Figure 4.

Characterization of purified STh mutants. (A) Structural model of STh highlighting residues that were targeted for mutation in red (L9, N12, A14) and Y19 in blue, which is the main epitope residue of the C30 epitope [16]. (B) The effect of binding of the STh mutant peptides to the C30 mAb was assessed using competitive ELISA. Three independent experiments were performed, and the mean calculated IC₅₀ values with standard deviations are shown for each mutant. (C) Dose-response analysis of the mutant STh peptides in the T84 cell assay. Four concentrations were used for each peptide (shown from left to right): 1627 nM, 163 nM, 16 nM, and 1,6 nM. Three independent experiments were performed, and the mean normalized cGMP response with standard deviations is shown for each mutant. The cGMP levels were normalized relative to native STh (100%) (D) The toxicities as observed in the T84 cell assay of the single mutant STh peptides relative to that of native STh. The relative toxicities were calculated from a standard curve of native STh, and the error bars depict the standard deviations from three individual experiments. L9S/A14T is not shown as it had no measurable residual toxicity.