膠原蛋白是人體內含量最豐富的蛋白，其之所以被廣泛用於生醫材料上是因為它對於人體的相容性佳，應用於人體醫學可在體內被酵素自然分解吸收，且無毒性。在生醫應用方面，分子大小過長的模擬膠原蛋白胜肽不僅製作成本較高，且其結構複雜而難以掌控，因此在研究上我們首要目標便是選用胜肽鏈越短越好，效果卻不變的模擬膠原蛋白胜肽鏈來研究。膠原蛋白是由三條多胜肽鏈所組成的，而此三條多胜肽鏈相互平行沿著同一方向軸相互纏繞成右手螺旋的三股螺旋結構，彼此利用氫鍵緊密地結合。三條螺旋結構的組成方式不僅有同源三股螺旋，也有異源三股螺旋。異源三股螺旋可細分為兩條α1和一條α2所組成的AAB型以及三條皆不同的ABC型。第一型膠原蛋白是人體內含量最多的膠原蛋白屬於AAB型，因此對於異源三股螺旋結構的探討，可以幫助我們更了解天然膠原蛋白，而本論文主要即為探討關於膠原蛋白異源三股螺旋結構的形成及其穩定性。膠原蛋白是由序列中包含三種主要的天然胺基酸重複排列而成的，取代其中幾個胺基酸可以大大的改變其特性。膠原蛋白本身可以進行自組裝，利用在序列中置換含有苯環結構以及帶有正電荷官能基的胺基酸，能使其產生陽離子−π作用力增加結構的穩定性。經由設計過的序列可以誘導異源三股螺旋的形成，再利用核磁共振儀分析其結構，並利用圓二色光譜儀進行動力學的實驗、示差掃描熱量分析儀進行熱力學實驗。
我們利用含有陽離子（精胺酸，賴胺酸）或芳香族（苯丙胺酸，酪胺酸）官能基的胺基酸來建立陽離子−π作用力。我們設計了三個異源三股螺旋系統，第一部分是將陽離子−π作用力平均分散在目標序列中，第二部分是將陽離子−π作用力集中於序列的碳端，第三個系統則是綜合了前兩者再加以設計。在第一個系統中，我們成功的使陽離子−π相互作用誘導AAB型異源三股螺旋的形成，並且通過控制陽離子和芳香肽在溶液中的混合比例，可以獲得不同類型的異源三聚體；熱力學的分析顯示，這些異源三股螺旋的摺疊是焓效應所主導的。在第二個系統中，光譜結果顯示在精胺酸（CR）-苯丙胺酸（CF）和賴胺酸（CK）-苯丙胺酸（CF）混合物中均可形成AAB型異源三股螺旋，顯示碳端陽離子−π相互作用力可以促進異源三股螺旋的摺疊；特別的是，在每種混合物中我們都只發現單一為主的異源三股螺旋。對於CR-CF混合物，取決於溶液中CR與CF的莫耳濃度比，我們可以製備具有兩條CR鏈和一條CF鏈的異源三股螺旋或是具有一條CR鏈和兩條CF鏈的異源三股螺旋，而CK-CF混合後則可形成兩條CK鏈和一條CF鏈的異源三股螺旋。此外，差示掃描量熱分析顯示這些異源三股螺旋的摺疊是受到熵效應所主導。而在第三個系統中，我們成功利用陽離子−π相互作用誘導ABC型異源三股螺旋的產生，並且佐以核磁共振光譜證明之。本論文成功的利用陽離子−π作用力誘導膠原蛋白模擬胜肽形成異源三股螺旋，並對它們的摺疊熱力學提供新的見解。

Collagen is the most predominant component of the extracellular matrix. Exploring the forces to assemble synthetic collagen mimetic peptides (CMPs) into trimers has been an attractive topic in preparing collagen-related biomaterials. Natural collagens consist of all identical (AAA, homotrimer), two different (AAB, heterotrimer), or three different (ABC, heterotrimer) peptide chains. Many natural collagens are either AAB- or ABC-type heterotrimers, making heterotrimeric helices better mimics for studying such collagen structures in nature. We prepared CMPs containing cationic (Arg, Lys) or aromatic (Phe, Tyr) residues to explore the folding of collagen heterotrimers via cation−π interactions. We designed three systems of CMPs, the first two systems focus on generating AAB-type heterotrimers by interspersed cation−π interactions and C-terminal multiple cation−π interactions. The third system shows how ABC-type heterotrimer was brought out by cation−π interactions.
In the first system, circular dichroism (CD), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR) measurements showed that the interchain cation−π interactions between cationic and aromatic peptides could induce AAB-type heterotrimer formation. By controlling the mixing molar ratios of cationic and aromatic peptides in solution, we could obtain the heterotrimers with various compositions. The thermodynamic results revealed that the folding of such heterotrimers is an enthalpy-driven process, which directly showed the contribution of cation−π interactions to the folding of an AAB-type heterotrimer. In the second system, CD and NMR spectroscopy showed that heterotrimers (CR-CF) and (CK-CF) could be induced by the C-terminal multiple pairwise Arg-Phe and Lys-Phe interactions, suggesting that the C-terminal cation−π interactions between cationic and aromatic residues could serve as a nucleation force and substantially promote the folding of heterotrimers. In particular, only one major heterotrimeric fold was found in this system. For CR−CF mixtures, either the heterotrimer with two CR chains and one CF chain or that with one CR chain and two CF chains could form, depending on the molar ratios of CR to CF in solution. By contrast, in CK−CF mixtures only the heterotrimer consisting of two CK chains and one CF chain was found in solution even increasing the ratio of CF, implying that the heterotrimer composed of one CK chain and two CF chains is highly unstable. Additionally, DSC analysis showed that the folding of these heterotrimers is governed by entropic effects. In the third system, we used NMR to identify the composition of different heterotrimers by inlaying different numbers of 15N-isotopically enriched glycine in each peptide. The formation of a cation−π interaction induced ABC-type heterotrimer, which the interacting pairs were concentrated at both the N- and C-termini, was confirmed by NMR.
Together, we have demonstrated the effectiveness of cation−π interactions as a force to fold collagen heterotrimers and revealed new insights into their folding thermodynamics. We also provide a new level of structural details that were not achieved for ABC-type collagen heterotrimers before.

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