膠原蛋白是人體中含量最豐富的蛋白質，是由三股第二型聚脯胺酸鏈組成，每股主要包含(Pro-Hyp-Gly)作為重複序列單元。三股螺旋可以是三條相同鏈形成的AAA型同源三股螺旋，或是以兩種或三種不同鏈組成的AAB型、ABC型異源三股螺旋。近年來，膠原蛋白模擬胜肽常被拿來探討膠原蛋白的結構與功能性，也被應用於超分子結構或生物材料中。我們實驗室先前的研究顯示了cation-π作用力可以促使異源三股螺旋的生成，為了進一步探討胺基酸變異對三股螺旋結構的影響，在第一部分，我們在以cation-π作用力形成的異源三股螺旋結構中，將胜肽鏈的甘胺酸置換為肌胺酸或丙胺酸，並研究對於三股螺旋穩定度的影響；在這項實驗中，我們也會討論氫鍵的貢獻與立體效應的影響。在第二部分，我們以(Pro-Hyp-Gly)9為模板，設計新的序列，在中間位置及碳端換上帶有陽離子側鏈的精胺酸與芳香環側鏈的酪胺酸組合，嘗試引入cation-π作用力，探討是否能夠有效形成同源及異源三股螺旋。另外，我們也在此序列氮端處將其中一個甘胺酸置換為肌胺酸與丙胺酸，觀察置換後對三股螺旋穩定度的影響。由實驗結果發現某些置換為肌胺酸的胜肽仍能夠形成三股螺旋，但置換為丙胺酸後結構變得不穩定，無法形成三股螺旋。由此顯示置換丙胺酸所造成的影響比起置換肌胺酸時來得大。

Collagen is the most abundant protein in the human body. It is a triple helix composed of three polyproline II (PPII) strands, each of which often consists of proline-hydroxyproline-glycine (Pro-Hyp-Gly) as a repetitive sequence. Triple helices can be homotrimers, which are comprised of three identical strands, or heterotrimers, with two or three different strands. In the recent years, collagen-mimetic peptides (CMPs) are commonly used to help understand the structures and functions of collagen, and have been applied to supramolecular structures and biomaterials. Our previous studies have shown that cation-π interactions can be used to induce the folding of heterotrimers.To understand the mutation effects on heterotrimers, in the first part, we replaced the glycine residue of the backbone with a sarcosine or alanine in the cation-π induced heterotrimeric structure to study the consequences on the triple helix. The contribution of hydrogen bonds and the impact of steric effects were also discussed in this work. In the second part, we used (Pro-Hyp-Gly)9 as the template, and installed arginine (with the cationic side chain) and tyrosine (with the aromatic ring side chain) in the center part and C-terminus to observe whether it would form homotrimers or heterotrimers via cation-π interactions. In addition, we also replaced one of the glycines at the N-terminal fragment of this sequence with a sarcosine or alanine to examine the effect of the replacement on the triple helix stability. The results showed that some peptides substituted with sarcosine could still form a triple helix, but being replaced with an alanine would make the peptides fail to form trimers. From the data, we found that the impact of replacing alanine is greater than replacing sarcosine.

中文摘要 I
Abstract II
序言 III
第一章 緒論 1
1.1. 膠原蛋白 1
1.1.1. 膠原蛋白組成 2
1.1.2. 膠原蛋白結構 2
1.1.3. 膠原蛋白作用力 4
1.1.4. 膠原蛋白模擬胜肽胺基酸置換的穩定性 6
1.1.5. 膠原蛋白中胺基酸的置換 7
1.2. Cation-π作用力(Cation-π interaction) 8
1.2.1. 影響Cation-π作用力的因素 9
1.2.2. 水溶液中的Cation-π作用力 9
1.2.3. 帶電荷與芳香環側鏈之胺基酸 11
1.2.4. 胺基酸與芳香環側鏈間的作用力 12
1.2.5. 徑向與軸向作用力 14
1.3. 研究動機 15
第二章 實驗部分 18
2.1. 實驗儀器 18
2.2. 實驗藥品 19
2.3. 實驗流程 20
2.4. 固相胜肽合成法(Solid-Phase Peptide Synthesis, SPPS) 21
2.4.1. 酯化/醯胺化反應 23
2.4.2. 去保護 23
2.4.3. 活化 25
2.4.4. 耦合 25
2.4.5. 切除 26
2.5. 胜肽合成 26
2.5.1. 用自動胜肽合成儀合成B1_Sar, B1_Ala, CR3_Ala 27
2.5.2. 手動合成B2_Sar, B2_Ala, B3, B4, CF3, D1, D2, D1_Sar, D1_Ala, B3* 28
2.5.3. 切除胜肽 28
2.5.4. 以HPLC純化胜肽 29
2.5.5. 以HPLC和MALDI-TOF MS鑑定胜肽純度 29
2.6. 圓二色光譜儀 30
2.7. 圓二色光譜儀之量測 31
2.7.1. 製備胜肽溶液 31
2.7.2. Far-UV CD光譜量測 32
2.7.3. 變溫CD光譜量測 32
2.7.4. 變溫實驗數據處理 32
2.8. 1H, 15N-HSQC(Heteronuclear single quantum coherence)光譜 33
2.8.1. 1H, 15N-HSQC樣品配製 34
2.9. 差式掃描量熱法(Differential scanning calorimetry, DSC) 35
2.9.1. DSC樣品配製 35
2.9.2. 熱力學實驗數據處理 35
第三章 結果與討論 37
第一部分、肌胺酸與丙胺酸置換之探討 37
3.1. 置換位置設計 37
3.2. CD光譜之探討 38
3.2.1. Far-UV之CD光譜 39
3.2.2. 變溫實驗之CD光譜 41
3.3. 1H, 15N-HSQC光譜 47
第二部分、中間位置與碳端置換精胺酸與酪胺酸之探討 50
3.4. 胜肽序列設計 50
3.5. CD光譜之探討 50
3.5.1. Far-UV之CD光譜 50
3.5.2. 變溫實驗之CD光譜 51
3.6. 熱力學性質之探討 58
第四章 結論 60
參考文獻 62
附錄 68

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