硫脯胺酸((2R)-4-thiaproline, Thp)為脯胺酸的衍生物，是以硫取代脯胺酸中吡咯烷環(pyrrolidine ring)的Cγ位而得的。在過去實驗室的研究已發現它有利於形成內環褶皺構形以及順式胜肽鍵，進而破壞PPII構形的穩定性。膠原蛋白是哺乳動物中含量最多的蛋白質，主要由(Xaa-Yaa-Gly)三聯體序列組成：其中Xaa通常為脯胺酸，Yaa通常為羥脯胺酸，二級結構上以PPII構形為主。因此在本研究中，我們將膠原蛋白模擬胜肽中的Xaa和Yaa替換為Thp並探討此替換下造成的構形及穩定性變化。研究中我們發現含有Thp的膠原蛋白肽比我們預期的更加穩定。這可能是由於環褶皺(ring pucker)在內環褶皺(endo)與外環褶皺(exo)相互轉換的容易性使得Thp的噻唑烷環(thiazolidine ring)具有高度柔韌性。此外，我們通過將Thp氧化為Thp’(N- Formyl-cysteine)及Thp’’(S,S- dioxide Thp)來製備衍生肽。我們的結果表明，Thp’在Xaa位置並不會顯著影響膠原蛋白的穩定性，但在Yaa位置確實會產生很大的影響。藉由分子建模的結果，我們認為這種現象可能是由於二面角的過度扭曲。最後，我們依Thp在Xaa位的高穩定性，另外製備了兩條置換多個Thp在Xaa的胜肽，並發現多個Thp仍會對胜肽的穩定造成一定的破壞。我們已經證明了置入Thp及其氧化衍生物對膠原蛋白摺疊熱力學和動力學的影響。實驗和模擬計算數據的結合使我們能夠對Thp、Thp’、Thp’’ 對膠原蛋白構形的影響獲得新的見解，並為膠原蛋白相關生物材料的設計提供有價值的信息。

(2R)-4-thiaproline (Thp) is a derivative of proline, replacing Cγ in the pyrrolidine ring with sulfur. It favors an endo ring pucker and a cis prolyl peptide bond destabilizing polyproline II conformation. Collagen, the most abundant protein in mammals, is mainly composed of (Xaa-Yaa-Gly) triplet sequence: where Xaa is often proline, and Yaa is frequently hydroxyproline. In this study, we substitute the Xaa and Yaa of the collagen peptides with Thp and examine the consequences of this replacement. Strikingly, we found that Thp-containing collagen peptides are more stable than we expected. It could be due to that the ease of the ring pucker interconversion makes the thiazolidine ring of Thp highly flexible. Moreover, we prepared the derivative peptides by oxidizing Thp to Thp’(N-Formyl-cysteine) or Thp’’(S,S-dioxide Thp). Our results showed that the oxidation of Thp at position Xaa does not significantly affect collagen stability, but that at position Yaa does impose a large impact. Based on the molecular modeling results, we suggested that this phenomenon could be due to the over-twist of dihedral angels. Finally, due to the high stability of Thp at position Xaa, we prepared two peptides that replaced multiple Thp at Xaa, and found that more than one Thp substitution destabilize the triple helices. We have demonstrated the consequences of incorporating Thp and its oxidative derivatives on the folding thermodynamics and kinetics of collagen triple helices. The combination of experimental and computational data allows us to gain new insights into the impact of Thp, Thp’, Thp’’ on collagen conformation and provides valuable information to the design of collagen related biomaterials.

中文摘要 I
Abstract II
目錄 III
圖索引 VI
表索引 IX
第一章、緒論 1
1-1膠原蛋白 1
1-1-1膠原蛋白結構 1
1-1-2膠原蛋白中的氫鍵作用力 2
1-2 PPII結構(Polyproline II helix) 3
1-2-1 PPII螺旋結構 3
1-2-2 PPII螺旋的二面角 4
1-2-3 PPII螺旋中的作用力 5
1-3脯胺酸(Pro)及其衍生物 6
1-3-1脯胺酸(Pro)的構形 6
1-3-2脯胺酸上的Cγ取代 6
1-3-3脯胺酸衍生物在膠原蛋白序列中的變化 9
1-4硫脯胺酸Thiaproline (Thp) 11
1-5本實驗室過去的研究成果 13
1-6研究動機及序列設計 14
第二章、實驗部分 15
2-1實驗儀器 15
2-2實驗藥品 16
2-3實驗步驟流程 17
2-4固相胜肽合成法 (Solid phase peptide synthesis, SPPS) 18
2-5胜肽合成 21
2-5-1利用手動合成法合成胜肽 21
2-5-2胜肽的純化與鑑定 22
2-5-3開環Thp (N-Formyl-cysteine)胜肽之合成 23
2-5-4 S,S-dioxide Thp胜肽之合成 23
2-6圓二色光譜儀 (Circular Dichroism Spectrometer, CD) 24
2-6-1儀器原理 24
2-6-2樣品配製 27
2-6-3光譜測量 27
2-6-4實驗數據處理 28
2-7差式掃描量熱儀 (Differential Scanning Calorimetry, DSC) 31
2-7-1儀器原理 31
2-7-2樣品配製 32
2-7-3實驗數據處理 32
2-8 Gaussian 16模擬計算方法與步驟 34
2-8-1研究系統以及計算方法 34
2-8-2研究步驟 34
2-9 Discovery Studio 模擬計算方法與步驟 35
第三章、實驗結果與討論 36
3-1 CD光譜測量與探討 37
3-1-1 Far-UV CD光譜 37
3-1-2變溫實驗光譜 39
3-1-3胜肽摺疊速率 42
3-2熱力學性質之探討 43
3-3脯胺酸衍生物Thp與Thp’’的構形 46
3-4脯胺酸衍生物Thp在膠原蛋白模擬胜肽中的構形變化 48
3-5多個Thp於膠原蛋白三股螺旋的影響 51
第四章、結論 54
參考文獻 56
附錄 63

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