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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Main Author: Tan, Cephas Li-Jie
Other Authors: Jaume Torres
Format: Final Year Project
Language:English
Published: Nanyang Technological University 2021
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Online Access:https://hdl.handle.net/10356/153267
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spelling sg-ntu-dr.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
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences
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
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.
author2 Jaume Torres
author_facet Jaume Torres
Tan, Cephas Li-Jie
format Final Year Project
author Tan, Cephas Li-Jie
author_sort Tan, Cephas Li-Jie
title 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_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_sort effects of lipid environment on aquaporin : exploration of styrene-maleic acid nanodiscs for single-particle cryo-em structural determination
publisher Nanyang Technological University
publishDate 2021
url https://hdl.handle.net/10356/153267
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