But the fact that this protein is present in the solid fraction of the culture and in the protein mixture after solubilization indicates that Cry1Ac′3 is an insoluble protein, or that it is accumulated into small inclusion bodies undetectable by contrast phase microscopy. Second instar

larvae of E. kuehniella were exposed to different doses of Cry1Ac, find more Cry1Ac′1 and Cry1Ac′3 δ-endotoxins. Whereas Cry1Ac showed an LC50 of 1300 μg g−1 of semolina after 10 days, Cry1Ac′1 and Cry1Ac′3 showed no mortality, indicating that the two mutations Y229P and F603S affected the toxicity of the proteins. In vitro processing of the two mutant δ-endotoxins Cry1Ac′1 and Cry1Ac′3 was carried out to study their stability. After 2 h incubation of crystals and inclusions in 50 mM Na2CO3 with trypsin at a concentration of 1/100 (trypsin/δ-endotoxins, Etoposide price w/w), Cry1Ac was totally converted to a doublet of 65 and 60 kDa. However, Cry1Ac′1 was converted to a weak band of 65 kDa and Cry1Ac′3 was totally degraded (Fig. 4). Therefore, the protein Cry1Ac′1 was able to persist in the processing but the truncated protein Cry1Ac′3

was affected by proteases. This could explain the abolishment of the toxicity of Cry1Ac′3, as the activation is a key step in the mechanism of toxicity of Cry proteins. To explore the effect of Y229P and F603S substitutions at a molecular level, a three-dimensional model of Cry1Ac was constructed on the basis of the crystal structure of 1CIY of B. thuringiensis kurstaki strain HD-1 (Grochulski et al., 1995). The analysis

of the generated model showed that Cry1Ac is made up of three distinct domains. The N-terminal domain, known as domain I, is a helical bundle of seven alpha helices in which the central helix 5, which is relatively hydrophobic, is surrounded by the helices. Domain II consists of three antiparallel β-sheets joined in a Greek key topology, Amrubicin arranged in a β-prism. Domain III is formed by two antiparallel β-sheets with a jelly roll topology. Figure 5 illustrates the overall structure of Cry1Ac and the positions of Y229 and F603. Residue Y229 is located near the bottom of the α7 helix; it is a partially surface-exposed residue with no intramolecular interaction. Y229P mutation shortens the α7 helix by seven residues and generates a huge loop of 12 amino acids (Fig. 5b and c). The resultant mutant is inactive and the crystals produced are very small, suggesting that these alterations have affected, in some way, the stability of this mutant. In fact, the Y229P mutation affects the structure and the stability of the loop connecting helices α6 and α7 of domain I. This part of the toxin is maintained exclusively by tightly packed hydrophobic residues centered on Y229 and connecting the α6 and α7 helices. The hydrophobic network is the consequence of the interaction between residues W226, Y229, F232 and R233 of the α7 helix and residues L215, V218 and W219 of α6.