Nanopore analysis of tethered peptides

Abstract

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.

Binding of bovine T194A PrPCby PrPSc-specific antibodies

“…34,35 In nanopore analysis, generally, type-I events have small blockade currents and short blockade times and correspond to individual molecules bumping into the pore before rapidly diffusing away. 36 Type-II events have larger blockade currents and longer blockade times and correspond to either translocation of a molecule through the pore after unfolding or transient insertion of a loop or strand of the protein into the pore before diffusing back to the cis side. 36 This latter type of event has been called intercalation and, for proteins it is often difficult to distinguish between translocation and intercalation.…”

Nanopore analysis reveals differences in structural stability of ovine PrPCproteins corresponding to scrapie susceptible (VRQ) and resistance (ARR) genotypes

“…36, 37 Intercalation/ translocation events have larger blockade currents and times as compared with type I events. 37 At present it is very difficult to distinguish between intercalation and translocation events for proteins.…”

“…37 At present it is very difficult to distinguish between intercalation and translocation events for proteins. Therefore we refer to events having a blockade current of <40 pA as type I (bumping) and >40 pA as type II (intercalation/ translocation).…”

“…Experiments were conducted with an applied potential of -100 mV at a bandwidth of 10 KHz. [36][37][38][39]43 A perfusion unit made up of a black perfusion chamber containing a white perfusion cup that divided two chambers (cis and trans) by a 150 µm aperture was used for nanopore analysis. A lipid bilayer was formed by applying a synthetic lipid, (1, 2-diphytanoyl-sn-glycero-3-phosphocholine [Avanti Polar Lipids]) on the 150 µm aperture (Fig.…”

“…[33][34][35] Nanopore analysis also allows investigation of structural characteristics of individual peptides or proteins via characterization of their interactions with a biological pore. [36][37][38][39] In nanopore analysis, single molecules are interrogated with the pore to give rise to distinct event parameters, blockade current (I) and blockade time (T).…”