Nair Manoj S, Dean Donald H
Biophysics Program, Ohio State University, Columbus, Ohio 43210, USA.
J Biol Chem. 2008 Sep 26;283(39):26324-31. doi: 10.1074/jbc.M802895200. Epub 2008 Jul 17.
A critical step in understanding the mode of action of insecticidal crystal toxins from Bacillus thuringiensis is their partitioning into membranes and, in particular, the insertion of the toxin into insect brush border membranes. The Umbrella and Penknife models predict that only alpha-helix 5 of domain I along with adjacent helices alpha-4 or alpha-6 insert into the brush border membranes because of their hydrophobic nature. By employing fluorescent-labeled cysteine mutations, we observe that all three domains of the toxin insert into the insect membrane. Using proteinase K protection assays, steady state fluorescence quenching measurements, and blue shift analysis of acrylodan-labeled cysteine mutants, we show that regions beyond those proposed by the two models insert into the membrane. Based on our studies, the only extended region that does not partition into the membrane is that of alpha-helix 1. Bioassays and voltage clamping studies show that all mutations examined, except certain domain II mutations in loop 2 (e.g. F371C and G374C), which disrupt membrane partitioning, retain their ability to form ion channels and toxicity in Manduca sexta larvae. This study confirms our earlier hypothesis that insertion of crystal toxin does not occur as separate helices alone, but virtually the entire molecule inserts as one or more units of the whole molecule.
了解苏云金芽孢杆菌杀虫晶体毒素作用模式的关键步骤是其在膜中的分配，特别是毒素插入昆虫刷状缘膜。“伞”模型和“小刀”模型预测，由于其疏水性，只有结构域I的α-螺旋5以及相邻的α-4或α-6螺旋插入刷状缘膜。通过使用荧光标记的半胱氨酸突变体，我们观察到毒素的所有三个结构域都插入昆虫膜中。利用蛋白酶K保护试验、稳态荧光猝灭测量以及丙烯罗丹标记的半胱氨酸突变体的蓝移分析，我们表明除了这两个模型提出的区域之外，还有其他区域插入膜中。根据我们的研究，唯一不分配到膜中的延伸区域是α-螺旋1。生物测定和电压钳制研究表明，除了环2中某些破坏膜分配的结构域II突变（例如F371C和G374C）外，所有检测的突变在烟草天蛾幼虫中都保留了形成离子通道和产生毒性的能力。这项研究证实了我们早期的假设，即晶体毒素不是单独以分离的螺旋形式插入，而是实际上整个分子作为一个或多个整体单元插入。
