## 3 Discussion
It is important to mention that BBMVs were prepared in absence of protease inhibitors and thus the cleavage of helix \(\alpha\)-1 may be catalyzed by BBMV-anchored proteases. This observation is consistent with the report that helix \(\alpha\)-1 and part of helix \(\alpha\)-2a of the Cry1 Ab N-terminal end were cleaved out as indicated by the N-terminal sequence of the oligomeric structure [15]. Interestingly, we still observed some FRET in the presence of BBMVs, which could be explained if not all toxin molecules interacted with the membrane or only some molecules were cleaved, also if helix \(\alpha\)-1 swings away from domains II-III but still remains associated to the toxin. However, oligomerization assays with BBMVs revealed that only oligomers were associated with BBMVs, supporting that after cleavage the helix \(\alpha\)-1 may swing away from the core toxin and remain associated with the toxin. The exact position of helix \(\alpha\)-1 remains to be demonstrated; in any case, our data support that an important conformational change in helix \(\alpha\)-1 is produced during oligomerization. In contrast, the analysis of the double labeled mutant Cry1 Ab S71C-DA, where the donor is located in helix \(\alpha\)-2b, in the presence of BBMVs, revealed that the fluorescence emission of D fluorophore was very similar to that observed in solution, just slightly higher, suggesting certain flexibility of helix \(\alpha\)-2b, which is also consistent with our previous report where it was proposed that helices \(\alpha\)-2b and \(\alpha\)-3 are arranged to form a unique extended helix \(\alpha\)-3 [19]. These results support that upon oligomerization helix \(\alpha\)-1 has to be removed from the core toxin to allow the conformational change of helix \(\alpha\)-2b, leading to an extended \(\alpha\)-3 helix to establish contacts with adjacent monomers. The second strategy used to analyze the conformational changes of Cry1 Ab toxin during its oligomerization was the introduction of two Cys residues to allow disulfide bridge formation that could restrict secondary structure mobility; thus, the separation of two adjacent \(\alpha\)-helices is expected to be limited by the introduced cross-linking due to disulfide bridge formation. Interestingly, we found that a single Cry1 Ab T239C mutant was affected in its crystal production in Bt and thus its toxicity was not analyzed, but the double Cry1 Ab S39C-T239C mutant produced crystals that were toxic to _M. sexta_,indicating that the disulfide bridge stabilized the double mutant protein, allowing its accumulation in crystal inclusions. These data also indicated that the cross-linking between helices \(\alpha\)-1 and \(\alpha\)-7 did not affect the conformational changes required for oligomer formation. The single mutants Cry1 Ab W73C and Cry1 Ab I97C were able to produce crystal inclusions in Bt and were toxic to the larvae, similar to the wild type Cry1 Ab toxin. In contrast, when helix \(\alpha\)-2b was linked with helix \(\alpha\)-3 in the double Cry1 Ab W73C-I97C mutant, this protein was severely affected in toxicity against _M. sexta_ and also in its capacity to form oligomer structures, which were observed only in the presence of reducing agents, DTT or 2-ME, during its incubation with BBMVs. These data suggest that a conformational change of helix \(\alpha\)-2b is required for oligomer formation, and restriction of its mobility severely affects oligomerization and insecticidal activity. This result also shows that disulfide bridges remain stable in vivo since the toxicity of this double Cry1 Ab W73C-I97C mutant was severely affected, regardless of the aforementioned reducing environment of lepidopteran larval gut [9]. It is possible that the introduced disulfide bridge is protected by the toxin structure or that the concentration of reducing agents in the midgut is not high enough to affect this particular bridge.