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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王彥士(Yane-Shih Wang) | |
| dc.contributor.author | Chien-Lung Li | en |
| dc.contributor.author | 李健隆 | zh_TW |
| dc.date.accessioned | 2021-07-09T15:54:14Z | - |
| dc.date.available | 2023-08-21 | |
| dc.date.copyright | 2018-08-21 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-14 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76549 | - |
| dc.description.abstract | 泛素化和類泛素化為細胞內重要之蛋白質轉譯後修飾作用，廣泛參與並影響生物體作用。USP7近期發現亦為泛素-類泛素降解酶，能將鍵結在類泛素2上的泛素移除，暗示泛素-類泛素的動態鍵結之間有著調控受質蛋白功能的生物機制。然而，調控類泛素-泛素異元聚合物轉譯後修飾的其餘調控因子，尚未進一步發現及探討。本研究利用化學生物學方法及非典型胺基酸體內嵌入的技術，合成特定鍵結的泛素-類泛素異元二聚體，做為深入探討相關蛋白間作用的基體。本研究中，類泛素2中的八個賴胺酸基因位點，分別以TAG基因密碼突變並以生物正交配對PylRS•tRNA_CUA^Pyl體內嵌入非典型胺基酸SeCbzK；類泛素2進一步經過硒亞碸脫去反應轉換成類泛素2-Dha，進而衍生化的泛素-ethylthio進行硫醇麥可加成反應，得到泛素-類泛素2異元二聚體產物。
此論文研究亦針對以雜環類非典型胺基酸中對環境敏感而有螢光強弱變化的特性，發展辨認特定鍵結泛素二聚體的分子探針。利用演化後的PylRS•tRNA_CUA^Pyl正交配對，在超折疊綠螢光蛋白的發色團中酪胺酸66及苯丙胺酸27分別取代為7種不同雜環胺基酸。在螢光光譜的測定實驗中，顯示放射光譜和控制組實驗相比，有著顯著發射光譜位移、斯托克斯位移、分子內多重FRET光學現象、增加螢光發光團，相關結果可做為非典型胺基酸分子探針分辨特定泛素二聚體的工具。 | zh_TW |
| dc.description.abstract | Protein Ubiquitination and SUMOylation are unique protein post-translational modifications involved in a myriad biological processes. The discovery of USP7 as exclusive SUMO deubiquitinase has manifested the cross-talk between Ubiquitin (Ub) and SUMO. However, the corresponding effectors and biological language of such hetero-linkage are still indecipherable. Here, we report an unbiased novel chemical method in synthesizing Ub-SUMO2 dimers. Se-alkylselenocysteine was incorporated into SUMO2 protein at eight different lysine positions individually and further converted to SUMO2-dehydroalanine (Dha) through selenoxide-elimination chemistry. Subsequently, SUMO2-Dha was covalently tethered with Ub-ethylthio by thiol-Michael addition yielding Ub-SUMO2 heterodimer.
This study also centers on the development of sensitive heterocyclic fluorescent probe which can sense Di-Ub or Ub-SUMO linkage in response to environmental changes. With the evolved pyrrolysyl-tRNA synthetase • pyrrolysyl-tRNA (PylRS•tRNA_CUA^Pyl) pairs, the tyrosine 66 and phenylalanine 27 positions of superfolder green fluorescent protein were efficiently substituted with seven heterocyclic noncanonical amino acids. The photophysical characterization of these proteins have unraveled the significant differences at the emission wavelengths, stoke shift, intramolecular FRET, and additional chromophore. These findings have demonstrated that ncAA-based tool was employed to establish molecular probe which can specifically recognize Ubiquitin dimers. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-09T15:54:14Z (GMT). No. of bitstreams: 1 ntu-107-R05b46025-1.pdf: 7554595 bytes, checksum: 30221ae69d987c72482ae743d967d023 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | Abstract I
