Structural elucidation upon binding of antimicrobial peptides into binary mixed lipid monolayers mimicking bacterial membranes

Findings: Surface pressure-area isotherm analysis revealed that all binary lipid systems mix non-ideally, but mixing is thermodynamically favoured. An increase in the surface pressure encourages demixing, resulting in phase separation and formation of clusters. Neutron reflectivity measurements were undertaken to study the binding of an antimicrobial peptide G(IIKK)4-I-NH2 (G4) to the binary DPPG/TMCL and DPPG/DPPE monolayer mixtures at the molar ratios of 6/4 and 3/7, respectively. The results revealed stronger binding and penetration of G4 to the DPPG/TMCL monolayer, indicating greater affinity of the antimicrobial peptide due to the electrostatic interaction and more extensive penetration into the more loosely packed lipid film. This work helps explain how AMPs attack different bacterial membranes, and Hypothesis: Antimicrobial peptides (AMPs) kill microorganisms by causing structural damage to bacterial membranes. Different microorganisms often require a different type and concentration of an AMP to achieve full microbial killing. We hypothesise that the difference is caused by different membrane structure and composition. Experiments: Given the complexities of bacterial membranes, we have used monolayers of the binary DPPG/TMCL mixture to mimic the cytoplasmic membrane of Gram-positive bacteria and the binary DPPG/DPPE mixture to mimic the cytoplasmic membrane of Gram-negative bacteria, where DPPG, TMCL and DPPE stand for 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1',3'-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, respectively. A Langmuir trough was specially designed to control the spread lipid monolayers and facilitate neutron reflectivity measurements.

Automated summary

Surface pressure-area isotherm analysis showed non-ideal mixing in all binary lipid systems, but it was thermodynamically favored. An increase in surface pressure leads to demixing, resulting in phase separation and cluster formation. Neutron reflectivity measurements indicated stronger binding and penetration of G4 to the DPPG/TMCL monolayer, suggesting greater affinity due to electrostatic interaction.