Investigation of the bacterial mechanosensitive ion channel of large conductance within bacterial membrane mimetics in response to simple antimicrobial molecules

Abstract

This thesis describes the use of neutron and X-ray scattering and reflectivity to characterize the structure of the Mechanosensitive Ion Channel of Large Conductance, MscL, within bilayer constructs. MscL is known to gate with increased surface tension in the lipid bilayer. In bacteria, mechanosensitive ion channels such as MscL protect cells against osmotic shock. The channel has also been shown to gate in the presence of amphiphilic molecules. By investigating the response of MscL to two different amphiphilic antimicrobials in three different bacterial membrane mimetics, we explore whether gating of the channels triggered by interaction with these molecules can play a role in their antimicrobial behaviour. Cell-free expression of MscL directly into 3:1 POPC:POPG liposomes was optimised to produce proteoliposomes, that are the basis of the membrane mimetics used in this study. Small angle scattering provided evidence that single MscL channels were successfully expressed into liposomes. The response of the proteoliposomes to the antimicrobial peptide pexiganan and the naturally occurring antimicrobial surfactant, lyso-PC was investigated. We have shown through scattering experiments that we can observe a conformational change in MscL in the presence of lyso-PC and pexiganan that could be a signature of MscL gating. Reflectivity measurements require a planar membrane of about 10 cm² and we have developed a novel membrane mimetic, in which a POPC:POPG bilayer is suspended beneath a cationic surfactant monolayer. Our neutron reflectivity experiments show that a high quality bilayer can be formed and that there is a water layer between the surfactant monolayer and the lipid bilayer. This water gap means that the suspended bilayer can fluctuate and there is sufficient space to allow for membrane proteins inserted into the suspended bilayer to protrud out from the bilayer. We have also developed an experimental system in which the POPC:POPG:MscL bilayer is tethered to a gold layer, which sits on top of a thin permalloy film coated onto a silicon block. The silicon block acts as a neutron window, and the permalloy layer means that we can exploit the two spin states of the neutron to measure polarised reflectivity from the tethered bilayer system, allowing two data sets, with slightly different structural sensitivities, to be measured simultaneously . Using our planar membrane mimetics, we were able to investigate changes in the membrane and the MscL on addition of pexiganan. We observed a decrease in the distance that the proteins protrude out from the membrane from 50-60 ˚A to 30 ˚A, which we suggest is evidence that the channel has gated in response to the interaction of pexiganan with the membrane mimetic. In addition to further insight into the gating mechanism of the MscL this highlights the potential benefit of further investigating the channel as an antimicrobial target.