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研究生:吳昀樺
研究生(外文):Wu, Yun-Hua
論文名稱:利用含組胺酸之膠原蛋白模擬胜肽進行異源三股螺旋摺疊和酯類水解催化反應的探討
論文名稱(外文):Study of Heterotrimeric Folding and Ester Hydrolysis Catalysis using Histidine-Containing Collagen-Mimetic Peptides
指導教授:洪嘉呈洪嘉呈引用關係
指導教授(外文):Horng, Jia-Cherng
口試委員:吳淑褓陳青諭
口試委員(外文):Wu, Shu-PaoChen, Chin-Yu
口試日期:2018-07-20
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學系所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:69
中文關鍵詞:膠原蛋白酯類水解催化異源三股螺旋組胺酸
外文關鍵詞:collagenester hydrolysis catalysisheterotrimerhistidine
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膠原蛋白是動物體內各種結締組織細胞外空間的主要結構蛋白,由三條聚脯胺酸第二型結構(PPII)所形成之三股螺旋,可依照組成的胜肽種類分為AAA型同源三股螺旋及AAB、ABC型異源三股螺旋。穩定三股螺旋結構的作用力有許多種,如氫鍵、靜電作用力、疏水作用、以及一些特殊鍵結等等,其中一種由正電荷與芳香環四極矩間的非共價鍵作用力:cation-π作用力,在我們過去的研究中也發現有重要的貢獻。
本研究第一部分以(His-Hyp-Gly)9為基底,透過設計胜肽序列,希望形成以帶正電胺基酸及組胺酸上的咪唑官能團(imidazole)之間的cation-π作用力,形成AAB型或ABC型異源三股螺旋。實驗結果顯示,在此胜肽序列中不論以組胺酸上的咪唑官能團或輔以苯丙胺酸上的芳香環,和精胺酸提供的正電荷所產生的cation-π作用力均無法形成AAB型或ABC型異源三股螺旋。
在第二部分的研究,參考實驗室的先前研究曾利用膠原蛋白三股螺旋與鋅離子配位,模擬天然碳酸酐酶的活性中心,我們將鋅離子與含有組胺酸的胜肽序列配位,觀察其催化效率。實驗結果顯示,當胜肽序列中的組胺酸越多時,催化效率會越高。此外,當我們加入金屬離子,使之與胜肽配位後發現,發現(His-Hyp-Gly)9的結構會由PPII轉變成β sheet並可形成大型的纖維結構,但其催化效率卻有些微下降,推測原因是因為構形轉變後,組胺酸及鋅離子被包覆在胜肽內部而不易與受質接觸,因此使催化效率反而更低。由此實驗可知,具有催化功能的胜肽並非形成較穩定有序的結構,就能提升催化效率,還是需要考慮其他因素,希望此一結果對未來研究人工水解酶的設計提供有用的資訊。
Collagen is the main structural protein in animals, and the structure of collagen is a right-handed triple helix formed by three polyproline-II helices. The triple helices can be AAA homotrimers, AAB heterotrimers, or ABC heterotrimers according to the composition of the peptides. Various forces have been found to stabilize the triple helix, such as hydrogen bonds, electrostatic interactions, hydrophobic effects, and specific bonding. Among these forces, cation-π interactions are a noncovalent force and regarded as an electrostatic attraction between a positive charge and a quadrupole electron-rich π system. Cation-π interactions have also been found to have a positive contribution to the triple helix stability in our previous studies.
The first part of this study was based on the peptide of (His-Hyp-Gly)9. Through the design of peptide sequences, we attempted to form AAB-type or ABC-type heterotrimers by using the cation-π interactions between positively charged amino acids and the imidazole groups of histidines. The results show that the cation-π interactions between the imidazole groups of histidine residues and the positive side chains of arginine residues are not strong enough to generate the AAB-type or ABC-type heterotrimers.
In the second part of this study, we mimicked the active site of zinc metalloenzymes by using histidine-containing peptides as catalysts for ester hydrolysis. We used the His residues as the ligands to coordinate with zinc(II) ions and serve as the catalytic active site for ester hydrolysis. The results show that a large number of His residues in the peptide sequence will increase the catalytic efficiency. In addition, upon the addition of metal ions to coordinate with the peptides, we found that the structure of (His-Hyp-Gly)9 would change from PPII helices to β sheets and higher-order fibrils, but its catalytic efficiency was slightly decreased. This is presumably due to that His residues and zinc(II) ions are buried in the peptide assemblies and less exposed to the substrates, leading to a lower catalytic efficiency. From this study, we found that forming stable structure like β sheet might not be the main factor to improve the catalytic efficiency, and thus other elements must be also considered. We hoped that the results provide useful information for the future design of artificial hydrolytic enzymes.
中文摘要 I
Abstract II
誌謝辭 IV
目錄 V
圖目錄 VIII
表目錄 XII
第一章 緒論 1
1-1 蛋白質結構 1
1-1-1 β摺疊(β sheet) 2
1-1-2 聚脯胺酸螺旋(polyproline helix) 2
1-2 膠原蛋白 4
1-2-1 膠原蛋白模擬胜肽單一置換穩定性的探討 5
1-2-2 異源三股螺旋(heterotrimers) 6
1-3 Cation-π作用力 8
1-3-1 帶電荷與芳香環側鏈的胺基酸 9
1-3-2 胺基與芳香環間的作用力 11
1-4 酯類水解反應(Ester hydrolysis reaction) 12
1-5 鋅酶的作用與簡介 13
1-6 研究動機 16
第二章 實驗部分 18
2-1 實驗儀器 18
2-2 實驗藥品 19
2-3 圓二色光譜儀(Circular Dichroism Spectrometer,CD) 21
2-4 Fmoc-His(Trt)-Hyp-Gly-OH之合成 24
2-4-1 Boc-(2S,4R)-hydroxyproline (1)之合成 25
2-4-2 Boc-Hyp-Gly-OBn (2)之合成 25
2-4-3 Fmoc-His(Trt)-Hyp-Gly-OBn (3)之合成 26
2-4-4 Fmoc-His(Trt)-Hyp-Gly-OH (4)之合成 27
2-5 固相胜肽合成法(Solid-phase peptide synthesis, SPPS) 28
2-6 胜肽的合成、純化與鑑定 33
2-7 光譜測量 35
2-7-1 CD光譜測量 35
2-7-2 催化水解活性實驗 35
2-8 光譜數據分析 36
2-8-1 CD變溫實驗之數據處理 36
2-8-2 催化水解活性實驗之數據處理 38
2-9 穿透式電子顯微鏡(Transmission electron microscopy, TEM) 40
2-10 穿透式電子顯微鏡之樣品配製 40
第三章 結果與討論 41
第一部分 利用含組胺酸之膠原蛋白模擬胜肽進行異源三股螺旋摺疊之探討 41
3-1 胜肽序列之設計 41
3-2 CD光譜探討 42
3-2-1 同源三股螺旋 42
3-2-2 AAB型異源三股螺旋 44
3-2-3 ABC型異源三股螺旋 47
第二部分 利用含組胺酸之膠原蛋白模擬胜肽進行酯類水解催化反應之探討 49
3-3 胜肽加入鋅離子之CD光譜探討 49
3-4 穿透式電子顯微鏡影像 52
3-5 胜肽加入銅離子之CD光譜探討 54
3-6胜肽對酯類水解反應的催化效率探討 58
3-7 胜肽加入鋅離子後對酯類水解反應的催化效率探討 60
第四章 結論 63
參考文獻 65
附錄 68
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