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 (FYP) |
| Language: | English |
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Nanyang Technological University
2021
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| Online Access: | https://hdl.handle.net/10356/153267 |
| _version_ | 1811694789518688256 |
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| author | Tan, Cephas Li-Jie |
| author2 | Jaume Torres |
| author_facet | Jaume Torres Tan, Cephas Li-Jie |
| author_sort | Tan, Cephas Li-Jie |
| collection | NTU |
| description | 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. |
| first_indexed | 2024-10-01T07:13:09Z |
| format | Final Year Project (FYP) |
| id | ntu-10356/153267 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T07:13:09Z |
| publishDate | 2021 |
| publisher | Nanyang Technological University |
| record_format | dspace |
| spelling | ntu-10356/1532672023-02-28T18:08:37Z Effects of lipid environment on aquaporin : exploration of styrene-maleic acid nanodiscs for single-particle cryo-EM structural determination Tan, Cephas Li-Jie Jaume Torres School of Biological Sciences JTorres@ntu.edu.sg Science::Biological sciences 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. Bachelor of Science in Biological Sciences 2021-11-17T03:12:07Z 2021-11-17T03:12:07Z 2021 Final Year Project (FYP) 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 https://hdl.handle.net/10356/153267 en Ministry of Education of Singapore Tier 1 thematic grant call 2019 RT13/19 and Tier 1 RG101/20 application/pdf Nanyang Technological University |
| spellingShingle | Science::Biological sciences Tan, Cephas Li-Jie Effects of lipid environment on aquaporin : exploration of styrene-maleic acid nanodiscs for single-particle cryo-EM structural determination |
| title | Effects of lipid environment on aquaporin : exploration of styrene-maleic acid nanodiscs for single-particle cryo-EM structural determination |
| title_full | Effects of lipid environment on aquaporin : exploration of styrene-maleic acid nanodiscs for single-particle cryo-EM structural determination |
| title_fullStr | Effects of lipid environment on aquaporin : exploration of styrene-maleic acid nanodiscs for single-particle cryo-EM structural determination |
| title_full_unstemmed | Effects of lipid environment on aquaporin : exploration of styrene-maleic acid nanodiscs for single-particle cryo-EM structural determination |
| title_short | Effects of lipid environment on aquaporin : exploration of styrene-maleic acid nanodiscs for single-particle cryo-EM structural determination |
| title_sort | effects of lipid environment on aquaporin exploration of styrene maleic acid nanodiscs for single particle cryo em structural determination |
| topic | Science::Biological sciences |
| url | https://hdl.handle.net/10356/153267 |
| work_keys_str_mv | AT tancephaslijie effectsoflipidenvironmentonaquaporinexplorationofstyrenemaleicacidnanodiscsforsingleparticlecryoemstructuraldetermination |