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標題: | 開發新型支架蛋白協助冷凍電子顯微鏡分析低分子量蛋白結構 Imaging small proteins with a novel scaffold protein for cryo-EM structural analysis |
作者: | Yi-Hsiang Chiu 邱怡翔 |
指導教授: | 徐尚德(Shang-Te Danny Hsu) |
關鍵字: | 冷凍低溫電子顯微鏡,支架蛋白,鳥胺酸氨甲醯基轉移酶,泛素,泛素羧基末端水解酶-1, cryogenic electron microscopy,scaffold protein,ornithine transcarbamylase,ubiquitin,ubiquitin carboxyl-terminal hydrolase isozyme L1, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 隨著電子顯微鏡影像感測器和分析軟體的革新,冷凍低溫電子顯微鏡(cryo-EM)成為近期結構生物學中重要的工具。利用冷凍低溫電子顯微鏡收集樣品影線後,可使用分析軟體重建出蛋白質分子結構,達到近似原子級的解析度,這項技術稱為cryo-EM single particle reconstruction(SPR)。儘管能夠觀察到大分子結構的變化,但由於小於100 kD的蛋白的訊號和背景雜訊的訊號強度相似,小分子無法在顯微鏡下被清除觀察到,故對於分子量小於 100 kD 的樣品仍有困難性。為克服此苦難,結構生物學家設計支架蛋白(scaffold protein) 來幫助在顯微鏡下更易觀察到小蛋白。支架蛋白通常是對稱的同源多聚蛋白,其包括多個結合位置能以共價或非共價鍵與標的蛋白結合。在此研究中,我們設計了一種 scaffold protein,由鳥胺酸氨甲醯基轉移酶 (OTC) 組成。首先我們嘗試將OTC的C端與另一種蛋白,MSG的N端結合。使用冷凍低溫電子顯微鏡收集與分析數據後,OTC的機構可達近似原子級的解析度,但MSG的解析度仍非常低。儘管如此,OTC可望成為具有發展性的支架蛋白。接著,我們將OTC與泛素接在一起,形成OTC-Ub scaf-fold。UCHL1為一25 kD的蛋白,能與泛素結合。我們利用此結合性質,使用cryo-EM重建出OTC-Ub與UCHL1的復合物模型,其中泛素羧基末端水解酶-1 (UCHL1)部分的區塊可清楚被看見,並達到6.5 Å 解析度。因此,此研究提供了一種新支架蛋白來助於觀察小於30 kD蛋白的結構。 The recent resolution revolution in cryogenic-electron microscopy (cryo-EM) made it an increasingly powerful tool in structural biology. The revolution came with advances in the microscope optics, the direct electron detectors, and the analytical software. With the cur-rent pipeline, protein structures could be reconstructed to atomic resolutions through a se-ries of cryo-EM micrographs. This technique is known as cryo-EM single-particle recon-struction (SPR). Although capable of detecting macromolecular assemblies and their con-formational changes, there remained a resolution limit for samples with molecular weight below 100 kD. To overcome the size limit, scaffold proteins were developed to study sub-100 kD proteins by cryo-EM. Scaffold proteins are generally symmetrical homo-oligomeric proteins that include multiple fusion sites to attach covalently or non-covalently to the target proteins. We designed a scaffold system based on a homo-trimeric ornithine transcarbamylase (OTC). OTC was genetically fused at its C-terminus to an 80 kD target protein, malate synthase G (MSG). The scaffold protein, OTC, was visualized at near-atomic resolution, while MSG showed a diffused density. Nonetheless, the refined OTC density map showed its potential to serve as the scaffold for other proteins. We next fused OTC to ubiquitin to build an OTC-Ub fusion scaffold to visualize Ub-binding protein, such as ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1). UCHL1 is a 25 kD protein. The cryo-EM map showed clearly defined regions that could precisely house Ubiquitin and UCHL1. The currently attainable local resolution of UCHL1 was 6.5 Å, still larger than the required resolution for de novo model building. However, our results demonstrate the potential of OTC-Ub to aid visualization of the atomic structures of small (< 30 kD) ubiquitin-binding proteins. a 26 kD protein indicated OTC-Ub may serve as a imaging scaffold for other ubiquitin-binding proteins. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76870 |
DOI: | 10.6342/NTU202003123 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科學研究所 |
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