倉鼠普昂胜肽113-127原纖絲與成熟纖維之分子結構比較

Comparison of Molecular Structures of Protofibrils and Mature Fibrils Formed by Residues 113-127 of Syrian Hamster Prion Protein

鄭馨湄(Hsin-Mei Cheng)

指導教授 ： 陳振中

國立臺灣大學/理學院/化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.10148

摘要

類澱粉樣纖維 (amyloid fibril) 是由錯誤折疊之蛋白質聚集而成，傳染性海綿樣腦病變 (TSE) 即為類澱粉樣纖維或其先驅物造成的疾病之一。普昂蛋白 (Prion protein,PrP) 正是引發傳染性海綿樣腦病的主因，當有致病性的普昂蛋白 (PrPSc) 存在，會誘使其他正常的普昂蛋白 (PrPC) 轉變為致病性結構，PrPSc單體通過分子間氫鍵與凡德瓦力作用而聚集成類澱粉樣纖維沉積於患者腦中，傷害患者的神經系統。探討類澱粉樣纖維的分子結構對於了解其致病機制甚至是找出治療方法十分重要，但類澱粉樣纖維為不溶性的非晶型物質，難以用一般解析蛋白質分子結構的液態核磁共振與X光繞射偵測，所以目前固態核磁共振技術仍是分析類澱粉樣纖維結構的最佳方法。
在論文中我們針對敘利亞倉鼠普昂蛋白第113至127的胜肽片段(Ac-AGAAAAGAVVGGLGG-NH2, SHaPrP113-127)進行研究，探討胜肽在培養過程中的分子結構。我們利用ThT螢光吸收、穿透式電子顯微鏡以及原子力顯微鏡辨識纖維分子的生成與構形，發現SHaPrP113-127在純化後已具備纖維絲狀形貌，也具有ThT螢光吸收特性，其纖維寬度約12 nm，高度約1 nm，長度約100 nm，邊界並不明顯。經培養後的成熟纖維其寬度及高度與未培養者相似，唯長度略長，約300 nm，並且ThT螢光吸收較強，顯示其結構較為完整。接著利用固態核磁共振去偵測培養前後的分子結構，透過二級化學位移可以確定培養前後都是β-strand結構，同時可由13C線寬得知SHaPrP113-127纖維分子從115到120殘基位置結構較規則。實驗結果比較發現，培養之後的成熟纖維有明顯的steric zipper結構，未培養的樣品則沒有此特性。由實驗數據我們可以推測結構不完全的原纖絲(protofibril)應是形成單層β-sheet結構後，再以層與層之間side chain的疏水作用力形成zipper結構。配合紅外線光譜之鑑定，得到β-sheet是反向平行排列。最後以分子動態計算 (MD simulation)，得到SHaPrP113-127類澱粉樣纖維的分子結構模型。

關鍵字

普昂蛋白 ； 類澱粉樣纖維 ； 固態核磁共振 ； 原纖絲

並列摘要

Amyloid fibrils are ordered aggregates of misfolded proteins, which associated with many neurodisorder diseases. Transmissible spongiform encephalopathy (TSE) is one of these fatal diseases. It is suggested that TSE is caused by the conversion of prion protein from its normal cellular form (cellular prion protein, PrPC) to the disease-specific form (scrapie prion protein, PrPSc).The presence of PrPSc will induce the misfolding of other PrPC to PrPSc, existing in the form of amyloid fibrils and damaging the nervous system of the victims. To elucidate the mechanism and pathways of fibril formation and to find the therapy, it is very important to analyze the molecular structure of amyloid fibrils. However, because amyloid fibrils are insoluble in most buffers and are non-crystalline, it is difficult to use conventional experimental techniques such as solution-state NMR or X-ray to obtain the detailed structure of amyloid fibrils. Solid state NMR spectroscopy is a unique method that can provide high-resolution, site-specific structural constraints for amyloid fibrils. In this thesis, we report the results of ThT fluorescence, TEM, AFM, FTIR, and solid-state NMR (SSNMR) data for protofibrils and mature fibrils formed by residues 113-127 of the Syrian hamster prion protein (SHaPrP113-127, Ac-AGAAAAGAVVGGLGG-NH2). We found that after purification, SHaPrP113-127 peptides have already had short fibrillar structure, and also an enhanced fluorescence upon binding to ThT. These properties confirm that the SHaPrP113-127 peptides right after purification are in protofibrillar state. After incubation, the length of the mature fibrils becomes longer and the intensity of the ThT fluorescence becomes significantly enhanced. The mature fibrils in general have a length over 300 nm, a width of 12 nm, and a height of 1 nm. From the chemical shift and the linewidth data obtained from SSNMR measurements, the β-sheet structure formed in both protofibrils and mature fibrils have significant ordered structure from the residue 115 to 120. Our data reveal that the molecular structure of mature fibrils adopts the motif of steric zipper, whereas, the structure of protofibrils dose not. From the FTIR spectra, the β-strands within each layer are anti-parallel. Consequently, a molecular model for the fibrils was constructed by molecular dynamics simulations incorporated with structural constraints obtained from solid-state NMR measurements. We suppose that the process of fibril formation takes the following pathway. First, monomeric SHaPrP113-127 molecules self-assemble into single layer that is predominated by anti-parallel β-sheet. After that, two layers mate tightly and form the steric zipper by the hydrophobic interaction of side chains.

並列關鍵字

prion protein ； amyloid fibril ； solid-state NMR ； protofibril

參考文獻

1. Soto, C., Prions: The New Biology of Proteins. Taylor & Francis Group: 2006.

2. Sipe, J. D.; Cohen, A. S., Review: History of the Amyloid Fibril. Journal of Structural Biology 2000, 130 (2-3), 88-98.

3. Walsh, P.; Simonetti, K.; Sharpel, S., Core Structure of Amyloid Fibrils Formed by Residues 106-126 of the Human Prion Protein. Structure 2009, 17 (3), 417-426.

6. Decatur, S. M., Elucidation of Residue-Level Structure and Dynamics of Polypeptides Via Isotopp-Edited Infrared Spectroscopy. Accounts of Chemical Research 2006, 39 (3), 169-175.

7. Dobson, C. M., Protein Folding and Misfolding. Nature 2003, 426 (6968), 884-890.