Ser170 of Bacillus thuringiensis Cry1Ab δ-endotoxin becomes anchored in a hydrophobic moiety upon insertion of this protein into Manduca sexta brush border membranes

摘要

Background: Three spin-labeled mutant proteins, mutated at the beginning, middle, and end of α-helix 5 of the Bacillus thuringiensis Cry1Ab δ-endotoxin, were used to study the involvement ofthese specific amino acid residues in ion transport and to determine conformational changes in thevicinity of these residues when the protein was translocated into a biological membrane.Results: Amino acid residue leucine 157, located in the N-terminal portion of α-helix 5, showedno involvement in ion transport, and the environment that surrounds the residue did not show anychange when transferred into the biological membrane. Serine 170, located in the middle of the α-helix, showed no involvement in ion transport, but our findings indicate that in the membrane-bound state this residue faces an environment that makes the spin less mobile, as opposed to themobility observed in an aqueous environment. Serine 176, located in the C-terminal end of the α-helix 5 is shown to be involved in ion transport activity.Conclusion: Ion transport data for L157, S170, and S176, along with the mobility of the spin-labels,structural characterization of the resulting proteins, and toxicity assays against a target insect,suggest that the toxin undergoes conformational changes upon protein translocation into themidgut membrane. These conformational changes result in the midregion of the α-helix 5 beingexposed to a hydrophobic-like environment. The location of these three residues in the toxinsuggests that the entire α-helix becomes inserted in the insect midgut membrane.