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標題: | 藉由冷凍電顯技術研究核蛋白結構 Structural studies of nucleoproteins by Cryo-EM |
作者: | 葉敏琪 Min-Chi Yeh |
指導教授: | 何孟樵 Meng-Chiao Ho |
關鍵字: | 冷凍電子顯微技術,RAD51,同源重組,Rpl23,核糖體生合成, Cryo-EM,RAD51,Homologous recombination,Rpl23,Ribosome biogensis, |
出版年 : | 2023 |
學位: | 博士 |
摘要: | 隨著冷凍電子顯微術的發展,許多過去無法利用X射線晶體學或核磁共振光譜學研究的蛋白質得以被解釋,也大大的提升了生物學家的視野。冷凍電子顯微術相較於其他研究蛋白質結構方法學有著四個優點。一,適用於分子量較大且複雜的蛋白聚合物;二,無需結晶;三,無需高濃度蛋白樣品;四,可看到動態變化。因此有越來越多的蛋白質結構藉由冷凍電子顯微術被解出,使其成為現今生物物理學界的主流。
在此篇論文中,我們藉由冷凍電子顯微術解開了兩種核蛋白的結構。其一為無活性的RAD51聚合物,在同源重組中扮演重要的角色。另一為Rpl23聚合物,而Rpl23為核糖體生合成中不可或缺的核糖體蛋白質。在研究RAD51的部分中,我們藉由冷凍電顯得到高解析度的ADP結合的RAD51聚合物,解釋了過去數十年中單分子實驗無法解釋的現象,也提出了當ATP水解時,RAD51聚合物的崩塌機制。而在研究Rpl23的部分中,我們藉由化學交叉鏈結、冷凍電顯和電腦模擬,揭開了Rpl23如何在核糖體生合成過程中被Bcp1和Rkm1保護。 With the development of Cryo-electron microscopy (Cryo-EM), more and more protein structures can be determined, which could not be resolved by X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy in the past, enhancing new insight into biophysical studies. Compared to the other structural determination methods, cryo-EM has four advantages: 1) appropriate for protein complex or marcoprotein, 2) no crystallization, 3) relatively low concentration of protein sample, and 4) the observation of protein dynamics. Hence, more and more structures of proteins or protein complexes can be determined via cryo-EM, leading it to a mainstream method in the biophysical field these days. In this dissertation, I determined two nucleoprotein structures by cryo-EM. One is RAD51-nucleopfilament, the key player in homologous recombination, the other is Rpl23 protein complex, the essential ribosomal protein (r-protein) during ribosome biogenesis. In the RAD51 section, we determine the high resolution of ADP-bound RAD51 filament to explain the ambiguous phenomena observed in single molecular experiments for decades and propose the collapsing mechanism during ATP hydrolysis. In the Rpl23 section, we combined chemical-crosslinked, cryo-EM, and computational simulation to reveal how Rpl23 is protected by both Bcp1 and Rkm1 during ribosome biogenesis process. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/87615 |
DOI: | 10.6342/NTU202300298 |
全文授權: | 同意授權(限校園內公開) |
電子全文公開日期: | 2028-02-01 |
顯示於系所單位: | 生化科學研究所 |
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