摘要 II Table of contents III List of figures VII List of tables XI Abbreviations XII Chapter 1 Introduction 1 1.1 Protein translation 1 1.1.1 Initiation 1 1.1.2 Elongation 2 1.1.3 Termination 3 1.2 Protein post-translational modifications 4 1.2.1 Protein SUMOylation 4 1.2.2 Protein Ubiquitination 8 1.3 Expanding genetic codes 12 1.3.1 Genetic code reassignment 12 1.3.2 Reside-specific incorporation of ncAAs 13 1.3.3 Site-specific incorporation of ncAAs 13 1.4 Protein ligation 17 1.4.1 Native chemical ligation 17 1.4.2 Selenoxide elimination 17 1.4.3 Michael addition 18 1.5 Specific aim of thesis 18 Chapter 2 Experimental materials and methods 23 2.1 DNA and Protein sequences 23 2.1.1 DNA sequences 23 2.1.2 Protein sequences 27 2.2 Plasmid construction 30 2.2.1 Primer list 30 2.2.2 Plasmids 31 2.2.3 Molecular cloning 35 2.3 Protein productions and purifications 37 2.4 Gel analysis 41 2.4.1 SDS-PAGE 41 2.4.2 Western blot analysis 42 2.5 Protein chemistry 43 2.5.1 Synthesis of Ub-cysteamine 43 2.5.2 Selenoxide β-elimination 43 2.6 Mass spectrometry characterization 44 2.6.1 Protein MALDI-TOF-MS analysis 44 2.6.2 Protein ESI-MS analysis 45 2.7 Protein Biophysical characterizations 45 2.7.1 UV/Visible absorption spectrum 45 2.7.2 Fluorescence spectrum 45 Chapter 3 Results 46 3.1 Synthesis of Ubiquitin-cysteamine 46 3.1.1 Purification of Ubiquitin 46 3.1.2 Purification of Ubiquitin-activating enzyme 49 3.1.3 ESI-MS analysis of Ubiquitin-cysteamine 53 3.1.4 Purification of Ubiquitin-cysteamine 57 3.2 Studying selenoxide β-elimination of SUMO2 variants 59 3.2.1 Purification of SUMO2 variants 59 3.2.2 ESI-MS analysis and MALDI-TOF-MS/MS analysis of SUMO2 variants 65 3.2.3 ESI-MS analysis of selenoxide β-elimination of SUMO2 variants 77 3.3 Protein ligation through thiol-Michael addition on SUMO2Dha 81 3.4 Engineering MmPylRS for heterocyclic ncAAs incorporation 94 3.4.1 Analysis of sfGFP variants 94 3.4.2 ESI-MS analysis and MALDI-TOF-MS/MS analysis of sfGFP variants 98 3.4.3 Incorporating heterocyclic ncAAs into chromophore of sfGFP 110 3.4.4 Photophysical characteristics of sfGFP variants with heterocyclic ncAAs 117 Chapter 4 Discussion 129 Chapter 5 Conclusion 132 Reference 133 Appendix 137 | |
| dc.language.iso | en | |
| dc.subject | 硫醇麥可加成反應 | zh_TW |
| dc.subject | 泛素-類泛素異元二聚體 | zh_TW |
| dc.subject | 非典型胺基酸體內嵌入 | zh_TW |
| dc.subject | 雜環非典型胺基酸 | zh_TW |
| dc.subject | PylRS‧tRNACUAPyl配對 | zh_TW |
| dc.subject | PylRS‧tRNACUAPyl pair | en |
| dc.subject | Expanding genetic code | en |
| dc.subject | Ubiquitin-SUMO2 dimer | en |
| dc.subject | thiol-Michael addition reaction | en |
| dc.subject | heterocyclic noncanonical amino acid incorporation | en |
| dc.title | 利用擴充基因密碼方法合成泛素-SUMO2異元二聚體與其功能分析之研究 | zh_TW |
| dc.title | Expanding genetic code for synthesis and functional studies of Ubiquitin-SUMO2 heterodimers | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蔡明道(Ming-Daw Tsai),謝俊結(Jiun-Jie Shie) | |
| dc.subject.keyword | 非典型胺基酸體內嵌入,泛素-類泛素異元二聚體,硫醇麥可加成反應,雜環非典型胺基酸,PylRS‧tRNACUAPyl配對, | zh_TW |
| dc.subject.keyword | Expanding genetic code,Ubiquitin-SUMO2 dimer,thiol-Michael addition reaction,heterocyclic noncanonical amino acid incorporation,PylRS‧tRNACUAPyl pair, | en |
| dc.relation.page | 154 | |
| dc.identifier.doi | 10.6342/NTU201803256 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2018-08-14 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| dc.date.embargo-lift | 2023-08-21 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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