## 2 Results
### Analysis of the Conformational Changes at the N-Terminal Region of Cry1 Ab Toxin by FRET Assays
FRET analysis under such conditions showed that the emission of the D fluorophore in the presence of A of the double labeled mutant Cry1 Ab S41C-DA was considerably higher (Figure 2B, green line), around 70% of the total fluorescence emission, when compared to the same mutant Cry1 Ab S41C-DA in the solution (Figure 2B, red line), supporting that the loss of FRET energy transfer between D and A in the oligomeric structure could be due to the cleavage of helix \(\alpha\)-1, which could have been lost or swung away from the toxin core. In contrast, when the double labeled mutant Cry1 Ab S71C-DA was analyzed with BBMVs, we observed that the fluorescence emission of D fluorophore (Figure 2C, green line) was very similar, just slightly higher than in the solution (Figure 2C, red line), supporting that helix \(\alpha\)-2b was not cleaved out upon toxin oligomerization, suggesting that only a subtle conformational change occurred in helix \(\alpha\)-2b upon oligomerization. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Toxin**} & \multicolumn{2}{c}{**LC\({}_{\text{50}}\) in ng/cm\({}^{2}\)**} \\ \cline{2-3}  & **(Fiducial Limits) * & **Localization of Mutation** \\ \hline Cry1 Ab & 1.81 (1.3–2.5) & \\ Cry1 Ab S41C & 1.84 (0.1–3.3) & Helix \(\alpha\)-1 \\ Cry1 Ab S71C & 2.5 (1.2–3.1) & Helix \(\alpha\)-2b \\ Cry1 Ab S39C-T239C & 0.86 (0.6–1.1) & Helices \(\alpha\)-1 and \(\alpha\)-7 \\ Cry1 Ab W73C-I97C & \(>\)2000 & Helices \(\alpha\)-2b and \(\alpha\)-3 \\ Cry1 Ab S39C & 0.93 (0.6–1.2) & Helix \(\alpha\)-1 \\ Cry1 Ab T239C & ND & Helix \(\alpha\)-7 \\ Cry1 Ab W73C & 1.4 (0.2–3.4) & Helix \(\alpha\)-2b \\ Cry1 Ab I97C & 1.5 (1.2–2.6) & Helix \(\alpha\)-3 \\ \hline \hline \end{tabular}  
* Values of 95% fiducial limits are shown in parenthesis. ND: Not determined.  
\end{table} 
Table 1: Toxicity of Cry1 Ab toxins against _Manduca sexta_ larvae. Restriction of the Conformational Changes at the N-Terminal Region of Cry1 Ab Toxin by Introducing Disulfide Bridges  
To further analyze the conformational changes in the N-terminal region of Cry1 Ab during oligomerization, intramolecular disulfide bridges were engineered among different helices of Cry1 Ab toxin to restrict their flexibility during oligomer assembling. Typically, the optimal distance of the \(\beta\)-carbons of two amino acids forming a disulfide bond is approximately 5.5 A [22]. Thus, to analyze if the removal of helix \(\alpha\)-1 and the helix \(\alpha\)-2b conformational change are required for oligomer assembly, we carefully selected two residues that were at the appropriate locations suitable for disulfide bridge formation between two Cys residues introduced by site directed mutagenesis. We selected to  
Figure 2: FRET analysis during oligomerization of Cry1 Ab toxins. (**A**) Model structure of Cry1 Ab toxin showing labeled residues. The introduced Cys residues S41C and S71C labeled with Alexa Fluor 350 (D) are colored in cyan, and the exposed Lys residues K403 and K490 labeled with Alexa Fluor 488 (A) are colored in green. (**B**) FRET assay with Cry1 Ab S41C mutant. (**C**) FRET assay with Cry1 Ab S71C mutant. Cyan lines show emission spectra of the donor fluorophore Alexa Fluor 350 (D) bound to the introduced Cys residues in the mutant toxins in solution. Red lines show emission spectra of the D fluorophore bound to the Cys residues when these mutants were also labeled with acceptor fluorophore Alexa Fluor 488 (A) bound to the Lys residues in solution (DA). Green lines were similar proteins labeled with donor and acceptor fluorophores (DA) analyzed after induction of oligomer formation by incubation with \(M\). _sexta_ BBMVs. Inserts show emission spectra of Alexa Fluor 488 (A) of the double labeled mutants (DA), which were excited with a wavelength of 488 nm to confirm that samples analyzed in the presence or in the absence of BBMVs had the same amount of the acceptor protein.