The Effect of Varying Membrane Fluidity on the Binding Affinity of LL-37

Abstract

Antibiotic resistance is a prominent issue, and the number of bacterial strains with resistance to conventional antibiotics are rising readily. Antimicrobial peptides (AMPs) are a promising alternative to antibiotics and exhibit antimicrobial activity against bacteria, fungi, and viruses. While antibiotics target bacteria through a specific mechanism, AMPs use electrostatics and differences in membrane composition to target bacteria, which is known as AMP selectivity. In this study, the effects of varying membrane fluidity on the binding affinity of the human AMP LL-37 to the membrane are investigated. Large unilamellar vesicles (LUVs) varying in mol% cholesterol, saturation, and tail length were prepared. All LUVs have a diameter of 100 nm and a 3:1 mole ratio of PC (Zwitterionic) and PG (negative) head groups. The negative PG lipids promote the binding of the positively charged, FAM-labeled LL-37 to the membrane, and fluorescence anisotropy was used to measure this binding. The effect of cholesterol was studied using LUVs with 20, 30, and 40 mol% cholesterol, where increasing mol% cholesterol correlates with decreasing fluidity. The effect of tail length was investigated using lipids with fatty acids containing 14, 16, and 18 carbons, where increased tail length correlates with lower fluidity. The effect of saturation was studied using lipids with zero, one, and two double bonds in their fatty acids, where increased saturation correlates with decreased fluidity. The binding affinity of LL-37 to the membrane generally increased with an increasing saturation and increasing mol% cholesterol, but it fluctuated with increasing tail length. The optimal bilayer for LL-37 to bind contains tails with no double bonds and 16 carbons. Non-cooperative binding was mostly observed. Notably, as mol% cholesterol increases, there is more negative cooperativity, which highlights the rigidity of membranes with cholesterol and makes it harder for the LL-37 to bind at other sites on the membrane. The mechanisms underlying AMP selectivity are not fully clear and include more than just electrostatics. Understanding the effect of physical membrane characteristics (like fluidity) can explain what directs AMP selectivity towards bacteria and predict how AMPs will target microbes that evolve new physical ways of resistance.