Figure 6:

The C-terminal boundary in EC12 for binding of CrylAb. Two deletion mutants, EC12_SQ and EC12_VS, at the C-terminal end of EC12 were generated. The mutants were purified by nickel affinity chromatography. Competition binding of the mutant dipeptides was assessed using fluorescently labeled EC12 probe and purified Cry1Ab toxin. (A) Sequences for EC12W and the two deletion EC12 mutant peptides. (B) Samples from the competitive binding assay resolved on a native gel. Fluorescent gel showing competition binding of EC12_sq and EC12_vs. The Cry1Ab-EC12 complex and unbound EC12 probe are indicated by arrows on the left side. P: EC12 probe only (lane 1,200 nM); P-C: EC12 probe plus 500 nM C ry1Ab (lane 2). Competitor peptides for EC12_wT (lanes 3–5 at 200, 400, and 800 nM respectively), EC12_sq (lanes 6–8 at 200, 400 and 800 nM, respectively) and EC12_vs (lanes 9–11 at 200, 400 and 800 nM, respectively) were incubated with 500nM Cry1Ab prior to the addition of the fluorescent EC12 probe as indicated at the top of the image. (C) Western blot analysis of the peptide competitors using anti-6xHis antibody demonstrates expression of the two peptides. (D) Sequence change in the C-terminus of EC12 in which the serine and glutamine residues were substituted by alanine is presented. Dashed lines represent unchanged EC12 amino acid sequences. The EC12_sq/aa mutant was expressed and purified by nickel affinity chromatography. Assays were conducted to test the mutant’s ability to compete with the EC12 probe for Cry1Ab binding. (E) Fluorescent gel displays competitive binding of the mutant EC12_sq/M. Fluorescent Cry1Ab-EC12 complex and unbound EC12 bands are indicated by arrows on the left side of the gel. P: 200 nM EC12 probe only (lane 1); P-C: 200 nM EC12 probe plus 250 nM CrylAb (lane 2); non-fluorescent EC12_wT (lanes 3–6: 50, 100, 200, and 400 nM, respectively) and EC12_sq/Aa (lanes 7–10: 50, 100, 200 and 400 nM, respectively) were incubated with 250 nM Cry1Ab prior to the addition of the fluorescent EC12 probe. (F) Western blot analysis using anti-6xHis antibody against EC12W (lane 1) and EC12_sq/Aa (lane 2) show expression of these peptides.