Effects of lipid environment on aquaporin : exploration of styrene-maleic acid nanodiscs for single-particle cryo-EM structural determination
Aquaporin Z (AqpZ) is a water channel protein native to Escherichia coli with lipid-dependent activity. However, AqpZ structural changes that may explain water flux changes have not yet been elucidated. Thus, my aim was to use styrene-maleic acid copolymers (SMAc) to produce AqpZ-nanodiscs suitable...
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| Format: | Final Year Project |
| Language: | English |
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Nanyang Technological University
2021
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| Online Access: | https://hdl.handle.net/10356/153267 |
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| Institution: | Nanyang Technological University |
| Language: | English |
| Summary: | Aquaporin Z (AqpZ) is a water channel protein native to Escherichia coli with lipid-dependent activity. However, AqpZ structural changes that may explain water flux changes have not yet been elucidated. Thus, my aim was to use styrene-maleic acid copolymers (SMAc) to produce AqpZ-nanodiscs suitable for single-particle cryo-EM reconstruction testing different lipid compositions. SMAc has typically been used to directly solubilize native cell membranes. Herein, we report a novel use of SMAc to solubilize proteins from proteoliposomes, thus allowing control of lipid composition within the nanodiscs. However, size-exclusion chromatography (SEC) of SMA-solubilized proteoliposomes suggests that nanodiscs are preferentially formed with-out protein. We also show that SEC alone cannot resolve protein-containing nanodiscs from lipodiscs, requiring an additional enrichment step. Ni2+NTA chromatography was found to be unsuitable, possibly by interference of the AqpZ-His tail with the polymer. Future endeavours should consider alternative protein or resin modifications. Lastly, to reduce the cost of future SMA usage, we show a successful SMAc hydrolysis of its cheaper anhydride precursor. |
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