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標題: | 描繪扭結蛋白之結構動力學與摺疊機制 Delineating the Structural Dynamics and Folding Mechanism of Topologically Knotted Proteins |
作者: | Yun-Tzai Lee 李耘在 |
指導教授: | 徐尚德(Shang-Te Danny Hsu) |
關鍵字: | 扭結蛋白,蛋白折疊, knotted proteins,protein folding, |
出版年 : | 2018 |
學位: | 博士 |
摘要: | Protein folding is driven by native interactions encoded within primary sequences in order to attain a defined three-dimensional structure to perform biological functions. Unlike a myriad of other proteins, knotted proteins exhibit relatively rugged folding energy landscapes and multiple folding pathways. In this thesis, we explore the folding energy landscape and characterize folding intermediates of two topologically knotted proteins, namely human ubiquitin carboxyl hydrolase UCH-L1 and bacterial tRNA methyltransferase YibK, which have Gordian 52- and trefoil 31-knotted topologies, respectively.
UCH-L1 is a monomeric protein with a 52-knotted fold. Using elastic light scattering coupled with size-exclusion chromatography (SEC) under denatured conditions we identified well-defined dimeric and tetrameric folding intermediates of UCH-L1 that are considerably disordered and dynamic while its folded core is retained. Moreover, we observed that the Parkinson’s disease-associated mutation I93M, which increases the proportion of partially unfolded forms in both native and folding intermediate state of UCH-L1, induces the formation of higher order oligomerization under the same denatured conditions we used. This result suggested a potential misfolding and aggregation pathway that aligns with previous observations that the I93M mutation can increase aggregation propensity of UCH-L1 implicated in Parkinson’s disease pathogenesis. The other part of this dissertation describes the application of post-translational protein engineering to study knotted proteins. Sortase mediated the end-to-end closure of the 31-knotted architecture of YibK, transforming a conventionally knotted protein into a true mathematical 31 knot without loose ends. The cyclization neither alters the overall structure nor protein function. However, it substantially increases the thermostability and remodels the folding pathway of YibK to prevent chemically induced aggregation. These results provided a strategy to characterize the productive knotted folding intermediates that are involved in knotted protein folding pathways. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69856 |
DOI: | 10.6342/NTU201800566 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科學研究所 |
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