Single-Molecule Dynamics and Discrimination between Hydrophilic and Hydrophobic Amino Acids in Peptides, through Controllable, Stepwise Translocation across Nanopores

Abstract

In this work, we demonstrate the proof-of-concept of real-time discrimination between patches of hydrophilic and hydrophobic monomers in the primary structure of custom-engineered, macro-dipole-like peptides, at uni-molecular level. We employed single-molecule recordings to examine the ionic current through the α-hemolysin (α-HL) nanopore, when serine or isoleucine residues, flanked by segments of oppositely charged arginine and glutamic amino acids functioning as a voltage-dependent “molecular brake” on the peptide, were driven at controllable rates across the nanopore. The observed differences in the ionic currents blockades through the nanopore, visible at time resolutions corresponding to peptide threading through the α-HL’s constriction region, was explained by a simple model of the volumes of electrolyte excluded by either amino acid species, as groups of serine or isoleucine monomers transiently occupy the α-HL. To provide insights into the conditions ensuring optimal throughput of peptide readout through the nanopore, we probed the sidedness-dependence of peptide association to and dissociation from the electrically and geometrically asymmetric α-HL.

Nanopore‐Based Protein Sequencing Using Biopores: Current Achievements and Open Challenges

“…By using a similar paradigm, we proved the ability of the α-HL nanopore to identify three-amino acids long patches of polar S (serine) and aliphatic I (isoleucine) residues in the primary structure of polypeptides. [73] Recently, the robustness of our approach was also confirmed by independent experimental results by Zhao et al [127] and Pérez et al [128] that employed a FraC nanopore, and by coarse-grained computational analysis by Ghosh and Chaudhury. [129] If combined with click addition of positive and negative tails to the termini of a short peptide, this approach for residues identification can be applied to any peptide construct, regardless of its charge.…”

“…Major hurdles that limit the accurate polypeptide sequencing at single amino acid resolution, include: i) the dimension of the sensitivity region inside the nanopore, usually the most constricted domain of the inner volume, does not scale with the size of individual amino acids, implying that current fluctuations schematically shown in Figure 1II,b, are not correlated with the passage of individual residues; [ 53 ] ii) with the exception of simplified, engineered peptide constructs, [ 72,73 ] the heterogenous charge distribution along the polypeptide's sequence hinders the controlled, electrophoretic‐driven translocation of the polypeptide through nanopore. To add to this complexity, peptide‐nanopore nonspecific interactions, as well as and the peptide length, are relevant factors during such peptide recognition attempts; [ 74,75 ] iii) the high velocity of the polymer's passage across the nanopore poses serious challenges to unraveling the identity of the moving monomers.…”

Insights into protein sequencing with an α-Hemolysin nanopore by atomistic simulations

“…It is worth noting that recent experiments indicate that α HL is able to distinguish among three-block peptides where the central neutral residues were Alanine and Triptophan 35 , or Isoleucine and Serine 49 . Moreover, very recently Piquet et al .…”

The Nanopore-Tweezing-Based, Targeted Detection of Nucleobases on Short Functionalized Peptide Nucleic Acid Sequences

“…Encouraged by the successful application of the nanopore-tweezer technique for single-molecule interrogation of the primary structure on model polypeptides [ 50 , 51 ], we embarked, herein, on a “proof-of-concept” attempt to demonstrate bases’ recognition and discrimination on engineered PNAs with a similar approach ( Figure 1 ).…”