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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王彥士 | |
| dc.contributor.author | Yi-Hui Wang | en |
| dc.contributor.author | 王伊慧 | zh_TW |
| dc.date.accessioned | 2021-07-09T15:52:06Z | - |
| dc.date.available | 2023-08-21 | |
| dc.date.copyright | 2018-08-21 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-07-25 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76420 | - |
| dc.description.abstract | 在合成生物學的蛋白設計應用中，利用蛋白轉譯系統中重新定義基因密碼，進而在體內將非典型胺基酸嵌入生合成蛋白的特定位置中，是探討酵素催化的機制以及設計含有新穎化學功能蛋白質的重要方法。雖然自然界存在D-胺基酸，但是核醣體中蛋白胜肽鏈合成的單體皆為L-胺基酸，且文獻報導中生物正交配對的體內嵌入非典型胺基酸皆為L-胺基酸。此項研究中，透過演化吡咯-轉核醣核酸合成酶(PylRS)的N端以及與活性中心，演化的PylRS，N-DFRS，能在大腸桿菌中分別辨認D-或L-非典型胺基酸，六種苯丙胺酸衍生物(D/LFA)，單個及多個位點位置特異嵌入DFA於超折疊綠螢光蛋白中，發現DFA影響蛋白內部疏水結構，造成單位螢光強度下降。N-DFRS含活性中心的C端蛋白與D/LFA六個共結晶的蛋白質X光晶體繞射結構顯示N-DFRS/DFA的有效活性來自於非穩定的多態鍵結模式，而非N-DFRS/LFA的固定鍵結模式。
DFA於蛋白質功能及蛋白設計的初步測試也近一步瞭解單一位點DFA置換在蛋白螢光光學物理，溫度變化結構效應，以及殼蛋白組裝及解構的影響。sfGFP-Y66DmClPhe蛋白的螢光光譜顯示DFA嵌入增加紅位移(510 nm)的放射波長；此蛋白在圓二色光譜於變溫實驗中在螺旋二級結構標示的波長(215及218 nm)顯示反轉趨勢。人類攜鐵蛋白hFerritin-L56DmCF3Phe在酸鹼解構實驗中，在pH = 4環境下，DLS分析中如同hFerritin-L56LmCF3Phe殼蛋白趨向包裝(assembling)結構鬆散而造成再包裝更大粒徑殼蛋白(~100 nm半徑)。 | zh_TW |
| dc.description.abstract | Expanding genetic codes research is an emerging field for incorporating noncanonical amino acids (ncAA) into proteins in vivo and studying mechanism of enzymatic reactions and designing novel enzymes with innovative chemistry. Yet native protein synthesis machinery precludes D-amino acid (D-aa) incorporation in polypeptide bond formation, protein chemists are assiduously developing the promiscuous amino acyl-tRNA synthetase with polyspecificity for the wide spectrum of ncAA. Here, we report an evolved pyrrolysyl-tRNA synthetase • pyrrolysyl-tRNA (PylRS•tRNA_CUA^Pyl) pair with collaborated mutations in N-terminus and active site, naming N-DFRS, that can charge D- or L-phenylalanine analogues (DFA or LFA) into superfolder green fluorescent proteins (sfGFP) in E. coli. The molecular insight of N-DFRS/DFA and N-DFRS/LFA co-crystal structures reveal multiple and dynamic binding modes for relaxing chiral selective of ncAA sidechain protruding in the enlarged binding pocket. Moreover, sfGFP incorporated with DFA at Y66 position exhibited additional fluorophore in 510 nm emission wavelength. It suggests D-aa may display crucial role in studying protein photochemical property and protein design.
