人類普昂蛋白127-147胜肽片段所形成含脯胺酸殘基之類澱粉樣纖維的分子結構研究

Abstract

普昂疾病（prion disease）為類澱粉樣疾病（amyloid disease）的一種，是會傷害腦神經細胞的感染性疾病，造成這種疾病的主要原因為結構有異變的普昂蛋白（prion protein, PrPSc）。利用電子顯微鏡測量PrPSc所形成的沉澱物，能夠觀察到很明顯的絲狀纖維結構。類澱粉樣蛋白纖維為一種非晶性的低水溶性物質，所以一般用於解出蛋白質分子結構的液態核磁共振與X光繞射都不適用，固態核磁共振技術為最合適的方法。在論文中，我們以人類普昂胜肽第127至147的多肽片段（Ac-GYMLGSAMSRPIIHFGSDYED-NH2, HuPrP127-147）所形成的類澱粉樣纖維來進行研究，利用固態核共振技術來解出其分子模型。HuPrP127-147普遍被認為是致病的主要胜肽片段之ㄧ，因此在普昂疾病的研究中是一個重要題目。除此以外，HuPrP127-147胜肽的中段含有一個脯胺酸殘基（P137），基於脯胺酸特別的空間構形，HuPrP127-147纖維的分子結構也是一個十分有趣的生物物理課題。我們成功的培養出HuPrP127-147的類澱粉樣蛋白纖維，並且利用ThT 螢光吸收、穿透式電子顯微鏡與原子力顯微鏡去確定其纖維分子的生成與形成的構形，我們量測出成熟的類澱粉樣纖維分子具有絞結（twist）的結構，絞結之間的長度約為70 至80 nm，高度約為3~4 nm。接著利用固態核磁共振去偵測其分子結構。我們由碳13訊號的線寬發現HuPrP127-147胜肽分子由第129號至142號殘基位置為結構性較高的區域，並且透過化學位移的偏移與背脊扭轉角psi的判定來確定此區域為b-strand的二級結構，再偵測出部分碳13之間的同核距離關係。實驗結果顯示，HuPrP127-147類澱粉樣蛋白纖維的基本結構為相互平行對齊的b-strand所組成，彼此之間的距離約為5~6 Å。由N端到C端的殘基訊號的強度發現， 以P137殘基位置為分界靠近C端的b-strand區域擁有較高動性結構。最後，使用紅外光吸收光譜觀測特定殘基位置的紅位移現象來偵測纖維分子中每條多肽之間的排列方式，實驗結果發現以P137殘基位置為分界靠近N端的b-strand區域的結構為cross-b的結構；靠近C端的b-strand區域雖有一定的結構性，但沒有背脊氫鍵鍵結，可能只有靠著支鏈之間的作用力來穩定其結構。

Prion disease is a kind of amyloid disease, which 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). PrPSc exists in the form of amyloid fibrils, which are inherent insoluble and non-crystalline solids. Due to this reason, the detailed structural information of PrPSc is difficult to obtain by conventional techniques such as X-ray crystallography or solution-state NMR. It has recently been shown that solid-state nuclear magnetic resonance (SSNMR) is a particularly useful method for the structural elucidation of amyloid fibrils. In our study, amyloid fibrils are prepared by incubating the peptide fragment of human prion protein (HuPrP127-147), Ac-GYMLGSAMSRPIIHFGSDYED-NH2, in PBS at 37°C. HuPrP127-147 is believed to be an infectious fragment of the amyloidogenic region. In addition, HuPrP127-147 contains a proline residue, whose structure is highly constrained. Therefore, studying the fibrillar structure of HuPrP127-147 is an interesting topic of biophysics. Accordingly, ThT fluorescence spectra are obtained to confirm the fibril formation and the fibril morphology is studied by TEM and AFM. The fibrils have twist morphology and the twist period is about 70 to 80 nm The fibril height is between 3 and 4 nm. SSNMR measurements are carried out to probe for the molecular structure of the fibril samples. Our data show that the fibrils are formed by parallel in-registered alignment of peptides adopting b-strand conformation. In particular, each peptide constituting the fibrils has a b-strand region, viz. 129-142. With the proline (P137) as the boundary, the b-strand region near the N terminal (b1) has higher structural order than that near the C terminus (b2), which is associated with considerable dynamic motions. We also use FT-IR measurements to detect the vibrational dipole coupling in isotope-edited polypeptides. The results show that the b1 region conforms to the cross-b structure, but not the b2 region. We speculate that the b2 region is mainly stabilized by the side-chain-side-chain interactions.