利用陽離子-π作用力和雙硫鍵誘導膠原蛋白異源三股螺旋摺疊之探討

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研究生：	陳怡礽 Chen, Yi-Reng
論文名稱：	利用陽離子-π作用力和雙硫鍵誘導膠原蛋白異源三股螺旋摺疊之探討 Using cation-π interactions and disulfide bonds to induce the folding of collagen heterotrimers
指導教授：	洪嘉呈 Horng, Jia-Cherng
口試委員:	朱立岡 Chu, Li-Kang 杜玲嫻 Tu, Ling-Hsien
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2021
畢業學年度：	109
語文別：	中文
論文頁數：	109
中文關鍵詞：	膠原蛋白、異源三股螺旋、陽離子-π 、雙硫鍵
外文關鍵詞：	collagen, heterotrimers, cation-π, disulfide
相關次數：	點閱：3 下載：0
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膠原蛋白是細胞外基質的主要成分，其參與組織的再生，並且有良好的生物相容性，可應用在藥物傳遞或是生醫材料。在最近的發展中，它可作為染色病理組織的肽探針，為涉及膠原的疾病提供了方便的工具。膠原蛋白結構主要是有三股左手螺旋的PPII沿著同一軸互相纏繞成一條三股螺旋，依照組成分為AAA 型的同源三股螺旋及AAB或ABC型的異源三股螺旋。倘若膠原蛋白的二級結構摺疊錯亂可能造成疾病，因此研究膠原蛋白股與股之間的作用力是相當重要的。

我們的研究主要是以(Pro-Hyp-Gly) 9為基底，將序列中的胺基酸以Arg及Tyr及Pro取代，利用陽離子-π作用力誘導異源三聚體的摺疊。圓二色光譜儀(CD)測量證實，由陽離子殘基和芳香族殘基的側鏈形成的陽離子-π相互作用可以幫助三聚體摺疊。第一部分探討(POG)n可以看作(X–Y-Gly)n，當陽離子殘基在Y位置，芳香族殘基在X位置時，可以通過陽離子-π相互作用形成三股螺旋。反之，如果相對的陽離子-芳香族位置互換則三聚體無法形成，實驗結果證實胺基酸在序列中的位置對形成膠原三螺旋中有著至關重要的影響。第二部分探討當Hyp替換為Pro是否影響膠原蛋白整體穩定性，由CD與差示掃描量熱儀(DSC)結果得知異源三股螺旋仍然可以形成但相較於未置換前有較低的穩定性，並經由異核單量子相關實驗(HSQC)進一步確認三聚體組成。第三部分為了進一步加強膠原蛋白鏈之間的相互作用力，我們在胜肽的碳末端添加了Cys，誘導其形成雙硫鍵的二聚體。結果證實雙硫鍵連接的二聚體可以促進穩定異源三股螺旋的形成，並捕獲變性膠原蛋白胜肽。

Collagen is the main component of extracellular matrix. It participates in tissue regeneration and has high biocompatibility. It can be used in drug delivery or biomedical materials. In a recent development, it was shown to serve as a peptide probe to stain pathological tissues, providing a convenient tool for detecting collagen-involved diseases. Collagen structure is mainly composed of three left-handed helices of PPII wound into a triple helix, which is classified into AAA-type homotrimers and AAB-type or ABC-type heterotrimers according to the composition. If the folding of collagen disorder, it may cause disease.Thus, it is very important to study the force between the collagen strands.
Our research mainly uses (Pro-Hyp-Gly)9 as the parent peptide, and replaces the amino acids with Arg, Tyr, and Pro in the sequence to induce the folding of heretotrimers via cation-πinteractions. Circular dichroism measurements showed the cation-π interactions formed by the side chains of cationic residues and aromatic residues can assist the heterotrimeric folding. The results show that when the cationic residue is at the Y position and the aromatic residue is at the X position, a heterotrimer can be formed through the cation-π interactions. In contrast, if the relative cationic-aromatic positions are reversed, the trimer cannot be formed, indicating the position in sequence plays a vital role on forming a collagen triple helix. When Hyp is replaced by Pro, the heterotrimers can still form but with lower stability. To further strengthen the interaction between the collagen strands, we also added Cys into the C-terminus of the peptide to form disulfide-linked dimers as a strategy to fold heterotrimers. The results show that the disulfide-linked dimers can promote the formation of a stable heterotrimer.

