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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 徐尚德 | zh_TW |
| dc.contributor.advisor | Shang-Te Danny Hsu | en |
| dc.contributor.author | 姜宗昇 | zh_TW |
| dc.contributor.author | Tsung-Sheng Chiang | en |
| dc.date.accessioned | 2025-09-10T16:19:11Z | - |
| dc.date.available | 2025-09-11 | - |
| dc.date.copyright | 2025-09-10 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-07-28 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/99448 | - |
| dc.description.abstract | 新型冠狀病毒(SARS-CoV-2)的棘蛋白(Spike protein)依賴其受體結合域(RBD)與血管張力素轉化酶2(ACE-2)結合，從而促進病毒進入宿主細胞引發感染。目前已知棘蛋白的 N-醣基化會影響病毒的感染力及免疫逃脫能力，但 N-醣基化對於其受體結合域 (RBD) 的結構與動力學特徵影響仍尚未釐清。為了定量探討這些效應，我們利用液相核磁共振光譜學以及多項生物物理分析技術，比較在人類細胞株(Expi293)表現的醣基化 RBD 與在大腸桿菌中表現的非醣基化RBD。
我們成功地在 Expi293 細胞表現的醣基化 RBD 中對含甲基之胺基酸進行了選擇性碳同位素(13C)標定，使我們能夠利用甲基核磁共振光譜分析，並發現 RBD的核心β-sheet 區域存在顯著化學位移改變。此外，核磁共振弛豫(Relaxation)測量顯示，醣基化 RBD 的橫向弛豫時間 (T2) 降低，此結果與其具較大的流體動力學半徑導致其較慢的分子滾動速率可相呼應，兩項結果為獨立核磁共振實驗測定，此結論也與熱穩定性及化學穩定性實驗測定吻合，皆顯示醣基化顯著增強了RBD的穩定性與結構完整性。然而，這些醣基化導致的結構與動態影響，並未提高RBD與ACE2的結合親和力，在我們的研究成果中顯示，醣基化並不直接幫助RBD與其受體的結合效率，而是透過影響其局部結構以及其動力學特性，在穩定其蛋白質折疊狀態方面扮演關鍵角色，本研究提供了定量分析N-醣基化對SARS-CoV-2 RBD影響的方法與方向，並在其他醣蛋白系統中具有高度運用潛力。 | zh_TW |
| dc.description.abstract | The SARS-CoV-2 spike protein relies on its receptor-binding domain (RBD) to engage angiotensin-converting enzyme 2 (ACE2), facilitating host cell entry and initiating viral infection. While N-glycosylation of the spike protein is known to influence viral infectivity and immune evasion, its impact on the structural and dynamic features of the RBD remains elusive. To quantitatively investigate these effects, we employed solution-state NMR spectroscopy and complementary biophysical techniques to compare glycosylated (expressed in human Expi293 cell line) and non-glycosylated (expressed in E. coli) forms of the SARS-CoV-2 RBD.
We have successfully introduced selective 13C labeling of methyl-containing amino acids in Expi293-expressed glycosylated RBD. This enables methyl NMR analysis, which reveals remarkable chemical shift perturbations within the core β-sheet region. Furthermore, NMR relaxation measurements showed a decrease in transverse relaxation times (T2) for the glycosylated RBD, consistent with its slower molecular tumbling due to a larger hydrodynamic radius, which was independently confirmed by diffusion NMR. These observations were corroborated by thermal and chemical stability assessments, demonstrating that glycosylation substantially enhances the stability and structural integrity of the RBD. However, despite these structural changes, glycosylation did not improve the binding affinity of the RBD for ACE2. Our findings suggest that N-glycosylation does not directly affect receptor-binding efficiency but plays a pivotal role in stabilizing the RBD’s folded state by influencing its local conformation and overall dynamic properties. These insights paved the way for quantitative analysis of the effects of N-glycosylation on the SARS-CoV-2 RBD and hold high potential for application to other glycoprotein systems. | en |
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| dc.description.provenance | Made available in DSpace on 2025-09-10T16:19:11Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 中文摘要 i
Abstract ii CONTENTS iv LIST OF FIGURES viii LIST OF TABLES xi Chapter 1 INTRODUCTION 1 1.1 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) 1 1.2 Structural characteristics of the spike (S) protein receptor binding domain (RBD) 2 1.3 Protein N-linked glycosylation 6 1.3.1 The N-linked glycosylation pathway in eukaryotic cells 6 1.3.2 N-Glycosylation as a key player in protein quality control and folding in the endoplasmic reticulum 8 1.3.3 Functional impacts of N-glycosylation: from modulating biophysical properties to orchestrating viral pathogenesis 10 1.4 Structural glycobiology: elucidating glycoprotein architecture 13 1.5 Nuclear Magnetic Resonance spectroscopy for glycoproteins 16 1.5.1 The unique potential of NMR in glycoprotein structural biology 16 1.5.2 Challenges in applying NMR to mammalian-derived glycoproteins 17 1.5.3 Innovative NMR