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Detergent-free approach to the studies of bacterial cell division membrane proteins using styrene maleic acid copolymer
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Teo, Alvin (2017) Detergent-free approach to the studies of bacterial cell division membrane proteins using styrene maleic acid copolymer. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3163767~S1
Abstract
Membrane proteins represent a subset of proteins embedded in or associated with the biological membrane. Despite accounting for 30% of the prokaryotic and eukaryotic proteomes and over 50% of current therapeutic targets, the structural and functional studies of membrane proteins still largely lag behind their soluble counterparts. This is predominantly due to the challenges in the isolation and purification of these proteins from their native mmembrane environment. Traditionally, detergents are employed for the extraction of membrane proteins from the native membrane, with subsequent solubilisation in mixed micelles. However, the surrounding lipids could be sequestered and lost during this process, which is potentially denaturing to the proteins.
A novel method exploiting the styrene maleic acid (SMA) copolymer for membrane solubilisation (in the total absence of detergents) results in the generation of SMA/lipid particles (SMALPs) or ‘native nanodiscs’ of polymer-encapsulated membrane proteins together with their surrounding lipid moieties. Besides allowing the direct solubilisation of target membrane proteins from their native environment, analyses of native protein-lipid interactions and the characterisation of membrane lipidomes could be implemented, which may provide pivotal information to underpin the development of the next-generation antimicrobial agents.
This detergent-free approach was successfully applied to the two cell division proteins, i.e. ZipA and FtsA and another integral membrane lipid phosphatase - PgpB from Escherichia coli. An analytically robust ‘SMALP lipidomics’ method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to elucidate the co-extracted membrane lipidomes of these proteins, resulting in the first comprehensive report of their respective native phospholipid compositions. Biophysical and biochemical characterisations were also conducted on PgpB in the context of SMALP and a detergent benchmark to facilitate the direct comparison between these two approaches for membrane protein studies.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QH Natural history > QH426 Genetics | ||||
Library of Congress Subject Headings (LCSH): | Cell division., Membrane proteins., Lipid membranes., Microcrystalline polymers., Copolymers., Polystyrene. | ||||
Official Date: | July 2017 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Roper, David I. | ||||
Extent: | xviii, 183 leaves : illustrations (chiefly colour | ||||
Language: | eng |
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