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.
Highlights
In nanopore-based resistive pulse sensing, fluctuations of ionic current flowing through a voltage-biased nanopore provide useful information about the identity and physico-chemical properties of a temporarily residing or translocating analyte [1,2,3,4,5,6]
Once captured inside the nanopore by an applied transmembrane potential, either peptide gets trapped in a metastable state with the nanopore’s constriction region temporarily occupied by the peptide’s middle domain residues (Figure 1), which constitute the main contributors to the recorded ionic current amplitude changes across the nanopore
The presented method demonstrates that α-HL represents a versatile single-molecule tool for identifying patches of polar and aliphatic residues in the primary structure of short peptides, via statistical analysis of ionic current fluctuations recorded during a peptide transit across the nanopore
Summary
In nanopore-based resistive pulse sensing, fluctuations of ionic current flowing through a voltage-biased nanopore provide useful information about the identity and physico-chemical properties of a temporarily residing or translocating analyte [1,2,3,4,5,6]. The system was successfully applied in a broad gamut of contexts, which started historically with detecting polynucleotides translocating through a nanopore [7,8,9,10], and eventually led to developing a rapid and inexpensive DNA sequencing technology [11] Another useful application of nanopore technology targets proteomics, for which it has been proven successfully to detect and distinguish between various conformations of proteins and peptides [12,13,14,15], and potentially sequence peptides or proteins [16,17,18,19,20,21,22]. One advantage of the α-HL for such purposes lies in its restricted geometry, meaning that ionic current blockades corresponding to reversible peptide-α-HL interactions reflect events associated to a captured peptide in the unfolded conformation, which is a prerequisite for the subsequent primary sequence readout
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