膠原蛋白是人體中含量最多的蛋白質，若是其有不正常的變異或重構會導致一些疾病的生成。而AAB型的異源三股螺旋佔人體膠原蛋白的大多數，因此若要研究此類膠原蛋白變異所造成的疾病、或是對這個領域的生醫材料開發，勢必得發展出AAB型異源三股螺旋的模板作為研究對象。穩定三股螺旋的作用力有很多種，陽離子-π 作用力即為其中一種，而根據實驗室的過往研究，可知其對於穩定度有著不小的貢獻。
在第一部分中，藉由引入四個Tyr與Arg來抑制同源三股螺旋的生成，並透過不同比例的混合使其形成不同的異源三股螺旋，且藉由1H, 15N-HSQC光譜驗證其不同的組成，而這樣的設計也再次證實了陽離子-π作用力能有效促使膠原蛋白異源三股螺旋之摺疊。
第二、三部分為第一部分的延伸，希望藉由雙股胜肽的結構提升三股螺旋的生成量與穩定度，再將其應用至胜肽的修復。從雙股胜肽c5ss與c6的比較可以得知，連接點的長度不同會影響三股螺旋結構的單一性與溶解度，就Lys與Cys來說，Cys為能夠進行預先組織成較適當距離之胺基酸。從c5ss與c7ss的比較可以發現，以Cys作為雙股結構的連接點確實皆可以提升預先組織的效率，而不同的連接環境帶來的是對於穩定度的影響，較暴露於溶液中的環境則會對溶解度帶來影響。
在胜肽修復方面，就以明顯有受到修復的[1]c7ss[1]B1_Sar來說，雙股結構確實能幫助第三股的接近，使其有機會生成三股螺旋，然而cation-π 作用力於此時也扮演重要的角色，幫助三股螺旋的穩定纏繞。在此也發現Ala比起Sar，立障所導致的不穩定，對於結構來說是較為致命的置換。

Collagen is the most abundant protein in the human body, of which type I is the majority. Type I collagen is an AAB-type heterotrimer. Many human diseases including cancers and fibrosis are associated with abnormal remodeling and mutation of collagen, making heterotrimers better mimics for studying collagen structures and developing related biomedical materials. In our previous studies, cation-π interaction was found to serve as a stabilizing force in the collagen triple helix. In the first part of this study, four arginine and four tyrosine residues were introduced in a single collagen-mimetic peptide (CMP), which successfully avoided the folding of homotrimers and only generated a single composition of heterotrimer. This results validated again that cation-π interactions can be used to induce formation of heterotrimers. In the second and third parts of this study, we further installed cysteine and lysine into these peptides to form dimeric CMPs through a covalent bond as an attempt to aid triple-association by preorganizing the linked strands into a PPII-like structure. From the comparison of dimeric CMPs, c5ss and c6, we found that the linker between two strands in a dimeric peptide would affect the unity and solubility of the triple helix structure, and using disulfide bond was a better strategy to tether two strands. From the comparison of dimeric CMPs, c5ss and c7ss, we found that the residues near the tethered point would affect the stability of the triple helix, and the residues exposed to solvent would affect the solubility. As to refolding a triple helix via dimeric peptides, our results indicated that dimeric peptides could help the third strand to approach, and cation-π interactions also played an important role helping heterotrimers fold. We also found that compared to N-methyl glycine (sarcrosine) , alanine caused a more destructive mutation to the collagen structure.

