Structural changes in wild-type Cry3A δ-endotoxin and its mutant forms in ethanolic solutions at pH 2–2.5

Circular dichroism, scanning microcalorimetry, electron microscopy, and proteolysis were used to study the ability of wild-type δ-endotoxin Cry3A and its three mutant forms with cysteine substitutions in helices α3 and α4 (domain I) to produce oligomeric structures in acidic alcohol solutions that model the perimembrane environment. At pH 2–2.2 and 20% ethanol, the mutant toxins with single substitutions E132C (α3) and S160C (α4), as well as the double mutant E132C/S160C with a cysteine bridge between α3 and α4, formed short linear oligomers typical of Cry3A, with a high content of β-structure and elevated sensitivity to pepsin. The data obtained indicate that the formation of oligomeric structures of this type does not require separation of helices α3 and α4 in domain I of the Cry3A toxin. It is shown that at higher pH values in 20% solution of ethanol the proteins studied are in a metastable state, and their ability to form oligomeric structures depends on temperature.