Biophysical characterisation of self-assembling nanoparticles
<p>Biophysical characterisation of membrane proteins has been revolutionised through the development of membrane protein extraction with the use of a random co- polymer of styrene:maleic acid (SMA). Since the development of SMA, several novel polymers have been developed, in addition to modifi...
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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2020
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| _version_ | 1797071567510831104 |
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| author | Sawczyc, HLD |
| author2 | Watts, A |
| author_facet | Watts, A Sawczyc, HLD |
| author_sort | Sawczyc, HLD |
| collection | OXFORD |
| description | <p>Biophysical characterisation of membrane proteins has been revolutionised through the development of membrane protein extraction with the use of a random co- polymer of styrene:maleic acid (SMA). Since the development of SMA, several novel polymers have been developed, in addition to modifications to the original SMA structure. However, a rigorous comparative characterisation of these polymers is missing.</p>
<p>This thesis presents the development of novel, high-throughput, solubilisation assays that can be used for the direct comparison of membrane solubilisation. These assays allow for the determination of optimal solubilisation conditions using the four polymers tested (SMA 3:1, SMA 2:1, DIBMA, and PMA). In addition, the effects of pH, ionic strength and divalent cation presence on the solubilisation of membranes by these polymers to form lipid-polymer nanoparticles was probed.</p>
<p>Characterisation of DMPC-polymer nanoparticles formed in ‘ideal’ conditions (pH 7.4, 100 mM NaCl) was performed to ascertain the lipid environment within these extracted bilayers. This showed that all lipid-polymer nanoparticles
possess a higher lipid order than that observed in large unilamellar vesicles (LUVs) and further the thermal phase transition of the lipids within these
nanoparticles is altered.</p>
<p>A previously discovered novel property of lipid-polymer nanoparticles, colli-
sional lipid transfer, was also explored. Through the novel use of asymmetric membrane models it was found that lipid mixing occurs during membrane solubilisation. The rate of this mixing is variable according to the temperature and polymer used, and is a native property of lipid-polymer nanoparticles. This thesis goes on to apply this novel property for the specific titration of lipids into protein containing SMA-nanoparticles (Lipodisqs), using the well characterised cytochrome oxidase c dependence on cardiolipin as a test case. This is the first recorded instance of specific lipid:protein titration without the use of detergent or organic solvent addition.</p>
<p>Finally, the thesis explores the generation of a self-assembling peptide, ST1- QAA, which has been designed to spontaneously form viral-like particles for the purpose of gene transfection. It present a robust, optimised, purification protocol for synthesised ST1-QAA and biophysical characterisation of peptide self-assembly to form viral-like particles.</p> |
| first_indexed | 2024-03-06T22:55:10Z |
| format | Thesis |
| id | oxford-uuid:601ebb0a-fd5e-4098-8d83-49ecc0124c17 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T22:55:10Z |
| publishDate | 2020 |
| record_format | dspace |
| spelling | oxford-uuid:601ebb0a-fd5e-4098-8d83-49ecc0124c172022-03-26T17:51:24ZBiophysical characterisation of self-assembling nanoparticlesThesishttp://purl.org/coar/resource_type/c_db06uuid:601ebb0a-fd5e-4098-8d83-49ecc0124c17membrane protein solubilisationMembrane biophysicsBiophysicsBiochemistryEnglishHyrax Deposit2020Sawczyc, HLDWatts, A<p>Biophysical characterisation of membrane proteins has been revolutionised through the development of membrane protein extraction with the use of a random co- polymer of styrene:maleic acid (SMA). Since the development of SMA, several novel polymers have been developed, in addition to modifications to the original SMA structure. However, a rigorous comparative characterisation of these polymers is missing.</p> <p>This thesis presents the development of novel, high-throughput, solubilisation assays that can be used for the direct comparison of membrane solubilisation. These assays allow for the determination of optimal solubilisation conditions using the four polymers tested (SMA 3:1, SMA 2:1, DIBMA, and PMA). In addition, the effects of pH, ionic strength and divalent cation presence on the solubilisation of membranes by these polymers to form lipid-polymer nanoparticles was probed.</p> <p>Characterisation of DMPC-polymer nanoparticles formed in ‘ideal’ conditions (pH 7.4, 100 mM NaCl) was performed to ascertain the lipid environment within these extracted bilayers. This showed that all lipid-polymer nanoparticles possess a higher lipid order than that observed in large unilamellar vesicles (LUVs) and further the thermal phase transition of the lipids within these nanoparticles is altered.</p> <p>A previously discovered novel property of lipid-polymer nanoparticles, colli- sional lipid transfer, was also explored. Through the novel use of asymmetric membrane models it was found that lipid mixing occurs during membrane solubilisation. The rate of this mixing is variable according to the temperature and polymer used, and is a native property of lipid-polymer nanoparticles. This thesis goes on to apply this novel property for the specific titration of lipids into protein containing SMA-nanoparticles (Lipodisqs), using the well characterised cytochrome oxidase c dependence on cardiolipin as a test case. This is the first recorded instance of specific lipid:protein titration without the use of detergent or organic solvent addition.</p> <p>Finally, the thesis explores the generation of a self-assembling peptide, ST1- QAA, which has been designed to spontaneously form viral-like particles for the purpose of gene transfection. It present a robust, optimised, purification protocol for synthesised ST1-QAA and biophysical characterisation of peptide self-assembly to form viral-like particles.</p> |
| spellingShingle | membrane protein solubilisation Membrane biophysics Biophysics Biochemistry Sawczyc, HLD Biophysical characterisation of self-assembling nanoparticles |
| title | Biophysical characterisation of self-assembling nanoparticles |
| title_full | Biophysical characterisation of self-assembling nanoparticles |
| title_fullStr | Biophysical characterisation of self-assembling nanoparticles |
| title_full_unstemmed | Biophysical characterisation of self-assembling nanoparticles |
| title_short | Biophysical characterisation of self-assembling nanoparticles |
| title_sort | biophysical characterisation of self assembling nanoparticles |
| topic | membrane protein solubilisation Membrane biophysics Biophysics Biochemistry |
| work_keys_str_mv | AT sawczychld biophysicalcharacterisationofselfassemblingnanoparticles |