Direct Observation of the Activation of MscL in Tethered Lipid Bilayers by an Antimicrobial Peptide

Data and Python code to support the manuscript "Direct Observation the Activation of MscL in Tethered Lipid Bilayers by an Antimicrobial Peptide". Abstract: Hypothesis Membrane proteins serve a wide range of vital roles in the functioning of living organisms. They account for approximately 20% to 30% of the genomes across bacterial, archaeal, and eukaryotic organisms. They are responsible for many cellular functions, such as signaling, ion and molecule transport, binding and catalytic reactions. Compared to other classes of proteins, determining membrane protein structures remains a challenge, in large part due to the difficulty in establishing experimental conditions that can preserve the correct conformation and function of the protein in isolation from its native environment. Many therapeutics target membrane proteins which are accessible on the surface of cells. Here we hypothesize that the observed efficacy of antimicrobial peptides (AMPs) that interact with bacterial membranes may in part be associated with their triggering of MscL (Mechansensitive Ion Channel of Large Conductance) gating. We further conjecture that the insertion of peptides into the membrane induces significant changes in membrane tension and/or curvature, leading to prolonged gating of the MscL channels.

Experiments We present realistic model membrane systems containing MscL. We investigated the ion channel in lipid vesicles and in a planar lipid bilayer. We developed a novel method for protein-lipid planar bilayer formation, avoiding the use of detergents. By using a polymeric tether our planar membrane mimetic was not constrained by the underlying solid substrate, making it sufficiently flexible to allow for increases in bilayer curvature and changes in membrane tension. We used quartz crystal microbalance with dissipation (QCM-D), and polarised neutron reflectivity (PNR) to show the formation of MscL containing phospholipid bilayers, tethered with a high density PEG layer onto gold substrates from vesicle rupture. The MscL containing vesicles were separately characterised with small angle neutron scattering (SANS).

Findings MscL was expressed into vesicles using cell free protein expression. Analysing these vesicles with small angle neutron scattering, the radius of gyration of the protein was determined to be between 26-29~\AA{}, consistent with the crystal structure of individual MscL channels. The MscL composition of the formed bilayer was 14\%v/v, close to the initial volume composition of the vesicles at ~13.6% and a protein protrusion extending ca. 46~\AA{} into the solvent was determined by PNR. Addition of 1.6 and 3.2 uM pexiganan resulted in a decrease in the protrusion of MscL (from ~46 to ~38~\AA{}). To our knowledge, these findings represent the first direct experimental evidence of a structural change in the C-terminus containing protrusion of MscL, triggered by an antimicrobial peptide. This adds to our understanding of antimicrobial peptide action in therapeutic treatments.