Nanopore analysis of tethered peptides

Peptides of 12 amino acids were tethered via a terminal cysteine to mono‐, di‐, tri‐, and tetrabromomethyl‐substituted benzene to produce bundles of one to four peptide strands (CY12‐T1 to CY12‐T4, respectively). The interaction of the bundles with the α‐hemolysin pore was assessed by measuring the blockade currents (I) and times (T) at an applied potential of ? 50, ? 100, and ? 150 mV. Three types of events could be distinguished: bumping events, with small I and short T where the molecule transiently interacts with the pore before diffusing away; translocation events, where the molecule threads through the pore with large I and the value of T decreases with increasing voltage; and intercalation events, where the molecule transiently enters the pore but does not translocate with large I and the value of T increases with increasing voltage. CY12‐T1 and CY12‐T2 gave only bumping and translocation events; CY12‐T3 and CY12‐T4 also gave intercalation events, some of which were of very long duration. The results suggest that three uncoiled peptide strands cannot simultaneously thread through the α‐hemolysin pore and that proteins must completely unfold in order to translocate. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.