Kasorn TiewsiriChanan AngsuthanasombatMahidol University2018-08-242018-08-242007-03-01Journal of Biochemistry and Molecular Biology. Vol.40, No.2 (2007), 163-17102191024122586872-s2.0-34047153317https://repository.li.mahidol.ac.th/handle/20.500.14594/24234Functional elements of the conserved helix 7 in the poreforming domain of the Bacillus thuringiensis Cry δ-endotoxins have not yet been clearly identified. Here, we initially performed alanine substitutions of four highly conserved aromatic residues, Trp243, Phe246, Tyr 249 and Phe264, in helix 7 of the Cry4Ba mosquito-larvicidal protein. All mutant toxins were overexpressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type. Bioassays against Stegomyia aegypti mosquito larvae revealed that only W243A, Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity. Further mutagenic analysis showed that replacements with an aromatic residue particularly at Tyr249 and Phe264 still retained the high-level toxin activity. In addition, a nearly complete loss in larvicidal activity was found for Y249L/F264L or F264A/Y249A double mutants, confirming the involvement in toxicity of both aromatic residues which face towards the same direction. Furthermore, the Y249L/F264L mutant was found to be structurally stable upon toxin solubilisation and trypsin digestion, albeit a small change in the circular dichroism spectrum. Altogether, the present study provides for the first time an insight into the highly conserved aromaticity of Tyr249 and Phe264 within helix 7 playing an important role in larvicidal activity of the Cry4Ba toxin.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS