Transport of α-Helical Peptides through α-Hemolysin and Aerolysin Pores

Abstract

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.

Disentangling Steric and Electrostatic Factors in Nanoscale Transport Through Confined Space

“…3,11,21,23,[28][29][30] Several biophysical parameters have been determined such as threading frequency, [31][32][33][34] orientation of strands, [35][36][37][38][39] velocity of DNA transport, 40 influence of pore geometry, 41 and interactions with the pore wall. 39 In addition to nucleic acids, translocation has also been investigated for peptides, [42][43][44][45] proteins, [46][47][48] and peptide-oligonucleotide conjugates, 49 which, unlike nucleic acid strands, frequently feature inhomogeneous charge distributions and vary considerably in diameter along the polymer sequence.…”

Binding of bovine T194A PrPCby PrPSc-specific antibodies

“…Antibody prion interactions were analyzed using an α-hemolysin pore as described previously. 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.…”

“…[33][34][35] After insertion of an α-hemolysin pore, 20 μl of protein solution (1 mg/ml) was added to the 1.5 ml cis chamber proximal to the aperture. 34,36 For some experiments the protein was partially unfolded (or conformationally-relaxed) by incubating with 1 M Gdn-HCl for 15 min at 22 °C. The protein was then incubated with antibody for 1 h before addition of the complete sample to the nanopore chamber.…”

“…Proteins separated by SDS-PAGE were transferred in Tris-Glycine transfer buffer at 250 mA for 1.5 h. The nitrocellulose blots were blocked duration distribution with a single exponential function. 34,35 Immunoprecipitation of recombinant prion protein. Tosyl-activated M-280 Dynabeads, were purchased from Invitrogen (14203; Life Technologies).…”

“…Through nanopore analysis, a change in current is observed when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. 33,34 Each molecule is interrogated individually, and thus, several conformations in the same sample can be analyzed simultaneously as long as each gives rise to distinct event parameters, namely the blockade current (I) and blockade time (T). Through nanopore analysis it was found that the SN6b antibody binds mutant bPrP(T194A), but not wild-type bPrP, under non-denaturing conditions.…”