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Title: Probing the effects of membrane mechanics on MscL activity
Author: Barriga, Hanna Maria Gabriella
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Biological membranes are a dynamic and complex mixture of lipids and proteins, all of which vary in size, shape and composition. The ability of cells to compartmentalise and regulate their contents is in large part due to the regulation of membrane stress and composition. This in turn can alter the behaviour of proteins associated with the membrane. For this reason, understanding membranes and how their fundamental properties can influence proteins in vitro is essential for improved in vivo knowledge, and is also of great interest to the pharmaceutical industry. In this thesis work is presented which investigates the effects of membrane stress on the mechanosensitive channel of large conductance (MscL) from E. Coli using a fluorescence based assay. Membrane compositional sweeps were used to control the stress and to prove that changes in membrane stress directly affect MscL activity. Corresponding X Ray scattering and solid state NMR measurements probed the fundamental properties of the membranes used during the MscL activity assay sweeps. Not only were buffers found to have a significant effect on the membranes but a unique result from the solid state NMR data showed that for a specific lipid composition the membranes were more susceptible to deformation within the magnetic field. This result correlates directly with a change in activity observed in the MscL activity assay. This is to the best of our knowledge the first direct correlation between MscL activity and a fundamental membrane parameter. One key result of this thesis is the applicability of the assay and the technology to other systems, lipids and proteins. We present preliminary data for the translation of this assay into droplet interface bilayer (DIB) format, thereby creating a platform technology enabling the study of the effects of membrane asymmetry. This assay is already being adapted for use by other researchers.
Supervisor: Ces, Oscar ; Templer, Richard Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available