Pulling Peptides across Nanochannels: Resolving Peptide Binding and Translocation through the Hetero-oligomeric Channel from <i>Nocardia farcinica</i>

We investigated translocation of cationic peptides through nanochannels derived from the Gram-positive bacterium <i>Nocardia farcinica</i> at the single-molecule level. The two subunits NfpA and NfpB form a hetero-oligomeric cation selective channel. On the basis of amino acid comparison we performed homology modeling and obtained a channel structurally related to MspA of <i>Mycobacterium smegmatis</i>. The quantitative single-molecule measurements provide an insight into transport processes of solutes through nanochannels. High-resolution ion conductance measurements in the presence of peptides of different charge and length revealed the kinetics of peptide binding. The observed asymmetry in peptide binding kinetics indicated a unidirectional channel insertion in the lipid bilayer. In the case of cationic peptides, the external voltage acts as a driving force that promotes the interaction of the peptide with the channel surface. At low voltage, the peptide just binds to the channel, whereas at higher voltage, the force is strong enough to pull the peptide across the channel. This allows distinguishing quantitatively between peptide binding and translocation through the channel.