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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王彥士(Yane-Shih Wang) | |
| dc.contributor.author | Jo-Chu Tsou | en |
| dc.contributor.author | 鄒若主 | zh_TW |
| dc.date.accessioned | 2022-11-25T03:05:37Z | - |
| dc.date.available | 2027-02-08 | |
| dc.date.copyright | 2022-02-18 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-02-09 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81877 | - |
| dc.description.abstract | 生物正交胺醯-核醣核酸合成酶‧轉核醣核酸(aaRS‧tRNA)配對為擴展遺傳密碼研究中的重要元素。吡咯-轉核醣核酸合成酶(PylRS)可催化非典型胺基酸的活化，將其透過醯化作用，接至同源tRNA上。在前行研究中，演化樹內另一群不具N端結構的ΔNPylRS，已被證實在大腸桿菌與哺乳類細胞中具有生物正交性。與Methanosarcina mazei(Mm)PylRS不同的是，MmPylRS必須同時具有與tRNA互相辨識的N端，和進行催化反應的C端，而不具N端結構的ΔNPylRS，仍然可成功將非典型胺基酸嵌入蛋白中。同實驗室之前的研究顯示MmPylRS•MmtRNA配對，和Methanogenic archaeon ISO4-G1 (G1) PylRS•G1tRNA配對，為相互生物正交。在此項研究中，更進一步發現野生株Methanogenic archaeon ISO4-G1 (G1) PylRS•G1tRNA配對，G1PylRS•Methanonatronarchaeum termitum (Mt) tRNA異種配對，可拓展其酵素的受質辨認領域，包含D-胺基酸和苯丙氨酸(Phenylalanine)類似物。此外，突變株G1PylRS-GQG (其變異位被發現可擴大PylRS酵素活性位)，能辨認色氨酸(tryptophan)類似物。為了分析G1PylRS催化口袋，我們透過蛋白質晶體學，解出G1PylRS的apo form，以及含AMP、AMPCPP的複合體(bound form)晶體結構，並進一步解出G1PylRS-GQG的晶體結構。此外，確立其催化口袋介於開放和閉合間的不同過渡型態之結構，進一步暗示MmPylRS與G1PylRS之間活性位點及催化機制的不同。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T03:05:37Z (GMT). No. of bitstreams: 1 U0001-0601202215214500.pdf: 8902932 bytes, checksum: 7fe330e0ec92d950888cfa1c3dd6ebc8 (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 摘要 I Abstract II Table of contents III List of figures V List of tables VII List of schemes VIII Abbreviations IX Chapter 1 Introduction 1 1.1 Expanding genetic code 1 1.2 PylRS family and engineering 2 1.3 ΔNPylRS•tRNAΔNPyl ― the newly found orthogonal pair 4 1.4 Specific aims 6 Chapter 2 Material and Method 8 2.1 DNA sequences 8 2.2 Protein sequences 10 2.3 Plasmid construction 12 2.3.1 Primer list 12 2.3.2 Plasmid 12 2.4 Suppression efficiencies measurement 14 2.4.1 Co-transform pCDF-PylRS variants and pET-pylT-sfGFP 14 2.4.2 Incorporation efficiency screening of PylRS variants 14 2.5 Protein expression and purification 15 2.6 Gel analysis 17 2.6.1 SDS-PAGE analysis 17 2.6.2 Western blot analysis 18 2.6.3 Native gel analysis 19 2.7 Protein biophysical characterizations 19 2.7.1 ESI-MS analysis 19 2.8 Protein crystallization and structural determination 20 2.8.1 G1PylRS crystallization (apo form) 20 2.8.2 G1PylRS crystallization (AMP bound form) 20 2.8.3 G1PylRS crystallization (AMPCPP bound form) 21 2.8.4 G1PylRS-GQG crystallization 22 Chapter 3 Results 23 3.1 Orthogonal PylRS•tRNAPyl pairs 23 3.1.1 Suppression efficiency screening of PylRS•tRNAPyl pairs 23 3.1.2 ESI-MS analysis of sfGFP variants 24 3.2 Distinctive conformational states of G1PylRS and G1PylRS-GQG 25 3.2.1 Preparation of G1PylRS and G1PylRS-GQG 25 3.2.2 Crystallization and data collection of G1PylRS 25 3.2.3 Crystallization and data collection of G1PylRS-GQG 27 Chapter 4 Discussion 29 Chapter 5 Conclusion 34 Chapter 6 Reference 65 Chapter 7 Appendix 70 | |
| dc.language.iso | en | |
| dc.subject | 蛋白質結晶體學 | zh_TW |
| dc.subject | 體內嵌入非典型胺基酸 | zh_TW |
| dc.subject | 吡咯-轉核醣核酸合成酶 | zh_TW |
| dc.subject | 蛋白質構型改變 | zh_TW |
| dc.subject | expanding genetic code | en |
| dc.subject | protein crystallography | en |
| dc.subject | conformational changes | en |
| dc.subject | pyrrolysyl-tRNA synthetase | en |
| dc.title | 吡咯離胺醯-tRNA合成酶酵素催化機制探討 | zh_TW |
| dc.title | Exploring the catalytic mechanism of Methanogenic archaeon ISO4-G1 pyrrolysyl-tRNA synthetase | en |
| dc.date.schoolyear | 110-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李宗璘(Hsin-Tsai Liu),梁博煌(Chih-Yang Tseng) | |
| dc.subject.keyword | 體內嵌入非典型胺基酸,吡咯-轉核醣核酸合成酶,蛋白質構型改變,蛋白質結晶體學, | zh_TW |
| dc.subject.keyword | expanding genetic code,pyrrolysyl-tRNA synthetase,conformational changes,protein crystallography, | en |
| dc.relation.page | 139 | |
| dc.identifier.doi | 10.6342/NTU202200019 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2022-02-10 | |
| 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 | 2027-02-08 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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