中文摘要    I
Abstract    II
誌謝辭    III
目錄    IV
圖目錄    VIII
表目錄    XIV
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 膠原蛋白序列中置換胺基酸的穩定性    5
1-1-3蛋白質中常見的作用力比較    6
1-2 陽離子-π作用力(Cation-π interaction)    7
1-2-1影響cation-π作用力的因素    8
1-2-2苯環上取代基對 cation-π作用力影響    9
1-2-3 胺基酸型cation-π作用的幾何結構    10
1-2-3正電荷胺基酸    12
1-2-4 芳香族胺基酸    12
1-2-5  Cation-π在生物體中的應用    14
1-3 Disulfide-bond    15
1-3-1 Cysteine    15
1-3-2形成雙硫鍵的條件    15
1-3-3氧化還原    16
1-3-4 Cys的置換位置及取代對膠原蛋白穩定性之影響    18
1-4 徑向(lateral)與軸向(axial)作用力    19
1-5 研究動機    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實驗流程    30
2-4 Fmoc-Pro-Hyp-Gly-OH之合成    31
2-5固相胜肽合成法 (Solid phase peptide synthesis, SPPS)    33
2-5-1酯化反應 (Esterification)/醯胺化反應 (Amidation)    35
2-5-2去保護 (Deprotection)    35
2-5-3 活化 (Activation)    36
2-5-4耦合 (Coupling)    36
2-5-5切除 (Cleavage)    36
2-6膠原蛋白模擬胜肽之合成、純化與鑑定    37
2-6-1 利用自動合成儀合成胜肽    38
2-6-2利用微波合成儀合成胜肽    38
2-6-3切除胜肽上樹脂    39
2-6-4高效能液相層析儀 (HPLC) 純化胜肽    39
2-6-5 利用HPLC 和 MALDI-TOF 鑑定胜肽純度、分子量    39
2-7圓二色光譜儀 (Circular dichroism spectrometer, CD)    41
2-8 1H,15N-HSQC (Heteronuclear single quantum coherence)    45
2-9差示掃描量熱儀 (Differential Scanning Calorimetry, DSC)    45
2-10光譜量測    46
2-10-1 UV光譜濃度量測    46
2-10-2 CD光譜量測    46
2-10-3 HSQC 光譜量測    47
2-10-4 DSC 光譜量測    47
2-11光譜數據分析    47
2-11-1 CD變溫實驗之數據處理    47
2-11-2 DSC熱力學實驗之數據分析    49
第三章、結果與討論    50
第一部分 陽離子與芳香環相對位置互換之胜肽探討    50
3-1 胜肽序列之設計    50
3-2 CD光譜探討    52
3-2-1 Far-UV CD 光譜    52
3-2-2 CD變溫實驗    53
第二部分Hyp置換為Pro對異源三股螺旋穩定性之探討    56
3-3 胜肽序列之設計    56
3-4 CD光譜探討    57
3-4-1 B1/A4 Far-UV CD 光譜    57
3-4-2 B1/A4 CD變溫實驗    59
3-4-3 B2/A3 Far-UV CD 光譜    61
3-4-4 B2/A3 CD變溫實驗    62
3-4-5 A3/A4 Far-UV CD 光譜    64
3-4-6 A3/A4 CD變溫實驗    65
3-5 1H, 15N-HSQC 2D-NMR 圖譜探討    68
3-5-1 B1*/A4* 1H, 15N-HSQC 光譜    68
3-5-2 B2*/A3* 1H, 15N-HSQC 光譜    71
3-5-3 A3*/A4* 1H, 15N-HSQC 光譜    73
3-6熱力學性質之探討    76
3-6-1 B1/A4 DSC光譜圖    76
3-6-2 B2/A3 DSC光譜圖    78
第三部分 雙硫鍵對胜肽熱穩定性影響之探討    82
3-7胜肽序列之設計    82
3-8 CD光譜探討    83
3-8-1 B1/A5/A5SS Far-UV CD 光譜    83
3-8-2 B1/A5/A5SS CD變溫實驗    84
3-8-3 A2/A6/A6SS Far-UV CD 光譜    86
3-8-4 A2/A6/A6SS CD變溫實驗    87
3-8-5 A5SS/A7 Far-UV CD 光譜    89
3-8-6 A5SS/A7 CD變溫實驗    90
3-8-7 A6SS/A7 Far-UV CD 光譜    91
3-8-8 A6SS/A7 CD變溫實驗    92
第四章、結論    94
參考文獻    96
附錄    101


                                

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