Identifying the selectivity of antimicrobial peptides to cell membranes by sum frequency generation spectroscopy.

Abstract

Cationic amphiphilic peptides have been engineered to target both Gram-positive and Gram-negative bacteria while avoiding damage to other cell types. However, the exact mechanism of how these peptides target, bind, and disrupt bacterial cell membranes is not understood. One specific peptide that has been engineered to selectively capture bacteria is WLBU2 (sequence: RRWVRRVRRWVRRVVRVVRRWVRR). It has been suggested that WLBU2 activity stems from the fact that when interacting with bacterial cell membranes the peptide assumes an α-helical structure and inserts itself into the membrane. Alternatively, in the presence of mammalian cell membranes, the peptide assumes an inert β-sheet structure. To test this hypothesis, the authors applied sum frequency generation (SFG) spectroscopy and surface tensiometry to identify the structure of WLBU2 as it interacts with model lipid monolayers that mimic mammalian and bacterial cell membranes. Model mammalian cell membranes were built upon zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine lipids while bacterial cell membranes were constructed with negatively charged 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) lipids. Observed changes in surface pressure at the peptide-lipid-air interface demonstrate that the peptide has a clear binding preference toward negatively charged bacteria-like lipids. The structure of both the lipids and peptides were characterized by SFG spectra collected at the monolayer interface. Changes in monolayer structure as the peptide binds were observed by tracking the intensities of SFG vibrational modes related to the acyl chains within the lipids. Peptide structures when bound to both types of lipids were determined by SFG spectra collected within the amide I vibrational band. The SFG spectra of WLBU2 interacting with the model mammalian lipid monolayer contain two peaks near 1642 and 1678 cm-1 indicative of an inactive β-sheet structure. SFG spectra collected from the peptide bound to a bacteria-like lipid monolayer contains just a single peak near 1651 cm-1 which corresponds to an active α-helix structure. Combined, the tensiometry and SFG results demonstrate that WLBU2 both possesses a higher binding affinity toward and is in an active α-helix structure when bound to bacterial cell membranes.