A series of negatively charged alpha-helical peptides of the general formula fluorenylmethoxycarbonyl (Fmoc)-D(x)A(y)K(z) were synthesized, where x and z were 1, 2, or 3 and y was 10, 14, 18, or 22. The translocation of the peptides through single pores, which were self-assembled into lipid membranes, was analyzed by measuring the current blockade i(block) and the duration t(block). The pores were either alpha-hemolysin, which has a wide vestibule leading into the pore, or aerolysin, which has no vestibule but has a longer pore of a similar diameter. Many thousands of events were measured for each peptide with each pore, and they could be assigned to two types: bumping events (type I) have a small i(block) and long t(block), and translocation events (type II) have a larger i(block) and shorter t(block). For type-II events, both i(block) and t(block) increase with the length of the peptides on both pores tested. The dipole moment and the net charge of each peptide has a major effect on the transport characteristics. The ratio of type-II/type-I events increases as the dipole moment increases, and uncharged peptides gave mostly type-I events. The structural differences between the two nanopores were reflected in the characteristic values of i(block), and in particular, the vestibule of alpha-hemolysin helps to orient the peptides for translocation. Overall, the results demonstrate that the nanopore technology can provide useful structural information but peptide sequencing will require further improvements in the design of the pores.

HPr is a small protein that must unfold to translocate the α‐hemolysin pore (see image). Single amino acid substitutions can cause large changes to the translocation parameters. A conservative mutation is sufficient to alter the event profile; either the mutant must unfold differently or it must interact with the pore differently. A negatively charged mutant is driven through the pore, which facilitates unfolding.

In the absence of Zn(II) a single Zn‐finger module such as Zif268 is unfolded and can translocate an α‐hemolysin pore (see image). Upon addition of Zn(II), the protein folds and is too large to translocate so that only bumping events are observed. In contrast, upon addition of Co(II) or Mg(II), there are only small changes to the event profiles.

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.

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.