strategies for glycoproteins: advancing isotope labeling in eukaryotic systems 18 1.6 Specific aims 20 Chapter 2 MATERIALS AND METHODS 21 2.1 Non-glycosylated SARS-CoV-2 RBD expression and purification 21 2.2 Glycosylated SARS-CoV-2 RBD expression and purification 22 2.3 Human ACE2 ectodomain (hACE2-8H) expression and purification 23 2.4 hACE2-Fc expression and purification 24 2.5 GST-PNGaseF expression and purification 25 2.6 Isothermal Titration Calorimetry (ITC) 26 2.7 Bio-Layer Interferometry (BLI) 27 2.8 Differential Scanning Calorimetry (DSC) 29 2.9 Chemical stability by intrinsic fluorescence spectroscopy 29 2.10 Nuclear Magnetic Resonance Spectroscopy (NMR) 32 2.10.1 Isotope-labeling in E. coli and Expi293 expression system 32 2.10.2 Pulsed Field Gradient NMR Spectroscopy for measuring translational diffusion coefficients (Dt) 33 2.10.3 Rapid acquisition of methyl 1H-13C fingerprints and dynamic monitoring using SOFAST-HMQC 35 2.10.4 Methyl 1H-13C Constant-Time HSQC for transverse relaxation (T2) estimation 37 2.10.5 Three-dimensional simultaneous 15N, 13C-edited NOESY-HSQC experiment 39 2.10.6 Time-resolved NMR analysis of SARS-CoV-2 RBD deglycosylation by GST-PNGaseF 40 Chapter 3 RESULTS 41 3.1 Production and purification of distinct SARS-CoV-2 RBD glycoforms 41 3.2 Production and purification of two types of Human ACE2 ectodomain 49 3.3 Assessing the impact of N-glycosylation on SARS-CoV-2 RBD accessibility through GlycoSHIELD computational modeling 52 3.4 Quantification of glycosylation impacts on SARS-CoV-2 RBD-ACE2 binding thermodynamics and kinetics 56 3.5 N-Glycosylation modulates the hydrodynamic properties of SARS-CoV-2 RBD in solution 62 3.6 Site-specific assignments of SARS-CoV-2 RBD methyl NMR spectra 69 3.7 N-Glycosylation induces methyl chemical shift perturbations in SARS-CoV-2 RBD 78 3.8 N-Glycosylation modulates the dynamic properties of SARS-CoV-2 RBD as probed by methyl NMR relaxation 86 3.9 Time-resolved NMR analysis of SARS-CoV-2 RBD deglycosylation kinetics and conformational change 94 3.10 N-glycosylation enhances the thermal stability of SARS-CoV-2 RBD 101 3.11 N-glycosylation enhances chemical stability and modulates the unfolding pathway of SARS-CoV-2 RBD 105 Chapter 4 DISCUSSIONS 111 4.1 N-Glycosylation as a critical determinant of SARS-CoV-2 RBD biophysical properties and structural integrity 111 4.2 Methyl NMR view of N-glycosylation-induced conformational changes in SARS-CoV-2 RBD 115 4.3 Linking global tumbling to local flexibility and protein stability in SARS-CoV-2 RBD 118 4.4 Re-evaluating the role of SARS-CoV-2 RBD N-Glycans in ACE2 receptor engagement 121 4.5 Conclusion, broader implications, and future directions 123 REFERENCES 127 APPENDIX 139 | - |
| dc.language.iso | en | - |
| dc.subject | SARS-CoV-2（新型冠狀病毒） | zh_TW |
| dc.subject | N-醣基化 | zh_TW |
| dc.subject | 核磁共振光譜學 | zh_TW |
| dc.subject | 蛋白質動力學 | zh_TW |
| dc.subject | 哺乳類細胞甲基同位素標定 | zh_TW |
| dc.subject | 蛋白質折疊穩定性 | zh_TW |
| dc.subject | NMR spectroscopy | en |
| dc.subject | SARS-CoV-2 | en |
| dc.subject | N-linked glycosylation | en |
| dc.subject | Protein folding stability | en |
| dc.subject | Mammalian cell methyl labeling | en |
| dc.subject | Protein dynamics | en |
| dc.title | 醣基化對新冠病毒棘蛋白受體結合結構域功能動態的影響 | zh_TW |
| dc.title | Impacts of glycosylation on the functional dynamics of the receptor binding domain of SARS-CoV-2 spike protein | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 余慈顏;吳昆峯 | zh_TW |
| dc.contributor.oralexamcommittee | Tsyr-Yan Yu;Kuen-Phon Wu | en |
| dc.subject.keyword | SARS-CoV-2（新型冠狀病毒）,N-醣基化,核磁共振光譜學,蛋白質動力學,哺乳類細胞甲基同位素標定,蛋白質折疊穩定性, | zh_TW |
| dc.subject.keyword | SARS-CoV-2,N-linked glycosylation,NMR spectroscopy,Protein dynamics,Mammalian cell methyl labeling,Protein folding stability, | en |
| dc.relation.page | 147 | - |
| dc.identifier.doi | 10.6342/NTU202502416 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2025-07-30 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科學研究所 | - |
| dc.date.embargo-lift | 2027-08-30 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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