Interactions of Antibacterial Peptides with Nanotubular Lipid Bilayers: Binding Kinetics and Distortions of the Bilayer Structure

Abstract

Interactions of peptides with membranes are central for understanding key cellular processes involving membrane proteins such as folding, signaling, transport, energy conversion, and immune response. These interactions also determine efficiency of antibacterial and cytotoxic peptides in disrupting the barrier function of cellular membranes. We employed macroscopically aligned tubular lipid bilayers confined inside cylindrical nanopores of anodic aluminum oxide (AAO) as a versatile nanotechnology platform to study membrane interactions of antibacterial peptides of melittin and alamecitin. Kinetics of peptide binding to lipid nanotubes and the eventual lipid removal/lysis (for melittin) were observed by quartz crystal microbalance (QCM) using a crystal with an in-house fabricated nanostructured surface while the structural changes in bilayers were monitored by solid state oriented sample NMR. Specifically, using 78 nm nanopores we have achieved exceptionally narrow (100-140 Hz or <1 ppm) 31P resonances of the lipid phosphate groups of macroscopically aligned nanotubular bilayers that indicated <1-2o mosaic spread in the lipid alignment in the absence of bioreactive peptides. 31P resonances broadened and shifted towards the isotropic values upon interacting with melittin with some lysis of lipids observed after several hours of exposure. The binding and lipid lysis from bilayers confined in essentially the same nanopores albeit at much smaller lipid quantity were further quantified by real-time QCM for bilayers of various lipid compositions. Taken together, the data relate peptide binding and lysis kinetics to structural changes in lipid bilayers, thus, shedding the light on the underlying multistep mechanism. The main advantage of the lipid nanotube AAO platform is in its versatile applicability to various biophysical methods such QCM and NMR under essentially the same environmental conditions such as pH, ionic strength, temperature, etc. and exceptionally broad range of lipid bilayer compositions.