Structure-function relationship of a rice small heat shock protein OsHsp16.9A

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Title:	Structure-function relationship of a rice small heat shock protein OsHsp16.9A
Authors:	林冠甫;Lin, Kuan-Fu
Contributors:	生命科學系
Keywords:	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;Small heat shock protein;Cryo-EM;SAXS;X-ray crystallography;Rice;Chaperone
Date:	2019-01-24
Issue Date:	2019-04-02 14:21:57 (UTC+8)
Publisher:	國立中央大學
Abstract:	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。;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.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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