探討高胺酸置換對於胜肽結構的影響及研究 Prion 蛋白片段作為醯基轉化催化劑之效益

The effect of (S)-piperidine-2-carboxylic acid on peptide structure and the catalytic activity of Prion protein fragments on acyl transfer reaction

林芷瑩(Chih-Ying Lin)

指導教授 ： 洪嘉呈

國立清華大學/理學院/化學系/碩士(2015年)

https://doi.org/10.6843/NTHU.2015.00084

摘要

(S)-piperidine-2-carboxylic acid (Pip) 為六員環的脯胺酸衍生物，其被認為與罕見疾病哌啶酸血症 (hyperpipecolic acidemia) 相關，該病症的特徵在於會增加高脯胺酸在血液中的濃度，導致神經疾病和肝腫大。本次研究第一部分即探討將 Pip 置換在脯胺酸 (Pro) 位置對於不同結構胜肽穩定度的影響，我們將 Pip 置換在 polyproline、HP7 及 Trp cage 胜肽上，以圓二色光譜儀 (Circular dichroism；CD) 來觀察其 Far-UV 光譜與熱熔化曲線的改變。結果發現 Pip 的置換對於 polyproline II (PPII) 結構較無影響，但會造成 polyproline I (PPI) 結構穩定度下降。在 HP7 胜肽置換 Pip 的實驗中，發現 Pip 不會對於β-harpin 結構產生太大的改變。最後將 Pip 置換在 Trp cage 上四個 Pro 的位置上，得到 Pip 的置換會造成 Trp cage 胜肽結構相當不穩定，判斷是因為 Pip 的六員環立體障礙過大而導致 α-helix 結構的崩解，也對於不同位置所造成的影響有所討論。
　　我們也利用理論計算試圖佐證我們在 polyproline 實驗中得到的結果，使用 Gaussian 軟體，以 DFT 方法進行結構最佳化，Ac-P2(Pip)P2-NH2 分子模型進行計算。計算所得到的結果為 Pip 主要的構形 boat up 會類似於 Pro 的 exo 構形。在 PPI 結構中四種構形的 Pip 都與 P5 PPI 有很大的差異，可能就是造成我們在 CD 光譜中 PPI 結構較不穩定的主要原因。
　　第二部分我們以 Prion octarepeat 片段來作為醯基轉化催化劑，發現將 Ac-XaaHGGGWGQP-NH2 和 Ac-GQXaaHGGGWGQ-NH2 中的 Xaa 位置置換成 Asp 對於催化醯基轉化有明顯地效果。而 Ac-GQDHGGGWFQ-NH2 (GQD) 的催化效果大於 Ac-DHGGGWGQP-NH2 (DP)，判斷是前者 Asp 位置位在胜肽鏈的內側較不易晃動，使得 Asp 和 His 間的距離較為固定，有比較強的作用造成催化效果較佳。

關鍵字

胜肽 ； 高胺酸 ； 脯胺酸

並列摘要

(S)-piperidine-2-carboxylic acid (Pip) is a six-membered ring of proline derivative. Pip has been reported to be associated with hyperpipecolic acidemia－one of the rare disorders, which an increase in pipecolic acid levels in the blood leads to neuropathy and hepatomegaly. In the fist part of this work, we prepared various peptides with Pip incorporated, including polyproline, HP7, and Trp cage. Circular dichroism spectroscopy (CD) was used to characterize their conformation. We found that the incorporation of Pip did not affect polyproline II structure but destabilized polyproline I conformation. When Pip was incorporated into HP7, it seemed to fit well in a β-harpin and did not significantly change the structure. However, the incorporation of Pip into Trp cage dramatically destabilized the α-helical structure, in particular replacing Pro12 to Pip destroyed the structure.
　　We also used a computational approach to study the effect of Pip on polyproline conformation. For each model compound and system, hybrid density functional theory (DFT) calculations, as implemented in Gaussian 09, were carried out to learn the energy difference between different conformations. We used Ac-P2(Pip)P2-NH2 as a model system for the computation. Pip prefers a boat up conformation similar to the Cϒ-exo pucker of Pro, and can also form the conformation of boat down, chair up, and chair down. The calculations indicate that all the conformers of Pip are not appropriate to a PPI helix, which may explain why Pip destabilizes PPI conformation as observed by the experiments.
　　In the second part, we chose Prion octarepeat fragments to study their potential catalytic activity on acyl transfer reaction. The results indicate that it has a high catalytic activity when the Xaa position in Ac-XaaHGGGWGQP-NH2 (GQX) or Ac-GQXaaHGGGWGQ-NH2 (XP) is Asp, and Ac-GQDHGGGWGQ-NH2 (GQD) is more efficient than Ac-GQDHGGGWGQ-NH2 (DP). In GQD, the Asp is at the middle of the peptide, making Asp interact with His more strongly, which might explain why GQD is more effective than DP on catalyzing the reaction.

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