## 1 Introduction
Interestingly, the monomeric structure of Cry4 Ba and Cry5 Ba toxins revealed by X-ray crystallography showed that the structural region corresponding to the helices \(\alpha\)-1 and \(\alpha\)-2a was cleaved out in a corresponding position located 50 residues upstream of the end of helix \(\alpha\)-3, resulting in a trimeric structure organization of the diffracted Cry4 Ba and Cry5 Ba crystals (pdb: 1W99 and 4D8M), even though these two \(\alpha\)-helices are present in the primary sequence of these proteins [8; 16; 17]. In these trimeric structures, the Cry4 Ba and Cry5 Ba proteins presented an extended helix \(\alpha\)-3 comprising the loop region and helix \(\alpha\)-2b. This long helix \(\alpha\)-3 is located at the central core of the trimeric conformation presented by both proteins, showing multiple contacts with helices \(\alpha\)-3, \(\alpha\)-4 and \(\alpha\)-6 from the adjacent monomers [16; 17]. We previously showed that helix \(\alpha\)-3 plays an important role in Cry toxin oligomerization, where intermolecular salt bridges were shown to participate in oligomerization and toxicity [18; 19; 20]. Furthermore, conserved charged residues found in the loop between helices \(\alpha\)-2b and \(\alpha\)-3 were shown to be involved in a salt bridge with adjacent monomers in several Cry toxins, implying that a conformational change of this loop into an \(\alpha\)-helix is required to allow oligomer assembly [19]. In fact, the residues D129 and K131 of Cry5 Ba toxin, located in this loop region, form an intermolecular salt bridge in the trimeric conformation of this protein, and mutations affecting these residues resulted in inactive toxins [19]. Similar evidence was observed by in silico modeling data of helix \(\alpha\)-3 of domain I of Cry1A toxin [21]. Therefore, we hypothesized that a conformational change at helices \(\alpha\)-2 and \(\alpha\)-3 forming an extended helix \(\alpha\)-3 is needed for oligomerization of Cry proteins. To further analyze the conformational changes that occur in domain I leading to oligomer formation, in this work we designed a series of mutants to perform Foster resonance energy transfer (FRET) distance analysis between some \(\alpha\)-helices of domain I and domains II-III of Cry1 Ab toxin. We selected to work with Cry1 Ab, since most of the work that has been done about the mechanism of action of Cry proteins has been done with this toxin. This protein also has an identical sequence to domain I of Cry1 Aa, which has a known structure. We also constructed mutants for introducing disulfide bridge formation to restrict conformational changes. Cry1 Ab activated toxin does not contain cysteine (Cys) residues, allowing the introduction of Cys residues at specific positions within helices \(\alpha\)-1 and \(\alpha\)-2b for specific labeling with extrinsic fluorophores or for the introduction of disulfide bridge formation. Our data support that helix \(\alpha\)-1 of domain I is cleaved and swings away from the toxin upon binding with BBMV of _Manduca sexta_. We also show that a conformational change in helix \(\alpha\)-2b is needed for oligomerization. These observations are consistent with the proposed hypothesis that helices \(\alpha\)-2b and \(\alpha\)-3 undergo a conformational change, forming an extended helix \(\alpha\)-3 for oligomer assembly of Cry toxins necessary for their insecticidal activity.