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.

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Nematodes exhibit a vast array of cys-loop ligand-gated ion channels with unique pharmacologic characteristics. However, many of the structural components that govern the binding of various ligands are unknown. The nematode cys-loop GABA receptor uncoordinated 49 (UNC-49) is an important receptor found at neuromuscular junctions that plays an important role in the sinusoidal movement of worms. The unique pharmacologic features of this receptor suggest that there are structural differences in the agonist binding site when compared with mammalian receptors. In this study, we examined each amino acid in one of the main agonist binding loops (loop E) via the substituted cysteine accessibility method (SCAM) and analyzed the interaction of various residues by molecular dynamic simulations. We found that of the 18 loop E mutants analyzed, H142C, R147C, and S157C had significant changes in GABA EC and were accessible to modification by a methanethiosulfonate reagent (MTSET) resulting in a change in In addition, the residue H142, which is unique to nematode UNC-49 GABA receptors, appears to play a negative role in GABA sensitivity as its mutation to cysteine increased sensitivity to GABA and caused the UNC-49 receptor partial agonist 5-aminovaleric acid (DAVA) to behave as a full agonist. Overall, this study has revealed potential differences in the agonist binding pocket between nematode UNC-49 and mammalian GABA receptors that could be exploited in the design of novel anthelmintics.

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Molecular detection via nanopore, achieved by monitoring changes in ionic current arising from analyte interaction with the sensor pore, is a promising technology for multiplex sensing development. Outer Membrane Protein G (OmpG), a monomeric porin possessing seven functionalizable loops, has been reported as an effective sensing platform for selective protein detection. Using flow cytometry to screen unfavorable constructs, we identified two OmpG nanopores with unique peptide motifs displayed in either loop 3 or 6, which also exhibited distinct analyte signals in single-channel current recordings. We exploited these motif-displaying loops concurrently to facilitate singlemolecule multiplex protein detection in a mixture. We additionally report a strategy to increase sensor sensitivity via avidity motif display. These sensing schemes may be expanded to more sophisticated designs utilizing additional loops to increase multiplicity and sensitivity.