Unidirectional single-file transport of full-length proteins through a nanopore

Abstract

Nanopore sequencing is one of only a few methods that can potentially determine the amino acid sequence of individual protein molecules as these are passed through a pore sensor. However, mechanisms for unfolding and translocation of proteins are still unavailable to date. Here we describe a general approach for realizing unidirectional transport of full-length proteins through nanopores. We combine a chemically resistant biological nanopore platform with a high concentration guanidinium chloride buffer to achieve unidirectional, single-file protein transport that is propelled by a giant electro-osmotic effect, as revealed by molecular dynamics simulations and confirmed experimentally. Remarkably, we observed that protein velocities are uniform regardless of the protein sequence, which allows the identification and discrimination among proteins based on their electrical signatures, as well as to distinguish protein signatures by their threading orientation (N-to-C vs. C-to-N terminus). With average transport velocities of 10 µs per amino acid, our method can enable direct, enzyme-free protein fingerprinting and protein sequencing when combined with a high-resolution pore and high-speed nanopore readout.

Engineering Biological Nanopore Approaches toward Protein Sequencing

“…To date, much of the nanopore research exploiting EOF has been devoted to controlling the translocation and trapping of peptides by (1) modulating the magnitude and polarity of the applied voltages or the charge distribution of the analytes, 115,162,163 (2) introducing oppositely charged segments at the N-and Ctermini of polypeptides to create a dipolar feature, 114 (3) tuning the solution pH, 164 (4) introducing electro-osmotic vortices into the inner surface of nanopores, 165 (5) immobilizing dihydrofolate reductase (DHFR) inside a ClyA biological nanopore, 166 and (6) binding guanidinium cations to the inner surface of the nanopore. 167 Sequencing by Controlled Movement of Peptides in a Nanopore. Nanopores have gained substantial recognition in genomics by demonstrating ultralong read lengths for DNA and RNA sequencing.…”

“…Advances in computing power and simulation algorithms continue to push the boundaries of what can be achieved for these complex systems. For example, atomistic simulations were able to attribute the steady, unidirectional transport of proteins in guanidinium chloride solution 167 to a strong electro-osmotic effect produced by the binding of guanidinium ions to the surface of the nanopore. Notwithstanding the ongoing progress in the development of software and hardware, enabling ever faster MD simulations, the predictive modeling of nanopore blockade signatures in the context of protein sequencing remains difficult.…”

An engineered electroosmotic flow transports unravelled proteins across nanopores

“…Notably, previous work showed that unfolded proteins might be captured by β-barrel nanopores such as α-hemolysin (homologous to CytK) or aerolysin, in the presence of denaturants. [11][12][13]24,25 Some work reported the translocation of unmodified proteins, 24,25 while other attached either a ssDNA 11,12 or a poly-10 aspartate tag 13 at the N-or C-termini to drive the EPF-driven transport across nanopores. We tested malE219a and malE219a-D10ssrA (malE219a expanded with a D10ssrA tag at the C-terminus) in the presence of 1.5 M GuHCl and using WT-CytK.…”

“…[7][8][9] It was found, however, that the neutral peptides cannot be fully stretched by this approach. 9 Other examples, proteins were elongated with DNA [10][11][12] or negatively charged polypeptide tags, 13,14 which then produced an electrophoretic drag to capture the polypeptide. Then, enzymes or the combination of the EPF and EOF induced the translocation of polypeptides.…”

Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses

“…Therefore, this approach shows promise in the wide-time bandwidth evaluation of single-protein dynamics. Nanopore sensors can also illuminate numerous structural and functional characteristics of proteins, including their shape and size, enzymatic activity, − mechanical stability, ,, cotranslocational unfolding, − and post-translational modifications. − For example, a significant benefit is an ability to unravel dynamic fluctuations of protein sizes and conformations in solution using glass and solid-state nanopores. ,,, In addition, nanopores are nowadays utilized to conduct peptide and protein profiling. − More recently, several studies showed prospects of nanopores in single-molecule protein sequencing. − …”