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|Title:||Effects of lipid environment on aquaporin : exploration of styrene-maleic acid nanodiscs for single-particle cryo-EM structural determination||Authors:||Tan, Cephas Li-Jie||Keywords:||Science::Biological sciences||Issue Date:||2021||Publisher:||Nanyang Technological University||Source:||Tan, C. L. (2021). Effects of lipid environment on aquaporin : exploration of styrene-maleic acid nanodiscs for single-particle cryo-EM structural determination. Final Year Project (FYP), Nanyang Technological University, Singapore. https://hdl.handle.net/10356/153267||Project:||Ministry of Education of Singapore Tier 1 thematic grant call 2019 RT13/19 and Tier 1 RG101/20||Abstract:||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.||URI:||https://hdl.handle.net/10356/153267||Schools:||School of Biological Sciences||Fulltext Permission:||restricted||Fulltext Availability:||With Fulltext|
|Appears in Collections:||SBS Student Reports (FYP/IA/PA/PI)|
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Updated on May 31, 2023
Updated on May 31, 2023
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