Elucidating the orientation of LL -37 in model cell membranes using sum frequency generation

Abstract

Elucidating the mode of action of antimicrobial peptides (AMPs) in cell membrane disruption is of interest in understanding the efficiency of different AMPs, which is essential to design antibiotic with desired potency and selectivity. As a key component of the innate immunity system, human cathelicidin LL-37 plays a crucial role in protecting human against infectious diseases. LL-37 kills cells by disrupting the membrane integrity through physical interaction with cell membranes. Structure, membrane orientation and target membrane selectivity of LL-37 were characterized by using a modern vibrational spectroscopic technique, Sum Frequency Generation (SFG). Recent developments in SFG have introduced it a powerful and unique biophysical technique in study the interaction between biological molecules and model cell membranes. A supported 1-palmitoyl-2-oleoyl- sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) bilayer was used as a model bacterial cell membrane. A supported 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) bilayer was used as a model mammalian cell membrane. It is found that, under our experimental conditions, the helical LL-37 molecules are associated with POPG/POPG bilayer surface with ~50o tilted from the bilayer normal, indicating a toroidal pore mechanism of action for the peptide at a concentration of 400 nM. In contrary, no interaction between LL-37 and a zwitterionic POPC bilayer was observed even at a much higher peptide concentration (∼1.2 μM). These results would explain a selective effect on bacteria over mammalian cells. Additionally, in order to deduce the orientation of LL-37 on model cell membranes, we have introduced a modification version in the calculation on data analysis, which is important in SFG study. In the data analysis, we proved that twistings along with tilting among helical segments are extremely important in orientation of the peptide. Our findings provide new approach to elucidate the orientation of AMPs with multiple helical structures, and thus demonstrate that SFG is a reliable technique which can provide insight into the molecular interaction of antimicrobial peptides and cell membranes in situ.