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  • 學位論文

固態核磁共振對於倉鼠普昂胜肽109—122形成類澱粉樣纖維之研究

Solid-state NMR Study of Amyloid Fibrils Formed by Residues 109—122 of the Syrian Hamster Prion Protein

指導教授 : 陳振中

摘要


普昂粒子(Prion)是帶有感染性蛋白粒子的簡稱,由於此類蛋白粒子會導致與海綿樣腦病變相關的疾病,在80年代連續爆發了羊騷症與狂牛症後,普昂粒子引起了科學家的高度注意。產生及傳播普昂疾病的原因目前仍未完全清楚,一般相信正常的普昂蛋白(PrPc)轉變為具致病性結構(PrPsc)後,會引致其他PrPc大量轉變為PrPsc,PrPsc胜肽單體因分子間氫鍵以及凡得瓦力作用力影響,會聚集成水溶性極低的類澱粉樣纖維,進一步傷害患者的神經系統。由於PrPsc形成類澱粉樣纖維後,樣品不溶於一般溶劑以及為非晶性粉末,故無法用液態核磁共振光譜學或X光繞射技術分析此等纖維分子之結構,因此固態核磁共振便成為對PrPsc極佳的分析工具。我們的目標主要在於利用固態核磁共振對普昂胜肽所產生的類澱粉樣纖維進行結構分析,以增進對此疾病的認識。 論文中,我們利用敘利亞倉鼠的普昂胜肽第109到122個殘基來做研究,實驗中所採用的氨基酸序列為 Ac-MKHMAGAAAAGAVV-NH2文献顯示,這個較短的序列容易形成纖維結構。我們成功的得到了培養類澱粉樣纖維的條件,同時以許多不同的方法辨識SHPrP109-122纖維分子之形成,分別有ThT 螢光吸收,穿透式電子顯微鏡(TEM)以及原子力顯微鏡(AFM)。除了證明纖維的生成外,還藉此分析SHPrP109~122纖維分子的型態,得到其寬約為11 nm,高約為0.9 nm 以及長度一般都大於400 μm。利用固態核磁共振技術,我們以樣品的半高寬鑑定其分子結構在疏水端序列區有良好的結構排序(structural order),這個結果和分析樣品殘基化學位移的二級偏移所得到的結論是相同的。我們也利用本實驗室發展的脈衝序列,具體偵測出其中七個殘基的扭轉角,並採用不同的脈衝序列測量系統中同核與異核之空間距離關係,以測量纖維樣品的超分子結構。結果顯示,SHPrP109~122纖維分子的基本結構單元是以兩組反向beta-sheet組成彼此相對應的距離為 6~7 Å。每組beta-sheet是以第117個殘基對齊排列而成。最後,我們以分子動態計算(MD simulations),以實驗數據為限制條件,得到SHPrP109~122纖維分子的結構模型。

並列摘要


Prion disease, a kind of neurodegenerative disease, is commonly believed to be caused by the conversion of the prion protein from its normal cellular form (PrPC) to the disease-specific form (PrPSc). However, the mechanism of fibrillization is still unclear. It is suggested that the presence of PrPSc will induce the misfolding of other PrPC to PrPSc, damaging the nervous system of the victims. Because PrPSc has a strong propensity to form amyloid fibrils, which are insoluble in common solvents and are non-crystalline in nature, it is difficult to use solution-state NMR or X-ray diffraction technique to study PrPSc. Consequently, solid-state NMR is well suited to study PrPSc. Our target is to employ solid-state NMR spectroscopy to investigate the molecular structure of the amyloid fibrils formed by the peptide fragment of prion protein. In this thesis, our target peptide sequence is the fragment 109 to 122 of Syrian hamster prion protein, MKHMAGAAAAGAVV, which is believed to be the most amyloidogenic region. We successfully obtained fibril sample from the target peptide and characterized the morphology of the fibrils by TEM and AFM. The fibrils in general have a length over 400 nm, a width of 11 nm, and a uniform height of 0.9 ± 0.1 nm. From the chemical shift and linewidth data , the beta-strands forming the fibrils have significant structural order in the hydrophobic region. We measured the backbone Ψ angle values and analysis the linewidth and second chemical shift and found the residues in the hydrophobic region form well-structure area. Furthermore, we found that each fibril comprises of two cross-beta layers. The beta strands within each layer are anti-parallel and aligned in such a way that the Ala 117 forms a linear chain along the fibril axis. Consequently, a molecular model for the fibrils was constructed by molecular dynamics simulations incorporated with structural constraints obtained from solid-state NMR measurements.

參考文獻


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