title: Urea denaturation of alpha-hemolysin pore inserted in planar lipid bilayer detected by single nanopore recording: loss of structural asymmetry.
abstract: 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 alpha-hemolysin channel and the channel formation of alpha-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 3.56M concentrations and vanishes abruptly down to 0 concentration exceeds 4M. The loss of current asymmetry through alpha-hemolysin is due to the denaturation of the channel's cap. We also show that the alpha-hemolysin pore inserted into a lipid bilayer is much more resistant to urea denaturation than the alpha-hemolysin monomer in solution: The pore remains in the lipid bilayer up to 7.2M urea. The pore formation is possible up to 4.66M urea when protein monomers were previously incubated in urea.
authors.affiliation: Laboratoire de Recherche sur les Polymères, équipe Matériaux Polymères aux Interfaces, CNRS-UMR 7581, Université d'Evry, Boulevard F. Mitterrand, 91025 Evry, France.
authors.forename: Manuela
authors.initials: M
authors.lastname: Pastoriza-Gallego
authors.forename: Ghani
authors.initials: G
authors.lastname: Oukhaled
authors.forename: Jérôme
authors.initials: J
authors.lastname: Mathé
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Thiebot
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Betton
authors.forename: Loïc
authors.initials: L
authors.lastname: Auvray
authors.forename: Juan
authors.initials: J
authors.lastname: Pelta
mesh_terms: D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D011489:Protein Denaturation / Q000187:drug effects; D014508:Urea / Q000494:pharmacology*
chemical_list: D006460:Hemolysin Proteins; D008051:Lipid Bilayers; D014508:Urea