore in a single-file manner. Compared to DNA, proteins have more complex secondary structures and non-uniform backbone charge, which makes single-file translocation of proteins through the pore difficult to achieve. Here we use a polymer-based membrane and a custom wedge-on-pillar aperture to allow high voltage and chemical denaturing of proteins for nanopore analysis. In comparison with a bilayer lipid membrane, the polymer membrane has superior mechanical and chemical stability under these conditions, which allows full-length protein translocation through alpha-hemolysin. By tagging full-length proteins (range from 200 - 800 aa) with a charged tail (10 aa), we demonstrate the efficient translocation of these proteins through the hemolysin pore, and further, we demonstrate that mean translocation times are linearly related to the protein length, irrespective of its native charge. Further, translocation times are slow enough to allow sufficient sampling (∼0.01 ms/aa). Simulations that probe the mechanism of such smooth translocations of these weakly-charged proteins reveal the role of denaturant-induced electroosmosis on the translocation process, which is key to successful translocations. When combined with a higher-resolution nanopore, we anticipate the ability to fingerprint unmodified protein molecules based on single molecule signals.