Urea denaturation of α-hemolysin pore inserted in planar lipid bilayer detected by single nanopore recording: Loss of structural asymmetry

The aim of this work is to study pore protein denaturation inside a lipid bilayer and to probe current asymmetry as a function of the channel conformation. We describe the urea denaturation of α-hemolysin channel and the channel formation of α-hemolysin monomer incubated with urea prior to insertion into a lipid bilayer. Analysis of single-channel recordings of current traces reveals a sigmoid curve of current intensity as a function of urea concentration. The normalized current asymmetry at 29 ± 4% is observed between 0 and 3.56 M concentrations and vanishes abruptly down to 0 concentration exceeds 4 M. The loss of current asymmetry through α-hemolysin is due to the denaturation of the channel's cap. We also show that the α-hemolysin pore inserted into a lipid bilayer is much more resistant to urea denaturation than the α-hemolysin monomer in solution: The pore remains in the lipid bilayer up to 7.2 M urea. The pore formation is possible up to 4.66 M urea when protein monomers were previously incubated in urea.