透過您的圖書館登入
IP:34.201.37.128
  • 學位論文

唾液蛋白片段與人類普昂蛋白胜肽之液態核磁共振光譜研究

Solution-State NMR Studies of Salivary Protein Fragment and Human Prion Peptide

指導教授 : 陳振中

摘要


近年來,液態核磁共振光譜技術已被廣泛的應用於蛋白質及巨胜肽分子的結構解析。本篇論文也將使用液態核磁共振的技術來分析唾液蛋白及普昂蛋白結構。人體的唾液是一種過飽和的磷酸鈣溶液,可以給與牙齒很好的保護,但同時也容易在牙齒的琺瑯質表面產生沉澱。此時唾液蛋白的存在就十分重要,這條由43個殘基組成的酸性磷酸化蛋白statherin,可藉由與磷酸鈣鹽類高度的親和力來抑制磷酸鈣的沉澱,使以氫氧基磷灰石 (HAp) 為主的牙結石不會在牙齒表面堆積。實驗證明其N端的前15個殘基 (DpSpSEEKFLRRIGRFG,簡稱SN-15) 對於HAp晶體的吸附力極強,也可以有效的抑制磷酸鈣的沉澱。為了探討staherin如何抑制磷酸鈣沉澱的機理,我們透過液態核磁共振技術,得到SN-15在水溶液中的結構,結果顯示SN-15具α螺旋結構。並證明在SN-15與HAp共合成的溶液中存在有奈米級未沉澱的HAp。由液態核磁共振數據可知在鈣離子或過飽和的磷酸鈣溶液中,SN-15的結構仍然維持原來的螺旋結構,顯示SN-15與磷酸鈣鹽類的親和力主要以簡單的電荷吸附為主。 錯誤摺疊的普昂蛋白 (簡稱PrPSc) 為類澱粉樣纖維疾病 (amyloid disease) 的致病原之一,此種疾病會造成中樞神經失調並且會在不同物種間相互感染,在感染時二級結構會由原本的α螺旋變成β摺板。普昂蛋白內大部分為疏水片段,所以容易聚集 (aggregate) 起來形成絲狀沉澱物,可於顯微鏡下觀察到。之前的研究顯示,第106-126片段為PrPSc主要的致病及毒性來源,其相關的分子結構亦已被成功解出。本論文將探討其前段第106-114胜肽片段 (Ac-KTNMKHMAG-NH2,簡稱HuPrP106-114) 。有別於一般普昂蛋白的疏水片段,HuPrP106-114為PrPSc中帶電密度高且極為親水的區段,想研究親水性胜肽是否也能形成類澱粉樣纖維結構。經培養後發現不會有沉澱物產生,但可在顯微鏡下觀察到絲狀的可溶性原絲纖維 (protofibril)。原絲纖維是類澱粉樣纖維形成時的中間物,我們利用液態核磁共振及CD光譜來觀察其二級結構,經實驗發現此段胜肽在溶液中以不規則堆疊 (random aggregate) 為主。

並列摘要


Solution-state NMR is a powerful method for the structural studies of polypeptides and proteins. Two types of polypeptides are discussed in this work. Human saliva is a supersaturated solution of calcium phosphates, which provides a protective and reparative environment for dental enamel and dentin. Precipitation of calcium phosphates in saliva is inhibited by an acidic phosphoprotein, statherin, which contains 43 residues. The function of statherin is mainly provided by a 15-residue (D1pSpSEEKFLRRIGRFG15) fragment near the N-terminus, viz. SN-15. In this work, we investigate the conformation of SN-15 in aqueous solution with solution-state NMR technique. High resolution 1H-1H NMR spectra (COSY, TOCSY, and NOESY) have been acquired in aqueous solution. Amide hydrogen exchange experiment indicates that SN-15 adopts an α-helical structure. The peptide structure of atomic scale resolution has been successfully calculated from the NMR constrains. The solution structure determined for SN-15 shows that the residues of K6, F7, L8, and R9 exhibit a helical structure, whereas the first 5 residues of the N-terminus are in helix-like conformation. Additional NMR measurements show that the structure of the SN-15 peptide in the supersaturated calcium ion and calcium phosphate solution is essentially the same as that determined in regular pH buffer. This interesting result indicates that the interactions between the SN-15 and calcium phosphates are mainly electrostatic in nature and do not involve any major conformational changes. Prion protein (PrP) is closely associated with prion diseases. PrP will become pathogenic when it undergoes structural change from its native α-helix to β-sheet form. In particular, we attempt to study the human PrP fragment compassing residue 106-114, which is highly hydrophilic. We find that the peptides do not form insoluble fibrils after incubation and therefore solution-state NMR is used to characterize its structure. Soluble protofibrils were observed for the HuPrP106-114 peptides from the electron microscopy. Analysis of CD and NMR spectra show that the secondary structure of the HuPrP106-114 peptide is random-coil in aqueous solution. This results indicate that fibril formation for HuPrP106-114 may involve a structural rearrangement of the random aggregate formed by the peptides.

參考文獻


13. Harrison, R. S.; Sharpe, P. C.; Singh, Y.; Fairlie, D. P., Amyloid peptides and proteins in review. Reviews of Physiology, Biochemistry and Pharmacology, Vol 159 2007, 159, 1-77.
Chapter 1
1. Mann, S., Biomineralization : Principles and Concepts in Bioinorganic Materials Chemistry. 2002.
2. Weiner, S.; Addadi, L., Design strategies in mineralized biological materials. Journal of Materials Chemistry 1997, 7, (5), 689-702.
3. Douglas, W. H.; Reech, E. S.; Ramasubbu, N.; Raj, P. A., Biochem. Biophys. Res. Commun. 1991, 180, 91-97.

延伸閱讀