Figure 1. Thermally stabilized maculatin.

(a) CD spectra show that wild-type (WT) maculatin starts to denature at elevated temperatures in the presence of 100 nm POPC LUVs (peptide-to-lipid ratio=1/100). (b) WT maculatin-induced LUV leakage is reduced for bilayers containing phosphatidylcholine (PC) lipids with longer hydrophobic tails (14:1 Δ9-cis; 16:1 Δ9-cis; 18:1 Δ9-cis; 20:1 Δ11-cis; 22:1 Δ13-cis). (c) The single-mutation P15A stabilizes maculatin against thermal denaturation, with no detectable loss of helicity even at 74 °C. The temperatures shown were the temperature measured for the cuvette, with 74 °C corresponding to a cell-holder temperature of 95 °C, the highest setting possible. (d) P15A induces similar but less liposomal leakage as WT maculatin, with similar lipid tail length dependence. (e) The pore-sizing assay measures the leakage of dyes of increasing size (400–10,000 Da) from 0.5 mM POPC LUVs (diameter=100 nm) after addition of 0.5 μM peptide (that is, P/L=1/1,000) using fluorescence spectroscopy. Hundred per cent leakage was determined using 10 vol.% Triton X-100. Pore size and leakage efficiency of the P15A single and P15A-E19Q double mutants are similar to that of WT. (f) Octameric pores formed by P15A-E19Q during assembly simulations were found to spontaneously conduct both ANTS and DPX dyes using unbiased conductance simulations. Error bars are s.e.m.