38th Annual Meeting of the Society for Invertebrate Pathology

The α-helices 4 and 5 of 130-kDa Bacillus thuringiensis Cry4Ba toxin have been demonstrated to be important determinants of mosquito-larvicidal activity, particularly in pore formation. In this study, E. coli cells harboring the mutant plasmid-pS136NSSRNP (T6) for the 130-kDa Cry4Ba mutant protoxin containing an additional proteolytic cleavage site in the loop between α3 and α4 were used for producing α4-α5 helical hairpins. The 130-kDa protein inclusions were solubilized in carbonate buffer, pH 9.0 and were activated by trypsin. The 65-kDa activated toxins were purified by the size-exclusion and further purified by reversed-phase HPLC using Jupiter C₁₈ column. N-terminal sequencing indicated a correct cleavage site (NPSYRT). A circular dichroism spectrum of these hairpins showed helical structure dissolved in methanol. Membrane permeation studies via calcein release assays revealed that the α4-α5 helical hairpin exhibited high perturbing activity against LUVs (50-70% release) whereas the 65-kDa activated Cry4Ba toxin or mutant toxin (T6) and 47-kDa elution fraction showed relatively low. These results suggested that α4-α5 helical hairpin of the Cry4Ba toxin is involved in pore formation in phospholipid membrane vesicles. ATR-FTIR spectroscopy measurement revealed that the α4-α5 helical hairpins are mainly buried into phosopholipid membranes and show a predominant α-helical structure. Taken together, the data indicate that the α4-α5 helical hairpins play a role in membrane penetration and support the pore-forming ‘umbrella’ model.