Molecular Determinants of Current Blockade Produced by Peptide Transport Through a Nanopore

Abstract

The nanopore sensing method holds the promise of delivering a single molecule technology for identification of biological proteins, direct detection of post-translational modifications, and perhaps de novo determination of a protein’s amino acid sequence. The key quantity measured in such nanopore sensing experiments is the magnitude of the ionic current passing through a nanopore blocked by a polypeptide chain. Establishing a relationship between the amino acid sequence of a peptide fragment confined within a nanopore and the blockade current flowing through the nanopore remains a major challenge for realizing the nanopore protein sequencing. Using the results of all-atom molecular dynamics simulations, here we compare nanopore sequencing of DNA with nanopore sequencing of proteins. We then delineate the factors affecting the blockade current modulation by the peptide sequence, showing that the current can be determined by (i) the steric footprint of an amino acid, (ii) its interactions with the pore wall, (iii) the local stretching of a polypeptide chain, and (iv) the local enhancement of the ion concentration at the nanopore constriction. We conclude with a brief discussion of the prospects for purely computational prediction of the blockade currents.

Resolving sulfation PTMs on a plant peptide hormone using nanopore sequencing

“…Sulfation on the same tyrosine residue similarly produced a signal peak but with a lower amplitude and an earlier onset when compared to phosphorylation (Figure 2A). This result is consistent with local salt modulation and charge-induced stretching of the polymer 3,20 . The di-anionic phosphotyrosine versus mono-anionic sulfotyrosine induces a higher local salt concentration and generates stronger stretching, and accordingly a delayed and higher signal peak.…”

“…The onset of this current increase occurs slightly before the tail starts translocating the nanopore (Figure 1F, dashed line). A recent molecular dynamics (MD) study discussed a similar effect from charged polymers during translocation 20 . A less densely-charged poly-T ssDNA tail resulted in only a minor ionic current elevation (Supp.…”

Sculpting conducting nanopore size and shape through de novo protein design

“…Unlike native pores, which are finite in number, there is no limit on the number of distinct designed pores that can be generated. With further optimization of synthetic TMB nanopore insertion into membranes in multichannel flow cells (e.g., by coupling the height of designed nanopores with that of matched thick synthetic membranes) ( 56 ), it should be possible to establish fast design-build-test loops to probe the relationship between the chemical properties of a nanopore and the detection of an analyte in the pore lumen ( 11 , 57 , 58 ). Our approach now enables the custom design of pore geometry and chemistry for applications ranging from detection and selective transport of a wide range of molecules of interest to biopolymer sequencing.…”

Sculpting conducting nanopore size and shape throughde novoprotein design

“…Further advances in understanding the relation between the chemical properties of a nanopore and the directed detection of an analyte in the pore lumen ( 11 , 53 , 54 ), coupled with our new ability to design custom pores, opens up exciting new directions for sequencing and sensing since the pore size and the side chains lining the pore can be designed specifically for the desired application. Unlike native pores, which are finite in number, there is no limit on the number of distinct designed pores that can be generated.…”