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研究生：	劉晨康 Lew, Sin-Hong
論文名稱：	利用磷脂奈米碟進行阿拉伯芥GTR1之結構研究 Reconstitution of phospholipid nanodiscs for structural studies of Arabidopsis thaliana GTR1
指導教授：	林士鳴 Lin, Shih-Ming
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生物科技與產業科學系 Department of Biotechnology and Bioindustry Sciences
論文出版年：	2023
畢業學年度：	111
語文別：	英文
論文頁數：	77
中文關鍵詞：	膜支架蛋白、奈米碟、阿拉伯芥硫代葡萄糖苷轉運蛋白1 、低溫冷凍電子顯微鏡、DoE實驗設計
外文關鍵詞：	Membrane Scaffold Protein (MSP), Nanodiscs, AtGTR1/NPF2.10, Cryo-Electron Microscopy (Cryo-EM), Design of Experiments (DoE)
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單分子低溫冷凍電子顯微鏡（cryo-EM）技術在結構生物學的研究中發揮了重要的影響，特別在穿膜蛋白結構解析領域。由於傳統的X光晶體學方法往往在蛋白質結晶化面臨著挑戰，而Cryo-EM 技術的使用提供了一種有潛力的替代方案。在研究中，我們即嘗試利用Cryo-EM技術來探討阿拉伯芥硫代葡萄糖苷轉運蛋白1（AtGTR1）的結構。我們選擇將AtGTR1重組於在膜支架蛋白（MSP）奈米碟中，這不僅提供了適宜的脂質環境，同時也減少了界面活性劑產生的微胞干擾。透過實驗設計策略（DoE），我們優化了重組條件，並且成功製備出高產率且大小均一的AtGTR1-GFP奈米碟。我們利用負染穿透式電子顯微鏡（nsTEM）驗證了這些奈米碟的尺寸均一性。此外，我們更使用Cryo-EM的單分子重組技術建構出AtGTR1的三維密度圖，結果顯示AtGTR1能在奈米碟中以單體和雙體的形式存在。值得注意的是，即使在無脂質存在的情況下，AtGTR1也能與MSP結合並維持其溶解度，這為未來探究穿膜蛋白結構提供了的新途徑。這些研究成果顯示了奈米碟組裝對於應用Cryo-EM解析穿膜蛋白結構的重要性。我們期望後續進一步針對Cryo-EM分析優化後，將能夠獲得更高解析度之結構資訊。

The advancement of single-molecule cryo-electron microscopy (cryo-EM) has significantly influenced structural biology research, especially in the realm of transmembrane protein structural analysis. While traditional X-ray crystallography often struggles with protein crystallization, cryo-EM provides a compelling alternative. In this study, we attempted to utilize cryo-EM to investigate the structure of Arabidopsis thaliana glucosinolate transporter 1 (AtGTR1). We reconstitute AtGTR1 within the nanodiscs of membrane scaffold protein (MSP), offering not only an appropriate lipid environment but also minimizing interference from detergent micelles. Employing a Design of Experiments (DoE) strategy, we optimized the reconstitution conditions and successfully produced high-yield and uniform AtGTR1-GFP nanodiscs. The size homogeneity of these nanodiscs was validated by using negative stain transmission electron microscopy (nsTEM). Furthermore, we conducted cryo-EM single particle reconstitution to generate the 3D maps of AtGTR1. The resulting maps revealed both monomeric and dimeric forms of AtGTR1 within the nanodiscs. Interestingly, even in the absence of lipids, AtGTR1 was able to bind to MSP and retain its solubility, suggesting new avenues for studying membrane protein structures. These results emphasize the importance of nanodisc assembly in determining membrane protein structure via cryo-EM. We are optimistic that continued refinement of the cryo-EM protocol will yield even higher resolutions in the future.

Chinese Abstract (中文摘要)  I
Abstract II
Acknowledgements V
Table of Contents VI
Contents of Tables X
Contents of Figures XI
Abbreviation List XIII
1. Research Background	 1
1-1 Structural studies of membrane proteins 1
1-2 MSP nanodiscs: A practical approach to membrane protein study 1
1-2-1 Types of MSP proteins 2
1-2-2 Choice of Lipids 3
1-2-3 Detergent removal 3
1-2-4 Ratio between MSPs, Lipids, MOI 4
1-3 MOI in this study: Arabidopsis thaliana GTR1/NPF2.10 4
1-4 Research objectives	5
2. Materials and Methods 6
2-1 Materials 6
2-1-1 Plasmids 6
2-1-2 Yeast and E. coli 6
2-2 Preparation of purified proteins 6
2-2-1 TEV protease 6
2-2-2 MSP1D1 7
2-2-3 AtGTR1-GFP and AtGTR1 8
2-3 Optimization and preparation of AtGTR1 nanodiscs 10
2-3-1 Design of Experiments (DoE) 10
2-3-2 Reconstitution of AtGTR1 nanodiscs 10
2-3-3 Elimination of empty nanodiscs 11
2-3-4 High Performance Liquid Chromatography-Size exclusion chromatography (HPLC-SEC) 11
2-4 TEV digestion for GFP-tag cleavage 11
2-5 Negative stain TEM analysis 12
2-6 Cryo-EM single particle reconstitution 12
2-6-1 Grid preparation 12
2-6-2 Data acquisition 12
2-6-3 Image processing	13
2-7 Graphene-oxide grid preparation 13
3. Results 15
3-1 Preparation of purified proteins for nanodiscs reconstitution	15
3-1-1 TEV protease preparation	15
3-1-2 MSP1D1 purification and His-tag removal 15
3-1-3 AtGTR1-GFP purification 16
3-2 Evaluation of optimized factors for nanodiscs reconstitution 17
3-2-1 Lipid composition 18
3-2-2 Lipid-to-MSP ratio	18
3-2-3 AtGTR1-GFP to MSP ratio 19
3-2-4 Detergent removal rate 19
3-2-5 Optimized conditions for nanodiscs reconstitution 19
3-3 Empty nanodiscs could be removed by using IMAC 20
3-4 Negative stain TEM suggested that the reconstituted AtGTR1-GFP nanodiscs is 12-13 nm discoidal particles 20
3-5 AtGTR1 nanodiscs could be formed by GFT-tag cleavage of AtGTR1-GFP nanodiscs 21
3-6 Cryo-EM analysis revealed monomer and dimer AtGTR1 was reconstituted into nanodiscs 22
3-7 AtGTR1 could be directly reconstituted into nanodiscs 23
3-8 Particles density on cryo-EM grid may be enhanced by coating GO layer on the grid 25
3-9 AtGTR1-GFP maybe stabilized by MSP1D1 without the existence of lipid 26
4. Discussion 28
4-1 The factors that affect nanodiscs reconstitution 28
4-1-1 Effects of lipid type 28
4-1-2 Effects of ratio between lipids, MSP and AtGTR1-GFP	 28
4-1-3 Effects of detergent removal speed 29
4-2 Challenges on solving AtGTR1 structure using cryo-EM 29
4-3 The formation of the AtGTR1-MSP1D1 complex without the existence of lipid	 32
4-4 Conclusion 32
References 34
Tables 39
Figures	41
Appendices 74
                                    

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2026-09-01公開

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