固態核磁共振技術研究倉鼠普昂胜肽109-122變異片段與人類普昂胜肽127-147片段之類澱粉樣纖維結構

Solid State NMR Study of Amyloid Fibrils Formed by Mutant Syrian Hamster PrP109-122 and Human PrP127-147

唐韻如(Yun-Ju Tang)

指導教授 ： 陳振中

國立臺灣大學/理學院/化學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02455

摘要

摘要
普昂粒子(Prion)是帶有感染性蛋白粒子的簡稱。普昂蛋白已被證實有兩種結構，正常的普昂蛋白(PrPC)受感染會轉變為具致病性結構的PrPsc，並且誘使其他PrPc大量轉變為PrPsc，當PrPsc透過分子間氫鍵與凡得瓦作用力聚集後，會形成類澱粉樣纖維沉積於生物體的大腦中，導致神經系統的退化，傳染性海綿樣腦病變就是由類澱粉樣纖維的沉積所引發的神經退化疾病。不同胺基酸組成之序列可能有不同之類澱粉樣分子的結構與致病性，因此，研究不同的類澱粉樣纖維之分子結構差異性，對於了解其結構轉變的機制甚至是治療方法就變得十分重要。由於類澱粉樣纖維為低溶解度的非結晶物質，無法用液態核磁共振與X光繞射技術研究其分子結構，所以目前固態核磁共振技術仍然為解析該類蛋白質結構最佳的方法。
論文中，使用敘利亞倉鼠的普昂胜肽109-122片段，由於前人的研究結果指出其結構為雙層的立障拉鏈(steric zipper)，並且單層的反向平行β-sheet是以Ala117對齊，故我們將117位置由丙胺酸(Alanine)變異為異亮胺酸(Isoleucine)當作研究對象—SHPrP109

關鍵字

固態核磁共振 ； 普昂胜肽

並列摘要

Abstract
Prion particles (prion) are proteinaceous infectious particles and it has been proved that there are two different structures exist for prion proteins. The normal cellular prion protein, denoted as PrPC, may convert into an abnormal scrapie cellular prion, denoted as PrPSc, through a process whereby a portion of its α-helical structure is refolded into β-sheet. The presence of PrPSc will catalyze the transformation of PrPC to PrPSc by the misfolding process. When PrPSc forms aggregate through hydrogen bonds and Van der Waals interactions, the amyloid fibril will be formed and cause the neurodegenerative disease. The transmissible spongiform encephalopathies (TSE) is one of the neurodegenerative diseases that caused by the amyloid fibril depositions. Different protein sequences may have different pathogenicities and distinctive amyloid fibril structures. Therefore, understanding structural conversion of amyloid fibril is very important. Owing to the low solublity and non-crystalline characteristics of amyloid fibrils, it is difficult to use conventional experimental techniques such as solution-state NMR and XRD to analyze the structure of amyloid fibrils. Hence, solid-state NMR is still the most suitable method to characterize structures of this kind.
In this study, we mutated the 117 position of Syrian hamster prion protein 109-122 fragment (SHaPrP109-122, A117I, Ac-MKHMAGAAIAGAVV-NH2) from Ala to Ile. The idea is to compare the structural difference induced by mutation on the aligned position of β-sheet in the native SHaPrP109-122. We have incubated the amyloid fibril formed by SHaPrP109-122, A117I successfully. We report the ThT fluorescence, TEM, AFM experiments to observe the formation of amyloid fibrils. From the TEM and AFM images, we have measured the matured fibrils about 24 nm in width, 1.1 nm in height and 380 nm in length. From the isotope-edited FTIR study, the fibril has an anti-parallel β-strand secondary structure and showed an obvious alignment at I117. The chemical shift and linewidth data obtained from ssNMR showed that β-sheet structure exists in the core region. The distance about 4.9±0.2Å between the two β layers is determined by fpRFDR-CT experiment. From the experimental data it has been inferred that the fibrils formed by A117I SHaPrP109-122 has the steric zipper structure. Finally, a molecular model for the fibrils was constructed by molecular dynamics simulations incorporated with structural constraints obtained from ssNMR measurements. The results point out that the fibrils still maintain the steric zipper structure after mutating alanine at residue 117 to isoleucine, which has a very bulky side chain.
In addition, we have also investigated the amyloid fibrils formed by the residues 127-147 of the human prion protein (HuPrP127-147, Ac-GYMLGSAMSRPIIHFGSDYED-NH2) by TEM to trace the initial stages of amyloid fibrils formation and observe the morphology of the fibril and spherical aggregates. The fibrils are about 6 nm in width from the sample incubated for 5 minutes only. The distance between the β-strands was determined by ssNMR to be 5±0.1 Å at the proline residue. It demonstrates that the side chain structure of P137 does not disrupt the β-sheet structure.

並列關鍵字

ssNMR ； Prion ； Amyloid fibril

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