中文摘要 i
Abstract ii
謝誌 iii
第一章、緒論 1
1-1 膠原蛋白 1
1-1-1 膠原蛋白的結構 1
1-1-2 同源三股螺旋與異源三股螺旋 3
1-1-3 膠原蛋白中的作用力 4
1-1-4 脯胺酸與羥脯胺酸穩定膠原蛋白的作用 5
1-1-5 膠原蛋白模擬胜肽單一置換的穩定性 6
1-2 陽離子-π 作用力 ( Cation-π interactions ) 7
1-2-1 影響cation-π 作用力的因素 8
1-2-2 帶正電荷側鏈與芳香族側鏈之胺基酸 8
1-2-3 蛋白質中的cation-π 作用力 10
1-3 本實驗室過去的研究結果 12
1-3-1 第一個針對膠原蛋白中cation-π 作用力的研究 12
1-3-2 透過cation-π 作用力自組裝成高階結構 13
1-3-3 Cation-π 作用力應用於膠原蛋白異源三股螺旋中 14
1-3-4 碳端的cation-π 作用力對異源三股螺旋摺疊的影響 15
1-3-5 徑向與軸向cation-π作用力對膠原蛋白三股螺旋的影響 17
1-3-6 集中置換Arg、Tyr於胜肽兩端所造成之影響 18
1-4 研究動機 19
1-4-1 實驗設計及流程 20
1-4-2 設計雙股膠原蛋白模擬胜肽之原因及其參考來源 21
第二章、實驗部分 26
2-1 實驗儀器 26
2-2 實驗藥品 27
2-3 實驗步驟流程 28
2-4 固相胜肽合成法（Solid Phase Peptide Synthesis, SPPS） 29
2-4-1 酯化反應 ( Esterification ) /醯胺化反應 ( Amidation ) 32
2-4-2 去保護 ( Deprotection ) 33
2-4-3 活化 ( Activation ) 34
2-4-4 耦合 ( Coupling ) 35
2-4-5 切除 ( Cleavage ) 35
2-5 胜肽之合成 36
2-5-1 利用微波合成儀合成c1、c2*、c3 36
2-5-2 利用自動合成儀合成c1*、c2、c4 36
2-5-3 利用手動合成法合成c1*、c2*、c5、c6、c7 38
2-5-4 胜肽的切除與純化 40
2-5-5 雙硫鍵膠原蛋白模擬胜肽之合成 40
2-5-6 HPLC 和 MALDI – TOF 鑑定胜肽鏈之純度 41
2-6 圓二色光譜儀 41
2-7 圓二色光譜儀實驗 44
2-7-1 配製 pH 7.4 、 pH 5.0 的磷酸鹽緩衝溶液 44
2-7-2 樣品配製 44
2-7-3 Far-UV CD光譜 47
2-7-4 變溫CD光譜測量 47
2-7-5 摺疊速率測量 47
2-8 圓二色光譜數據之處理 48
2-8-1 變溫實驗數據之處理 48
2-8-2 動力學實驗數據之處理 49
2-9 1H, 15N-HSQC ( Heteronuclear single quantum coherence ) 50
2-9-1 樣品配製 51
2-10 差式掃描量熱儀 ( Differential Scanning Calorimetry, DSC ) 52
2-10-1 樣品配製與測量 53
2-11 熱力學實驗之數據處理 53
第三章、結果與討論 55
3-1 第一部分序列設計及鑑定 55
3-2 CD光譜量測和探討 56
3-2-1 Far-UV CD光譜 56
3-2-2 變溫實驗 57
3-2-3 胜肽摺疊速度 59
3-3 1H,15N-HSQC光譜 60
3-4 徑向與軸向cation-π作用力對此部分異源三股螺旋之影響 63
3-5 熱力學性質之探討 66
3-6 此部分胜肽序列設計之優缺點探討 67
3-7 第二部分序列設計及鑑定 68
3-8 CD光譜量測和探討 – 未摺疊胜肽 70
3-8-1 Far-UV CD光譜 70
3-8-2 變溫實驗 70
3-9 CD光譜量測和探討 – c5 / c5ss / c6 71
3-9-1 Far-UV CD光譜 71
3-9-2 變溫實驗 72
3-10 第三部分序列設計及鑑定 81
3-11 CD光譜圖探討 - c7 / c7ss部分 81
3-11-1 Far-UV CD光譜 81
3-11-2 變溫實驗 82
3-12 第二及第三部分胜肽序列設計之優缺點探討 89
第四章、結論 91
參考文獻 93
附錄 98

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