Neutron Reflectometry Reveals Conformational Changes in a Mechanosensitive Protein Induced by an Antimicrobial Peptide in Tethered Lipid Bilayers

Abstract

Membrane proteins serve a wide range of vital roles in the functioning of living organisms. 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. We investigated the ion channel in lipid vesicles and in a planar lipid bilayer. 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). 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~Å, consistent with the crystal structure of individual MscL channels. The MscL composition of the formed bilayer was 14\%$v/v$, close to the initial composition of the vesicles, and a protein protrusion extending ca. 46~Å into the solvent was determined by PNR. Addition of 1.6 and 3.2 $μ$M pexiganan resulted in a decrease in the protrusion of MscL (from $\sim$46 to $\sim$38~Å). 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.