Urea denaturation of alpha-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 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.
Pastoriza-Gallego
Manuela
M
Laboratoire de Recherche sur les Polymères, équipe Matériaux Polymères aux Interfaces, CNRS-UMR 7581, Université d'Evry, Boulevard F. Mitterrand, 91025 Evry, France.
Oukhaled
Ghani
G
Mathé
Jérôme
J
Thiebot
Bénédicte
B
Betton
Jean-Michel
JM
Auvray
Loïc
L
Pelta
Juan
J
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*
D006460:Hemolysin Proteins; D008051:Lipid Bilayers; D014508:Urea