## 3 Discussion  
The final structure of Cry toxin oligomers when inserted into the membrane remains unsolved. It was proposed that the interaction of Cry1 Ab with membrane receptors triggers cleavage of helix \(\alpha\)-1, facilitating the rearrangement of \(\alpha\)-helices at the N-terminal end of domain I and leading to oligomer assembly [15; 19]. Evidence based on disulfide cross-linking of various \(\alpha\)-helices from domain I among them or between \(\alpha\)-helices from domain I with domain II indicated that domain I swings out from domains II and III, resulting in insertion of helix \(\alpha\)-5 into the membrane [10]. These studies  
Figure 3: Mutagenesis to restrict mobility of Cry proteins by disulfide bridges. (**A**) Structure of Cry1 Ab showing the distance between residues chosen for disulfide bridging. (**B**) Oligomerization assay of cross-linked mutants by disulfide bridges. Oligomer formation was analyzed by western blot using anti Cry1 Ab-antibody after incubation with BBMVs isolated from _M. sexta_ larvae. These assays were done in the absence and in the presence of reducing agents: DTT, dithiothreitol; or 2-ME, 2-mercaptoethanolhanol. Pre-stained molecular weight markers PageRank Plus (ThermoFisher Scientific, Waltham, MA, USA) are shown in the first lane. showed that some cross-linked mutants resulted in the loss of pore formation activity in black lipid bilayers, which could be restored by reduction of the disulfide bonds, leading to the proposal that conformational changes involving several helices of domain I are required for pore formation [10]. In addition, protease protection analysis of Cry1 Ac protein inserted into the membrane identified that only helix \(\alpha\)-1 is highly susceptible to proteolysis, suggesting that this region is not inserted into the membrane [23]. Finally, studies using synthetic peptides corresponding to domain I \(\alpha\)-helices indicated that helix \(\alpha\)-1 peptide was the only one of the seven fluorescently labeled \(\alpha\)-helices peptides that did not bind to phospholipid vesicles [24]. In this work, we analyzed the conformational changes of Cry1 Ab upon oligomerization in the presence of BBMVs from _M. sexta_. We used two different strategies to perform these studies. First, we analyzed the conformational changes by FRET. FRET is an excellent assay to measure molecular distances between two fluorescent molecules that are located in a range of 20-100 A when the emission spectrum of the donor molecule (D) overlaps with the excitation spectrum of the acceptor molecule (A). We selected two fluorescent dyes (Alexa Fluor 350 and Alexa Fluor 488) with a Forster distance (\(R_{0}\)) value of 50 A, indicating that if two residues labeled with these D and A fluorophores are separated by 50 A, the reduction in fluorescence of the D molecule in the presence of the A molecule will be 50% due to their FRET interaction. We labeled two residues from domain I (one located in helix \(\alpha\)-1 and the other in helix \(\alpha\)-2b) with the donor Alexa Fluor 350 and performed FRET analysis on Lys residues located in domains II or III at approximately 50 A distance. Our data showed that in solution the reduction in fluorescence of D fluorophore in the presence of the A molecule corresponded to the expected 50% reduction from both mutant toxins. When we performed the same FRET analysis in the presence of BBMVs, which induced oligomerization of the Cry1 Ab toxin, we observed that fluorescence of the D bound to helix \(\alpha\)-1 (Cry1 Ab S41C) in the presence of the A bound to Lys residues was considerably higher in the oligomer conformation of this mutant when compared to the same protein in solution, supporting that the loss of FRET energy transfer between D and A in the oligomeric structure could be attributed to a conformational change that is compatible with the cleavage or the swinging away of helix \(\alpha\)-1.