原生型人類普昂蛋白127–147胜肽片段與P137A變異型所形成之類澱粉樣纖維分子之比較

Comparison of Amyloid Fibrils Formed by Wild-Type and P137A Mutant of 127–147 Fragment of Human Prion Protein

連惠瑩(Huei-Ying Lian)

指導教授 ： 陳振中

國立臺灣大學/理學院/化學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02857

摘要

普昂蛋白(PrP)為一種感染性蛋白質，此蛋白質與普昂疾病的產生有極大的關聯性。當普昂蛋白由含α-helix較多的自然結構(cellular PrP, PrPc)轉變成β-sheet較多的結構(scrapie PrP, PrPsc)時，會使普昂蛋白產生致病性。由原生型普昂蛋白127到147胜肽片段(wild-type HuPrP127–147)所形成之類澱粉樣纖維，分子結構利用殘基間相互對齊，而β-sheet間同向平行排列形成。這樣的纖維結構中含有一個由脯胺酸支鏈造成的空間限制，而此空間限制可能會影響蛋白質結構的形成。因此，對生物物理領域而言，將具有空間限制的P137位置脯胺酸由其他胺基酸進行點變異，是個值得探討的題目。
本實驗中，我們發表了原生型與P137A變異型人類普昂蛋白127–147胜肽片段所形成的類澱粉樣纖維之穿透式電子顯微鏡、ThT螢光光譜、圓二色光譜、紅外線吸收光譜與固態核磁共振光譜的數據。由TEM的數據可發現，P137A變異型胜肽在不同培養條件下可看見不同的纖維構形。令人驚訝的是，P137A變異型在透析條件培養下，會形成尺度較大的奈米管狀類澱粉樣纖維。此奈米管結構只出現在特定的透析培養條件中，表示此種培養條件適合多層β sheets的生成。此外，我們可以由FT-IR數據發現靜置培養的P137A變異型樣品中含有β-sheet彎曲折疊(bend structure)的結構資訊。這種彎曲折疊的結構應無法形成多層β sheets延伸的奈米管。因此，培養條件對於類澱粉樣纖維結構的形成為非常重要的關鍵。由固態核磁共振的數據可以發現，在靜置培養條件中，無論是原生型或是變異型的胜肽，皆會形成同向平行且有殘基對齊的cross-β結構，但是只有變異型的樣品會出現結構的多樣化。由ThT螢光與CD實驗可知，P137A變異型纖維生長速率比原生型纖維來得快許多，並且會形成β sheets含量較多的纖維結構。另外在實驗中還發現經過細胞破碎儀的振盪後，P137A變異型的β-sheet結構維持不變，但原生型纖維結構則會遭受破壞。我們可以利用脯胺酸會阻止β sheets生成的已知事實來說明以上觀察到的有趣現象。

關鍵字

普昂蛋白 ； 類澱粉樣纖維 ； 固態核磁共振

並列摘要

Prion protein (PrP) is an infectious species that is closely associated with prion diseases. PrP will become pathogenic when it undergoes structural change from its native α-helix (cellular PrP, PrPc) to β-sheet conformation (scrapie PrP, PrPsc). The molecular structure of the amyloid fibrils formed by the wild-type 127–147 peptide fragment of human prion protein (wild-type HuPrP127–147) adopts parallel β-sheet organization with in-registered alignment. There is a spatial constraint due to the side chain of proline, and this constraint may alter the molecular structure of the fibrils. Consequently, the mutation of the highly constrained P137 into other amino acids is a very interesting topic in biophysics.

In this study, we report the results of ThT fluorescence, TEM, CD, FT-IR, and solid-state NMR (SSNMR) data for the fibrils formed by both the wild type and P137A mutant of human prion protein 127–147 fragment. From the TEM data, various morphologies were observed in P137A mutant sample, depending on the incubation conditions. Surprisingly, the P137A mutants exhibit an additional self assembly at a relatively long length scale, viz. the formation of nanotube. The formation of nanotube is only observed under the dialysis conditions, which may facilitate the formation of β-sheet laminates. For the fibril samples prepared under the quiescent conditions, on the other hand, we find the evidence of bend-structure formation from the FT-IR data. Such bending structure most likely would preclude the formation of the nanotube. Consequently, the incubation conditions are crucial in the morphologenesis of our fibril systems. According to the SSNMR data, both the wild type and P137A mutant form in-registered parallel cross-β structures under quiescent incubation conditions, but morphology polymorphism have been observed for the mutant samples only. P137A mutant exhibits higher content of β sheets and faster fibrillization kinetics than the wild-type sample based on the CD and ThT experiments. In addition, the β-sheet structure of P137A mutant remains intact after sonification, whereas the wild-type sample becomes unstructured. These observations may be rationalized by the fact that proline would hinder β-sheet formation.

並列關鍵字

prion protein ； amyloid fibril ； solid state NMR

參考文獻

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