Fig. 3

Effect of amino acid residue double combinations. a Examples of double amino acid residue combinations resulting in small (top), large positive (middle), and large negative (bottom) ∆AUC values. Left: to calculate the AUC of a single amino acid residue substitution (AUC_AA1), all peptides are ranked on the horizontal axis according to their antimicrobial activity (as shown in Fig. 2a). Middle: the AUC of the same residue substitution is recalculated for the subset of peptides with a fixed second AA residue at another position (AUC_{AA1 | AA2}). Right: the influence of the second amino acid on the effect on antimicrobial activity of the first amino acid is calculated by subtracting the two previous calculations, resulting in ∆AUC. Indicators ~ and ^ are used to link the values to Fig. 3b. b ∆AUC values for all 4800 amino acid residue combinations. For each of the 20 amino acid residues at each of the four positions, there are 60 (20 amino acid residues at the remaining three positions) combinations with a second amino acid residue. Non-additive effects are estimated if a second amino acid residue changes the effect that a first amino acid residue has on antimicrobial activity, resulting in either larger positive or negative ∆AUC values. Cooperativity is measured when ∆AUC is larger than 0.09. Antagonism is measured when ∆AUC is smaller than −0.09 (=outliers of a boxplot containing all results; IQR ± 1.5 *IQR). Exemplary ∆AUC values: *P19 and C18, ~F19 and Y18, ^P19 and P18