Role of Helix 3 in Pore Formation by the Bacillus thuringiensis Insecticidal Toxin Cry1Aa

详细信息    查看全文

• 作者：Vincent Vachon ; Gabrielle Pré ; fontaine ; Florence Coux ; Cé ; cile Rang ; Lucie Marceau ; Luke Masson ; Roland Brousseau ; Roger Frutos ; Jean-Louis Schwartz ; and Raynald Laprade
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：May 14, 2002
• 年：2002
• 卷：41
• 期：19
• 页码：6178 - 6184
• 全文大小：161K
• 年卷期：v.41,no.19(May 14, 2002)
• ISSN：1520-4995

文摘

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, andR115), were mutated individually either to a neutral or to an oppositely charged amino acid. The mutatedgenes were expressed, and the resultant, trypsin-activated toxins were assessed for their toxicity to Manducasexta larvae and their ability to permeabilize M. sexta larval midgut brush border membrane vesicles toKCl, 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 minoreffects 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 formationthan 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 -helix 3 plays an important role inthe mechanism of pore formation without being directly involved in determining the properties of thepores.