Effect of charge, topology and orientation of the electric field on the interaction of peptides with the α-hemolysin pore.

Nanopore analysis is an emerging technique of structural biology which employs nanopores, such as the α-hemolysin pore, as a biosensor. A voltage applied across a membrane containing a nanopore generates a current, which is partially blocked when a molecule interacts with the pore. The magnitude (I) and the duration (T) of the current blockade provide an event signature for that molecule. Two peptides, CY12(+)T1 and CY12(-)T1 with net charges + 2 and - 2, respectively, were analysed using different applied voltages and all four possible orientations of the electrodes and pore. The four orientations were vestibule downstream (VD), vestibule upstream (VU), stem downstream (SD) and stem upstream (SU) where vestibule and stem refer to the side of the pore on which the peptide was placed and downstream and upstream refer to the application of a positive or negative electrophoretic force, respectively. For CY12(+)T1, the effect of voltage on the event duration was consistent with translocation in the VD and SD configurations, but only intercalation events were observed in the VU and SU configurations. For CY12(-)T1, translocations were only observed in the VD and VU configurations. The results are interpreted in terms of two energy barriers on either side of the lumen of the pore. The difference in height of the barriers determines the preferred direction of exit. Electroosmotic flow and current rectification due to the pore as well as the dipole moment and charge of the peptide also play significant roles. Thus, factors other than simple electrophoresis are important for determining the interaction of small peptides with the pore.

Divalent cations induce a compaction of intrinsically disordered myelin basic protein.

Central nervous system myelin is a dynamic entity arising from membrane processes extended from oligodendrocytes, which form a tightly-wrapped multilamellar structure around neurons. In mature myelin, the predominant splice isoform of classic MBP is 18.5kDa. In solution, MBP is an extended, intrinsically disordered protein with a large effective protein surface for myriad interactions, and possesses transient and/or induced ordered secondary structure elements for molecular association or recognition. Here, we show by nanopore analysis that the divalent cations copper and zinc induce a compaction of the extended protein in vitro, suggestive of a tertiary conformation that may reflect its arrangement in myelin.

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.