Use of scattering techniques to probe molecular interactions between novel Trp- and Arg-rich antimicrobial peptides (AMPs) in lipid model membranes (LMMs)

Antibiotics, one of the most important medical developments of the twentieth century, are losing effectiveness as diverse bacteria become resistant to standard and last resort commercial drugs. To address this challenge and provide alternatives to traditional antibiotics, we have rationally designed AMPs with different lengths, charges, hydrophobicities (H) and hydrophobic moments(µH). Five AMPs (out of 86) with the lowest minimum inhibitory concentrations (MICs) and <25% toxicity to mammalian cells were selected for biophysical studies. Their secondary structure was determined using circular dichroism (CD), which found that % α-helicity of AMPs depends on lipid composition. There was decreasing helicity with these LMMs: G(-) inner membrane (IM)> G(+) > Euk33 (eukaryotic with 33 mol% cholesterol). E2-35 (16-mer) and E2-05 (22-mer) are largely helical in G(-) IM LMM, (PE:PG:CL,7:2:1) and G(+) LMM (PG:DOTAP:PE:CL,6:1.5:1.5:1). LE-53 (12-mer) and LE-55 (16-mer) are mostly random coil when interacting with both G(-) IM and G(+) LMMs. The higher α-helical content of E2-35 and E2-05 correlated with a lower MIC and higher toxicity, suggesting that helicity is important for designing new AMPs. Other aspects of AMP/membrane interactions such as membrane elasticity, chain order parameter and location of the peptides in the membrane were investigated by low angle and wide angle x-ray diffuse scattering (XDS). A headgroup or interfacial location correlated with toxicity. The membrane bending modulus Kc displayed a non-monotonic change due to E2-35 and E2-05 perturbation in LMMs. The chain order parameter (Sxray) paralleled Kc in most cases. We suggest that juxtaposition of domains with different bending moduli could lead to leakage of ions and water along domain walls between softer and more rigid domains, thus killing the bacteria.