博碩士論文 962204016 詳細資訊




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姓名 林冠甫(Kuan-Fu Lin)  查詢紙本館藏   畢業系所 生命科學系
論文名稱
(Structure-function relationship of a rice small heat shock protein OsHsp16.9A)
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★ 水稻小分子量熱休克蛋白質- OsHSP16.9A在水稻種子耐熱性之功能分析
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摘要(中) 小分子量熱休克蛋白質可以透過與變性蛋白質結合,避免其在細胞內沈澱而傷害細胞活性,為細胞維持恆定所不可或缺。水稻內有一群在種子發育時期大量累積的小分子量熱休克蛋白質,其中表現量最多的為OsHsp16.9A,過去研究指出OsHsp16.9A可以幫助水稻抵抗熱逆境。OsHsp16.9A的胺基酸序列與小麥內同源蛋白質TaHsp16.9蛋白質胺基酸序列高度相似,卻會形成截然不同的聚合體構型。為了釐清小分子量熱休克蛋白質形成不同構型聚合體的機制,我們利用冷凍電子顯微鏡與小角度X光散射建構OsHsp16.9A聚合體的低解析度結構,並搭配圓二色光譜、蛋白質—受體凝集試驗與濁度試驗來探究N端的功能。我們發現除了N端上不同胺基酸的電荷與疏水性分佈會影響其形成不同聚合體外,N端的長度也是影響聚合體構型一個很重要的因素。刪除N端的OsHsp16.9A除了會喪失伴護功能外,也會影響聚合體的完整度。我們意外地發現OsHsp16.9A可以耐受高達攝氏95度的高溫,並且在溫度降低後維持原有的二級結構。以上發現都表示OsHsp16.9A為一俱有伴護功能的蛋白質,並且在水稻對熱的耐受性上扮演重要的角色。
摘要(英) Small heat shock proteins (sHsps) maintain the protein homeostasis by interacting with the unfolded proteins, preventing them from aggregation under stress conditions. We took an interest in OsHsp16.9A, a cytosolic sHsps crucial for thermo-resistance in rice, which shares similar amino acid sequences with a known structure, wheat TaHsp16.9, but with different N-terminal region. To bring a scheme of the structure-function relationship of OsHsp16.9A and clarify the N-terminal sequence difference, we used cryo-EM and small-angle X-ray scattering to generate a structural model and combined with circular dichroism, aggregation, and chaperone activity assay to further investigate the N-terminal effect. Our data suggest that only ten amino acids deleted from N-terminal of OsHsp16.9A would lead to severe loss of chaperone activity. Longer N-terminal region truncation results in the aggregation itself. Furthermore
, the N-terminal region also affects the thermal sustainability of OsHsp16.9A. The structural rigidity of intact OsHsp16.9A is higher than the N-terminal truncated variants. The secondary structure of intact OsHsp16.9A can even remain unchanged after the incubation at 95 °C. It is revealed that OsHsp16.9A is a thermal stable chaperone protein and N-terminal region also play a vital role in the thermostability.
關鍵字(中) ★ 小分子量熱休克蛋白質
★ 低溫電子顯微鏡
★ 小角度散射
★ X光晶體繞射學
★ 水稻
★ 伴護功能
關鍵字(英) ★ Small heat shock protein
★ Cryo-EM
★ SAXS
★ X-ray crystallography
★ Rice
★ Chaperone
論文目次 中文摘要 i
Abstract ii
Acknowledgments iii
Table of Contents iv
List of Figures v
Introduction 1
Material and Methods 6
Cloning, Expression, and Purification of OsHsp16.9A oligomer 6
Aggregation assay and chaperone activity assay 7
Negative EM and Cryo-EM 7
Single Particle Reconstruction 8
Small-angle X-ray Scattering (SAXS) 9
Circular Dichroism (CD) Spectrum 9
Results 11
OsHsp16.9A is a dodecamer 11
Three-dimensional reconstruction of OsHsp16.9A 11
The N-terminal regions are important to chaperone function and oligomerization 14
Thermal durability of OsHsp16.9A variants 17
Discussion 20
OsHsp16.9A forms a tetrahedron-like dodecamer 20
Effect of N-terminal region of OsHsp16.9A 22
Conclusion 25
Bibliographies 39
Appendices 47
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指導教授 葉靖輝 審核日期 2019-1-24
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