| dc.description.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. | en |
