title: Unfolding events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel.
abstract: The insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis undergo several conformational changes from crystal inclusion protoxins to membrane-inserted channels in the midgut epithelial cells of the target insect. Here we analyzed the stability of the different forms of Cry1Ab toxin, monomeric toxin, pre-pore complex, and membrane-inserted channel, after urea and thermal denaturation by monitoring intrinsic tryptophan fluorescence of the protein and 1-anilinonaphthalene-8-sulfonic acid binding to partially unfolded proteins. Our results showed that flexibility of the monomeric toxin was dramatically enhanced upon oligomerization and was even further increased by insertion of the pre-pore into the membrane as shown by the lower concentration of chaotropic agents needed to achieve unfolding of the oligomeric species. The flexibility of the toxin structures is further increased by alkaline pH. We found that the monomer-monomer interaction in the pre-pore is highly stable because urea promotes oligomer denaturation without disassembly. Partial unfolding and limited proteolysis studies demonstrated that domains II and III were less stable and unfold first, followed by unfolding of the most stable domain I, and also that domain I is involved in monomer-monomer interaction. The thermal-induced unfolding and analysis of energy transfer from Trp residues to bound 1-anilinonaphthalene-8-sulfonic acid dye showed that in the membrane-inserted pore domains II and III are particularly sensitive to heat denaturation, in contrast to domain I, suggesting that only domain I may be inserted into the membrane. Finally, the insertion into the membrane of the oligomeric pre-pore structure was not affected by pH. However, a looser conformation of the membrane-inserted domain I induced by neutral or alkaline pH correlates with active channel formation. Our studies suggest for the first time that a more flexible conformation of Cry toxin could be necessary for membrane insertion, and this flexible structure is induced by toxin oligomerization. Finally the alkaline pH found in the midgut lumen of lepidopteran insects could increase the flexibility of membrane-inserted domain I necessary for pore formation.
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca 62250, Morelos, México.
authors.forename: Carolina
authors.initials: C
authors.lastname: Rausell
authors.forename: Liliana
authors.initials: L
authors.lastname: Pardo-López
authors.forename: Jorge
authors.initials: J
authors.lastname: Sánchez
authors.forename: Carlos
authors.initials: C
authors.lastname: Muñoz-Garay
authors.forename: Claudia
authors.initials: C
authors.lastname: Morera
authors.forename: Mario
authors.initials: M
authors.lastname: Soberón
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D001413:Bacillus thuringiensis / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000737:chemistry* / Q000378:metabolism*; D004735:Energy Transfer; D006460:Hemolysin Proteins; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D010713:Phosphatidylcholines / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D013696:Temperature; D014508:Urea / Q000494:pharmacology
chemical_list: D000817:Anilino Naphthalenesulfonates; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D010713:Phosphatidylcholines; D011506:Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D011188:Potassium