## 2 Results
### Analysis of the Conformational Changes at the N-Terminal Region of Cry1 Ab Toxin by FRET Assays  
A structural model of the Cry1 Ab was obtained based on the coordinates of the Cry1 Aa structure (pdb:1 CIY) as described in the methods section. These two proteins shared the same domain I amino acid sequence and structure. With the aim to analyze possible conformational changes at the N-terminal end of Cry1 Ab domain I during its transition of monomer to oligomer conformation, we decided to use the FRET assays strategy. Activated Cry1 Ab monomer lacks Cys residues and contains only three lysines (Lys), where two of them are located in domain III and one in domain II. Residues K403 and K490 are exposed to the solvent, while the third, K470, is buried within domain III. In order to perform FRET assays we constructed two mutants with a single Cys residue localized either in helix \(\alpha\)-1 (Cry1 Ab S41C) or in helix \(\alpha\)-2b (Cry1 Ab S71C) of domain I. The resulting mutant toxins showed similar insecticidal activity to Cry1 Ab against _M. sexta_ larvae, indicating that these mutants retained their binding ability with receptors and did not have major conformational constrains (Table 1). Figure 1: Analysis of the amino acid sequences of helices \(\alpha\)-2b and \(\alpha\)-3 of Cry1 Ab. (**A**) Alignment of the amino acid sequence of regions containing helices \(\alpha\)-2b and \(\alpha\)-3 from Cry toxins, which have available structures. The \(\alpha\)-helix structures observed in their three-dimensional structures are shown in gray. (**B**) Prediction of the secondary structure of the Cry1 Ab hairpin regions among all \(\alpha\)-helices of domain I, by using the i-TASSER server. H, \(\alpha\)-helix; S, \(\beta\)-strand; C, coil (**C**) Prediction of the secondary structure of the coiled toxin regions between helices \(\alpha\)-2b and \(\alpha\)-3 of different Cry toxins by using the i-TASSER server, showing high probability to form an extended helix \(\alpha\)-3. H, \(\alpha\)-helix; S, \(\beta\)-strand; C, coil. Cys-substituted residues S41C and S71C localized in helix \(\alpha\)-1 or \(\alpha\)-2b, respectively. These residues were labeled with the fluorescent dye Alexa Fluor 350 to act as donor (D), while intrinsic K403 and K490 were labeled with Alexa Fluor 488, acting as acceptor (A) in the same mutant toxin. According to the built structural model of Cry1 Ab toxin, the average molecular distance between S41 or S71 with K403 and K490 was lower than 50 A (Figure 2A), which corresponds to the Foster radius distance (\(R_{0}\)) of the two selected fluorescent dyes (Alexa Fluor 350 as D, and Alexa Fluor 488 as A). At this distance, 50% of FRET energy transfer theoretically occurs. We first analyzed emission spectra of D fluorophore in the absence of A fluorophore of Cry1 Ab S41C-D and Cry1 Ab S71C-D mutants when these proteins were in the solution as monomeric structures (Figure 2B,C, cyan lines). FRET measurements were subsequently performed with the double labeled Alexa Fluor 350 and Alexa Fluor 488 (DA) Cry1 Ab S41C-DA and Cry1 Ab S71C-DA mutants in the same solution conditions. The fluorescence of D in the presence of A (DA) decreased approximately 50% of the total fluorescence emission observed in absence of A (Figure 2B,C, red lines), which is consistent with the molecular distance observed in the structural model of Cry1 Ab protein. To determine whether helix \(\alpha\)-1 is cleaved out or wrapped off upon oligomerization, the two mutants Cry1 Ab S41C-DA and Cry1 Ab S71C-DA were incubated with _M. sexta_ brush border membrane vesicles (BBMVs) to induce their oligomerization.