title: Single molecule fluorescence study of the Bacillus thuringiensis toxin Cry1Aa reveals tetramerization.
abstract: Pore-forming toxins constitute a class of potent virulence factors that attack their host membrane in a two- or three-step mechanism. After binding to the membrane, often aided by specific receptors, they form pores in the membrane. Pore formation either unfolds a cytolytic activity in itself or provides a pathway to introduce enzymes into the cells that act upon intracellular proteins. The elucidation of the pore-forming mechanism of many of these toxins represents a major research challenge. As the toxins often refold after entering the membrane, their structure in the membrane is unknown, and key questions such as the stoichiometry of individual pores and their mechanism of oligomerization remain unanswered. In this study, we used single subunit counting based on fluorescence spectroscopy to explore the oligomerization process of the Cry1Aa toxin of Bacillus thuringiensis. Purified Cry1Aa toxin molecules labeled at different positions in the pore-forming domain were inserted into supported lipid bilayers, and the photobleaching steps of single fluorophores in the fluorescence time traces were counted to determine the number of subunits of each oligomer. We found that toxin oligomerization is a highly dynamic process that occurs in the membrane and that tetramers represent the final form of the toxins in a lipid bilayer environment.
authors: Groupe d'Étude des Protéines Membranaires (GÉPROM), Université de Montréal, Montréal, Québec H3C 3J7, Canada; Department of Physics, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
authors: Nicolas
authors: N
authors: Groulx
authors: Groupe d'Étude des Protéines Membranaires (GÉPROM), Université de Montréal, Montréal, Québec H3C 3J7, Canada; Department of Physics, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
authors: Hugo
authors: H
authors: McGuire
authors: Groupe d'Étude des Protéines Membranaires (GÉPROM), Université de Montréal, Montréal, Québec H3C 3J7, Canada; Department of Physics, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
authors: Raynald
authors: R
authors: Laprade
authors: Groupe d'Étude des Protéines Membranaires (GÉPROM), Université de Montréal, Montréal, Québec H3C 3J7, Canada; Centre SÈVE, Université de Montréal, Montréal, Québec H3C 3J7, Canada; Department of Physiology, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
authors: Jean-Louis
authors: JL
authors: Schwartz
authors: Groupe d'Étude des Protéines Membranaires (GÉPROM), Université de Montréal, Montréal, Québec H3C 3J7, Canada; Department of Physics, Université de Montréal, Montréal, Québec H3C 3J7, Canada; Department of Physiology, Université de Montréal, Montréal, Québec H3C 3J7, Canada. Electronic address: rikard.blunck@umontreal.ca.
authors: Rikard
authors: R
authors: Blunck
mesh_terms: D001413:Bacillus thuringiensis / Q000378:metabolism*; D001426:Bacterial Proteins; D001703:Biophysics / Q000379:methods; D003460:Crystallization; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D019476:Insect Proteins / Q000737:chemistry*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D016012:Poisson Distribution; D011336:Probability; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D001426:Bacterial Proteins; C095416:Cry toxin receptors; D019476:Insect Proteins; D008051:Lipid Bilayers; D011956:Receptors, Cell Surface