Vachon Vincent, Préfontaine Gabrielle, Coux Florence, Rang Cécile, Marceau Lucie, Masson Luke, Brousseau Roland, Frutos Roger, Schwartz Jean-Louis, Laprade Raynald
Groupe de recherche en transport membranaire, Université de Montréal, P.O. Box 6128, Centre Ville Station, Montreal, Quebec, H3C 3J7, Canada.
Biochemistry. 2002 May 14;41(19):6178-84. doi: 10.1021/bi011572e.
Helix 3 of the Cry1Aa toxin from Bacillus thuringiensis possesses eight charged amino acids. These residues, with the exception of those involved in intramolecular salt bridges (E90, R93, E112, and R115), were mutated individually either to a neutral or to an oppositely charged amino acid. The mutated genes were expressed, and the resultant, trypsin-activated toxins were assessed for their toxicity to Manduca sexta larvae and their ability to permeabilize M. sexta larval midgut brush border membrane vesicles to KCl, sucrose, raffinose, potassium gluconate, and N-methyl-D-glucamine hydrochloride with a light-scattering assay based on osmotic swelling. Most mutants were considerably less toxic than Cry1Aa. Replacing either E101, E116, E118, or D120 by cysteine, glutamine, or lysine residues had only minor effects on the properties of the pores formed by the modified toxins. However, half of these mutants (E101C, E101Q, E101K, E116K, E118C, and D120K) had a significantly slower rate of pore formation than Cry1Aa. Mutations at R99 (R99C, R99E, and R99Y) resulted in an almost complete loss of pore-forming ability. These results are consistent with a model in which alpha-helix 3 plays an important role in the mechanism of pore formation without being directly involved in determining the properties of the pores.
苏云金芽孢杆菌Cry1Aa毒素的螺旋3含有8个带电荷的氨基酸。除了参与分子内盐桥形成的氨基酸（E90、R93、E112和R115）外，这些残基分别被突变为中性氨基酸或带相反电荷的氨基酸。对突变基因进行表达，并通过基于渗透肿胀的光散射测定法，评估所得的经胰蛋白酶激活的毒素对烟草天蛾幼虫的毒性，以及它们使烟草天蛾幼虫中肠刷状缘膜囊泡对氯化钾、蔗糖、棉子糖、葡萄糖酸钾和盐酸N-甲基-D-葡糖胺通透的能力。大多数突变体的毒性比Cry1Aa低得多。用半胱氨酸、谷氨酰胺或赖氨酸残基取代E101、E116、E118或D120，对修饰毒素形成的孔的性质只有轻微影响。然而，这些突变体中的一半（E101C、E101Q、E101K、E116K、E118C和D120K）形成孔的速率明显比Cry1Aa慢。R99处的突变（R99C、R99E和R99Y）导致几乎完全丧失成孔能力。这些结果与一个模型一致，即α-螺旋3在成孔机制中起重要作用，但不直接参与决定孔的性质。
