Unfolding Events in the Water-soluble Monomeric Cry1Ab Toxin during Transition to Oligomeric Pre-pore and Membrane-inserted Pore Channel

作者：Carolina Rausell, Liliana Pardo‐López, Jorge Julian Sanchez, Carlos Muñóz-Garay, Claudia Morera, Mário Soberón, Alejandra Bravo · 发表于：Journal of Biological Chemistry · 年份：2004 · DOI：10.1074/jbc.m406279200 · 被引用次数：54 · 研究领域：Insect Resistance and Genetics、Insect and Pesticide Research、Bacillus and Francisella bacterial research

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 p...