## 3 Discussion
In contrast, when we linked helix \(\alpha\)-1 with helix \(\alpha\)-7 in the Cry1 Ab S39C-T239C mutant, this mutant was still able to form oligomer structures without reducing agents and retained toxicity to _M. sexta_ larvae. These results suggest that helix \(\alpha\)-1 remained attached to the toxin but did not impair the conformational change of helix \(\alpha\)-2b upon oligomerization, probably because it is cleaved, allowing the helix \(\alpha\)-2b conformational change to occur even though helix \(\alpha\)-1 remains attached to the toxin by the disulfide bridge. Our FRET analysis suggested that helix \(\alpha\)-1 may be cleaved out from the toxin upon oligomerization, but remains attached to the toxin, which is compatible with these results obtained with the restriction of helix \(\alpha\)-1 mobility by disulfide bridge, showing that cross-linking helix \(\alpha\)-1 with helix \(\alpha\)-7 does not affect oligomerization. In silico prediction suggested that the hairpin between helices \(\alpha\)-2b and \(\alpha\)-3 h in Cry1 Ab toxin is likely to have \(\alpha\)-helix structure. A similar observation was previously shown when this region was analyzed for its capacity to form coiled coil structures by using a program designed to identify regions prone to form such coiled coils among putative \(\alpha\)-helices [18]. The analysis by the coiled coils prediction program showed that this region was the only one within the Cry toxin core that has high potential to form such structures [18]. These two analyses performed with different algorithms support that this region has the flexibility and biochemical characteristics required for the conformational change leading to an extended \(\alpha\)-3 helix formation required for facilitating oligomer formation. In agreement with these data, we previously showed that at least two conserved salt bridges may be formed between the predicted extended helices \(\alpha\)-3 from adjacent monomers in the Cry family during oligomerization. The most conserved salt bridge in the Cry family is the one located in the loop region between helix \(\alpha\)-2b of one monomer and helix \(\alpha\)-3 from the adjacent monomer [19]. It is important to mention that such a salt bridge could only be formed if helix \(\alpha\)-2b and the loop between this helix and helix \(\alpha\)-3 changed their conformation, forming an extended helix \(\alpha\)-3. In the case of Cry5 Ba, mutations of the salt bridge located in the loop region between helix \(\alpha\)-2b and helix \(\alpha\)-3 affected toxicity to nematodes, supporting the functionality of this salt bridge for toxicity [19]. The Cry1 Ab does not contain this salt bridge but has another salt bridge formed by two residues in helix \(\alpha\)-3 from adjacent monomers, which has been shown to be essential for toxicity since mutations in that salt bridge (Cry1 Ab R99E or Cry1 Ab E101K) severely affected oligomerization and toxicity, while reversed charged mutagenesis in the double mutant Cry1 Ab R99E-E101R recovered both oligomerization and toxicity [19]. Interestingly, the introduction of the salt bridge located in the loop between helices \(\alpha\)-2b and \(\alpha\)-3 to a Cry1 Ab mutant lacking the other nearby salt bridge (Cry1 Ab R99E-N85D-R87K) also recovered toxicity and oligomer formation, supporting that this loop region must be structured as \(\alpha\)-helix for oligomer assembly and toxicity [19]. Based on all results presented here and the previously published data, we propose a model to explain the first steps in oligomerization of Cry toxins, where the proteolytic cleavage between helix \(\alpha\)-2a and helix \(\alpha\)-3 releases helix \(\alpha\)-2b, allowing it to move and rearrange to make an extended helix \(\alpha\)-3 that is needed for oligomer formation (Figure 4). It has been proposed that additional changesmust occur during insertion of a Cry oligomer into the membrane, such as the movement of helices \(\alpha\)-4 and \(\alpha\)-5 to finally be inserted into the membrane [10; 11; 12].