Discrimination of L- and D-chirality in oligoarginine peptides using the wild-type and R220s mutant aerolysin pores.

Abstract

Amino acid chirality may have significant roles in diseases such as Alzheimer's and certain forms of cancer. Today, LC-MS/MS and circular dichroism are used to distinguish L and D forms. However, no single molecule techniques are available to analyze chiral variability of peptides. Here, nanopores formed by the bacterial toxin aerolysin (AeL) were used to differentiate the three octaarginines RRRR-RRRR (R8), rrrr-rrrr (r8) and rrrr-RRRR (r4-R4), where R: L-R and r: D-R. Trans negative potentials were applied to pores reconstituted in DPhPC-bilayers in 4 M KCl. Using wild-type (wt) AeL, we found distinctive behaviours of the relative residual conductance of the peptide-blocked state (I/Io) with an increase in negative bias (−30 to −70 mV): I/Io decreased for R8 (0.357±0.002 to 0.352±0.002), remained constant for r8 (0.360±0.002) and increased for r4-R4 (0.353±0.001 to 0.359±0.001). As a consequence, the sequence of I/Io values was r8>R8>r4-R4 at −30 mV with R8 and r4-R4 trading places at about −45 mV. In contrast, with the R220S mutant of AeL, I/Io for R8 and r8 decreased in parallel from −30 to −70 mV (0.332±0.002 to 0.322±0.002 for R8 and 0.340±0.004 to 0.333±0.002 for r8) but remained constant for r4-R4 (0.332±0.002). Characteristic durations of blocked states were more than 2-fold enhanced with R220S vs. wt-AeL, were similar for R8 and r8 but, surprisingly, about 2.5-fold longer for r4-R4 in both pores, with maxima occurring at −30 mV (R220S) and −40 mV (wt). Ionic current through the aerolysin pore thus appears to be differentially modulated by L- and D-residues of peptides. Differential voltage-dependencies of I/Io and prolonged dwell time of the heterochiral peptide might be clues to a mechanism involving peptide conformation and warrant further experimental and theoretical study.