To investigate mirror property and bio-orthogonal chemistry of encoded ncAA, we are exploring DFA incorporated ferritin for protein/drug encapsulation and delivering for therapeutic application. Our research indicated D/LFA incorporated into ferritin at L56 position slightly distort structure at pH 4.0. The establishment of encoding D/LFA into ferritin C2 interface emphasizes its potential to explore as engineered protein cage in therapeutic and diagnostic tools. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-09T15:52:06Z (GMT). No. of bitstreams: 1 ntu-107-R05b46007-1.pdf: 58971595 bytes, checksum: 2ffaa8e594805073ab4d7b8c241c24e9 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 摘要 I
Abstract II Table of contents III List of figures VI List of tables X Abbreviations XI Chapter 1 Introduction 1 1.1 Protein translation mechanism 1 1.2 Expanding genetic code research 3 1.2.1 Residue-specific incorporation of ncAA 3 1.2.2 Site-specific incorporation of ncAA 4 1.3 D-amino acid incorporation progress 7 1.3.1 Mirror and mixed chiral protein design 8 1.4 Properties of Ferritin cage 8 1.4.1 Design and application of Ferritin cage 9 1.5 Specific aim of thesis 12 Chapter 2 Materials and Methods 13 2.1 DNA and Protein sequences 13 2.1.1 DNA sequences 13 2.1.2 Protein sequence 15 2.2 Plasmid construction 16 2.2.1 Primer list 16 2.2.2 Plasmids 18 2.3 Screening incorporation efficiency of PylRS variants. 23 2.4 Protein productions and purifications 24 2.5 Gel analysis 27 2.5.1 SDS-PAGE analysis 27 2.5.2 Western blot analysis 28 2.5.3 Native gel analysis 28 2.6 Protein Biophysical characterizations 29 2.6.1 X-crystal structural analysis 29 2.6.2 Protein ESI-MS and MALDI-TOF-MS/MS analysis 30 2.6.3 Tm analyzed by CD 32 2.6.4 DLS analysis 32 2.6.5 TEM analysis 33 2.7 Emission and Excitation analysis 33 2.7.1 UV/Visible absorption spectrum 33 2.7.2 Fluoresces spectrum 33 Chapter 3 Results 35 3.1 PylRS designing for high efficiency incorporating D-aa 35 3.1.1 Engineering MmPylRS and sfGFP 35 3.1.2 High throughput screening of pylRS variants 40 3.1.3 Catalytic activity of PylRS with L/DFA 55 3.1.4 Analysis of N-DFRS catalytic activity at different position in protein 63 3.1.5 ESI-MS and MALDI-TOF-MS/MS analysis of sfGFP variants 68 3.2 Crystallization revealing L/DFA binding mode in N-DFRS 83 3.2.1 Purification of N-DFRS binding pocket for crystallization 83 3.2.2 D/LFA soaking with N-DFRS binding pocking 86 3.3 Studying biophysical property of sfGFP variant with D/LFA 93 3.3.1 Incorporating D/LFA into chromophore of sfGFP 93 3.3.2 Photophysical characteristics of sfGFP-Y66 with D/LFA 101 3.3.3 Thermally induced conformational changes of sfGFP with Y66 D/LFA 108 3.4 Application of D/LFA into protein cage design 114 3.4.1 Shape and biophysical property of Ferritin with D/LFA 121 Chapter 4 Discussion 126 Chapter 5 Conclusion 131 Reference 132 Appendix 138 | |
| dc.language.iso | en | |
| dc.subject | PylRS‧tRNA_Pyl 配對 | zh_TW |
| dc.subject | 體內嵌入非典型胺基酸 | zh_TW |
| dc.subject | D-非典型胺基酸 | zh_TW |
| dc.subject | 蛋白質設計 | zh_TW |
| dc.subject | D-noncanonical amino acids | en |
| dc.subject | Expanding genetic code | en |
| dc.subject | protein design | en |
| dc.subject | pyrrolysyl-tRNA synthetase‧tRNA_Pyl pair engineering | en |
| dc.title | 吡咯離胺醯-tRNA合成酶對於掌性非典型胺基酸嵌入蛋白之研究 | zh_TW |
| dc.title | Releasing chiral selectivity of pyrrolysyl-tRNA synthetase for encoding D-noncanonical amino acids into proteins | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 王健家,蔡明道 | |
| dc.subject.keyword | 體內嵌入非典型胺基酸, D-非典型胺基酸, PylRS‧tRNA_Pyl 配對, 蛋白質設計, | zh_TW |
| dc.subject.keyword | Expanding genetic code, D-noncanonical amino acids, pyrrolysyl-tRNA synthetase‧tRNA_Pyl pair engineering, protein design, | en |
| dc.relation.page | 184 | |
| dc.identifier.doi | 10.6342/NTU201801926 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2018-07-25 | |
| 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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