Panapat UawithyaTipparat TuntitippawanGerd KatzenmeierSakol PanyimChanan AngsuthanasombatThe Institute of Science and Technology for Research and Development, Mahidol UniversityMahidol University2018-07-042018-07-041998-01-01Biochemistry and Molecular Biology International. Vol.44, No.4 (1998), 825-832103997122-s2.0-0031923098https://repository.li.mahidol.ac.th/handle/20.500.14594/18318The possible role of α-helices 3 and 4 in toxicity of the dipteran-active Bacillus thuringiensis Cry4B δ-endotoxin was investigated by employing proline substitutions via site-directed mutagenesis. Similar to the wild-type Cry4B, the mutant toxins were over-expressed in Escherichia coli as cytoplasmic inclusions and were structurally stable upon solubilization and trypsin activation. The substitution of glutamine 149 by proline in the center of helix 4 (Q149P) resulted in a nearly complete loss of toxicity against Aedes aegypti mosquito-larvae. However, single proline replacements near the center of helix 3 (V1 19P) and at the N-terminus of helix 4 (Q140P) did not decrease larvicidal activity. The toxicity of E. coli cells expressing the wild-type toxin was significantly reduced by two-hour preincubation with the non-toxic mutant (Q149P), thus indicating that the primary binding step was not affected by the proline substitution in helix 4. The results therefore reveal a crucial role for helix 4 of the Cry4B toxin in toxicity, possibly in membrane insertion and pore formation rather than in receptor recognition.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS