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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80596完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳佩燁(Rita P.-Y. Chen) | |
| dc.contributor.author | Chih-Hsuan Lee | en |
| dc.contributor.author | 李至璿 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:10:19Z | - |
| dc.date.available | 2021-11-03 | |
| dc.date.available | 2022-11-24T03:10:19Z | - |
| dc.date.copyright | 2021-11-03 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-10-22 | |
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Molecular biology of prion diseases. Science, 252(5012), 1515-1522. https://doi.org/10.1126/science.1675487 Riek, R., Hornemann, S., Wider, G., Billeter, M., Glockshuber, R., Wüthrich, K. (1996). NMR structure of the mouse prion protein domain PrP(121–231). Nature, 382(6587), 180-182. https://doi.org/10.1038/382180a0 Rudd, P. M., Endo, T., Colominas, C., Groth, D., Wheeler, S. F., Harvey, D. J., Wormald, M. R., Serban, H., Prusiner, S. B., Kobata, A., Dwek, R. A. (1999). Glycosylation differences between the normal and pathogenic prion protein isoforms. Proc. Natl. Acad. Sci. U.S.A., 96(23), 13044. https://doi.org/10.1073/pnas.96.23.13044 Rudd, P. M., Wormald, M. R., Wing, D. R., Prusiner, S. B., Dwek, R. A. (2001). Prion glycoprotein: Structure, dynamics, and roles for the sugars. Biochemistry, 40(13), 3759-3766. https://doi.org/10.1021/bi002625f Scheres, S. H. (2012). RELION: Implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. 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M., Vidal, E., Rosa, I., Renault, L., Ramos, A., Peters, P. J., Fernández, J. J., van Heel, M., Young, H. S., Requena, J. R., Wille, H. (2016). The structural architecture of an infectious mammalian prion using electron cryomicroscopy. PLoS Pathog., 12(9), e1005835. https://doi.org/10.1371/journal.ppat.1005835 Wüthrich, K., Riek, R. (2001). Three-dimensional structures of prion proteins. In Adv. Protein Chem. (Vol. 57, pp. 55-82). Academic Press. https://doi.org/10.1016/S0065-3233(01)57018-7 Wang, L. Q., Zhao, K., Yuan, H. Y., Wang, Q., Guan, Z., Tao, J., Li, X. N., Sun, Y., Yi, C. W., Chen, J., Li, D., Zhang, D., Yin, P., Liu, C., Liang, Y. (2020). Cryo-EM structure of an amyloid fibril formed by full-length human prion protein. Nat. Struct. Mol. Biol., 27(6), 598-602. https://doi.org/10.1038/s41594-020-0441-5 Zhang, K. (2016). Gctf: Real-time CTF determination and correction. J. Struct. Biol., 193(1), 1-12. https://doi.org/10.1016/j.jsb.2015.11.003 Zheng, S. Q., Palovcak, E., Armache, J. P., Verba, K. A., Cheng, Y., Agard, D. A. (2017). MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods., 14(4), 331-332. https://doi.org/10.1038/nmeth.4193 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80596 | - |
| dc.description.abstract | 普利昂疾病是一類致命且具傳染性的神經退化性疾病，其臨床上的症狀包含失智、肌肉抽搐、共濟失調…等等。其發生的原因主要為普利昂蛋白質之二級結構由α結構轉變為β結構，形成類澱粉纖維堆積，造成腦部神經細胞死亡，因此，普利昂疾病是由蛋白質錯誤折疊引起的疾病。一種自發性普利昂疾病有基因序列突變會產生截短的普利昂蛋白，這種截短的普利昂蛋白可在接近生理條件之緩衝液內形成類澱粉纖維。 本論文中我們研究小鼠普利昂蛋白mPrP(23-144)產生的類澱粉纖維結構，我們選擇了五個位點並將它們單獨突變為 Cys 以進行自旋標記，兩種含 Cys 的蛋白質用 MTSSL 標記以獲得自旋標記的蛋白質，接著我們使用mPrP(23-144)形成的類澱粉纖維作為晶種去誘使各突變蛋白形成類澱粉纖維，最後再以電子自旋光譜ESR來解析類澱粉纖維中自旋標記之間的交互作用，探討mPrP(23-144)形成之類澱粉纖維的結構特性。實驗結果發現，113號殘基不在類澱粉纖維的cross-β結構中。此外，本研究中亦使用冷凍電子顯微鏡之技術，嘗試解析倉鼠普利昂胜肽haPrP(108-144)形成的類澱粉纖維的結構，我們成功得到高品質的電子顯微鏡影像，並且以單粒子分析之方式，進行類澱粉纖維之三維結構重組，已獲得解析度2.72 Å的三維結構。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:10:19Z (GMT). No. of bitstreams: 1 U0001-2210202116300000.pdf: 9597121 bytes, checksum: 1e40192fbd0682423df51c07a24e8bd5 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "中文摘要 i Abstract ii Abbreviations iii Table of contents iv List of figures vii List of tables x Chapter 1 Introduction 1 1.1 Prion diseases 1 1.2 Prion protein 3 1.3 Prion protein Y145Stop mutation 5 1.4 Electron Spin Resonance (ESR) 7 1.5 The aim of this study 10 Chapter 2 Materials and Methods 11 2.1 Materials 11 2.1.1 Water 11 2.1.2 Chemicals 11 2.1.3 Laboratory instruments 12 2.2 Methods 14 2.2.1 Site-directed mutagenesis 14 2.2.2 Expression of recombinant mouse PrP in E.coli and cell lysis 16 2.2.3 Immobilized metal-ion affinity chromatography (IMAC) 16 2.2.4 HPLC purification and protein identification 17 2.2.5 MTSSL labeling, purification and identification 17 2.2.6 Seed preparation 17 2.2.7 Amyloid Fibril formation 18 2.2.8 ThT binding assay 18 2.2.9 TEM grid preparation and observation 19 2.2.10 ESR sample preparation and spectroscopy measurement 19 Chapter 3 Results 20 3.1 Construction of mPrP(23-144) and the mutants 20 3.2 Mouse prion protein (23-144) expression and identification 21 3.3 Immobilized metal-ion affinity chromatography (IMAC) purification 21 3.4 High-performance liquid chromatography (HPLC) purification 22 3.5 Spin-labeling and purification 24 3.6 Spontaneous fibril formation 25 3.7 Seeded fibril formation 29 3.8 Transmissible Electron Microscopy 33 3.9 ESR spectra of A113R1 fibrils 36 Chapter 4 Cryo-EM study of hamster haPrP(108-144) peptide fibrils 38 4.1 Application of Cryo-EM in studying prion fibril structure 38 4.2 haPrP(108-144) fibril formation 40 4.3 Cryo-EM grid preparation 41 4.4 Cryo-EM data collection 42 4.5 Cryo-EM data processing 43 4.5.1 Import micrographs 43 4.5.2 Motion correction 44 4.5.3 CTF Estimation 46 4.5.4 Manual picking of fibrils 48 4.5.5 Particle extraction 49 4.5.6 2D classification 50 4.5.7 Selection of good particles 54 4.5.8 Initial model generation for amyloids 56 4.5.9 3D classification 59 4.5.10 3D Refinement 62 Chapter 5 Conclusion and discussion 73 Reference 75" | |
| dc.language.iso | en | |
| dc.subject | 冷凍電子顯微鏡 | zh_TW |
| dc.subject | 普利昂疾病 | zh_TW |
| dc.subject | 普利昂蛋白 | zh_TW |
| dc.subject | 類澱粉纖維 | zh_TW |
| dc.subject | 電子自旋光譜 | zh_TW |
| dc.subject | amyloid fibrils | en |
| dc.subject | electron spin resonance spectroscopy | en |
| dc.subject | prion diseases | en |
| dc.subject | prion protein | en |
| dc.subject | cryo-electron microscopy | en |
| dc.title | 以電子自旋共振光譜及冷凍電子顯微鏡探討普利昂蛋白生成的類澱粉纖維之結構 | zh_TW |
| dc.title | Exploring the structures of prion amyloid fibrils by electron spin resonance spectroscopy and cryo-electron microscopy | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳昆峯(Hsin-Tsai Liu),陳振中(Chih-Yang Tseng) | |
| dc.subject.keyword | 普利昂疾病,普利昂蛋白,類澱粉纖維,電子自旋光譜,冷凍電子顯微鏡, | zh_TW |
| dc.subject.keyword | prion diseases,prion protein,amyloid fibrils,electron spin resonance spectroscopy,cryo-electron microscopy, | en |
| dc.relation.page | 78 | |
| dc.identifier.doi | 10.6342/NTU202104042 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-10-25 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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