膠原蛋白是由三條聚脯胺酸第二型結構(PPII)所形成之三股螺旋，可依照纏繞的胜肽種類分為AAA型同源三股螺旋及AAB、ABC型異源三股螺旋；天然的膠原蛋白一般為異源三股螺旋，人體中含量最多的蛋白質：第一型膠原蛋白即為AAB型異源三股螺旋。穩定三股螺旋結構的作用力有許多種，其中一種由正電荷與芳香環四極矩間的非共價鍵作用力：cation-π作用力，在我們過去的研究中也發現有重要的貢獻。
本研究以(POG)9為基底，將帶正電荷(Lys)及芳香環胺基酸(Phe)置於胜肽的碳端，合成胜肽CK3與CF3，藉由Lys-Phe間的 cation-π作用力來促進AAB型異源三股螺旋之形成，並與含Arg胺基酸之CR3/CF3系列比較，探討不同的帶正電胺基酸對異源三股螺旋穩定性的影響。由CD與DSC實驗結果發現CK3與CF3混合後可藉由cation-π作用力形成(CK3)2(CF3)1異源三股螺旋，且經2D-NMR證實Lys-Phe間cation-π作用力的存在。與CR3/CF3系列比較後發現：由於Arg側鏈的胍團較疏水，使得Arg-Phe間cation-π作用力較強，形成的AAB型異源三股螺旋也較穩定。
我們更進一步將CR3及CK3中間位置POG重複序列中的一個甘胺酸(Gly)置換成肌胺酸(Sar)，合成出胜肽CR3_Sar與CK3_Sar，同樣與CF3進行混合，探討將Gly置換成Sar後能否藉由cation-π作用力形成AAB型異源三股螺旋。實驗結果發現CR3_Sar/CF3系列仍能形成(CR3_Sar)3同源三股螺旋及(CR3_Sar)2(CF3)1異源三股螺旋，且經2D-NMR證實Arg-Phe間cation-π作用力的存在；而CK3_Sar/CF3系列則因Lys-Phe間cation-π作用力較弱，而無法形成穩定的三股螺旋。

Collagen is a right-handed triple helix, and each helix is a left-handed polyproline type II structure containing many (X-Y-Gly) repeats. Many natural collagens consist of two or three different peptide chains. Type I collagen, the most abundant protein in the human body, is an AAB-type heterotrimer. Many forces were shown to have great contributions to collagen triple helix stability. Cation-π interaction, one of the forces, was found to be important in our previous studies.
In this work, we prepared the collagen-mimetic peptides containing cationic or aromatic residues by incorporating Lys or Phe into the C-terminal end of the peptide chains. We attempted to use cation-π interactions to assist the folding of heterotrimers. Circular dichroism (CD), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR) measurements showed that the cation-π interactions could effectively induced the formation of AAB-type heterotrimers. Compared to the results of Arg-containing CR3/CF3 series, (CK3)2(CF3)1 heterotrimers were less stable due to the weaker cation-π interactions between Lys and Phe.
In addition, we prepared CR3_Sar and CK3_Sar in which a Gly residue near the center of CR3 and CK3 was substituted to sarcosine (Sar). Our results revealed that CR3_Sar could form homotrimers and AAB-type heterotrimers with CF3. The existence of cation-π interactions between chains has been confirmed by NMR measurements. Due to the weaker cation-π interactions between Lys and Phe, CK3_Sar, by contrast, could form neither homotrimers nor AAB-type heterotrimers with CF3.

序言....................................................................................................I
中文摘要.............................................................................................II
Abstract............................................................................................III
第一章、緒論...............................................................................................................1
1.1 膠原蛋白................................................................................................................1
1.1.1 膠原蛋白.............................................................................................................1
1.1.2 膠原蛋白結構......................................................................................................2
1.1.3 穩定膠原蛋白的作用力..........................................................................................2
1.1.4 異源三股螺旋(heterotrimers)................................................................................4
1.1.5 膠原蛋白中胺基酸的置換.......................................................................................5
1.2 Cation-π作用力(Cation-π interactions).................................................................7
1.2.1 影響cation-π作用力的因素..................................................................................8
1.2.3 苯環的四極矩(quadrupole moment).....................................................................9
1.2.4 Cation-π作用力在水溶液中的研究......................................................................10
1.3 生物系統中的cation-π作用力................................................................................12
1.3.1 帶電荷與芳香環側鏈的胺基酸..............................................................................12
1.3.2 胺基與芳香環間作用力........................................................................................14
1-4 研究動機.............................................................................................................17
第二章、實驗步驟.......................................................................................................18
2.1實驗儀器...............................................................................................................18
2.2 實驗藥品..............................................................................................................19
2.3 實驗流程..............................................................................................................21
2.4 固相胜肽合成法(Solid-phase peptide synthesis, SPPS)............................................22
2.4.1 酯化反應(Esterification)或醯胺化反應(Amidation)................................................24
2.4.2 去保護(Deprotection)........................................................................................24
2.4.3 活化(Activation)................................................................................................25
2.4.4 耦合(Coupling).................................................................................................26
2.4.5 切除(Cleavage).................................................................................................26
2.5 圓二色光譜儀(Circular dichroism spectroscopy, CD)..............................................27
2.6 Fmoc-Pro-Hyp-Gly-OH之合成.............................................................................31
2.6.1 Boc-(2S,4R)-hydroxyproline (1)之合成..............................................................31
2.6.2 Boc-Hyp-Gly-OBn (2)之合成.............................................................................32
2.6.3 Fmoc-Pro-Hyp-Gly-OBn (3)之合成...................................................................32
2.6.4 Fmoc-Pro-Hyp-Gly-OH (4)之合成.....................................................................33
2.7 胜肽之合成、純化與鑑定........................................................................................34
2.7.1 胜肽合成...........................................................................................................35
2.7.2 胜肽純化與鑑定.................................................................................................35
2.8 CD光譜實驗.........................................................................................................36
2.8.1 far-UV CD光譜 (Wavelength scans)...................................................................36
2.8.2 變溫CD光譜量測 (Thermal denaturation)...........................................................37
2.8.3 測量摺疊速率 (Refolding kinetics).....................................................................37
2.8.4 變溫實驗資料處理.............................................................................................37
2.8.5 動力學實驗資料處理..........................................................................................39
2.9 摺疊熱力學之測量(DSC)........................................................................................39
2.9.1 熱力學實驗資料處理..........................................................................................40
2.10 2D-NMR光譜測量..............................................................................................41
2.10.1 1H, 15N-HSQC光譜........................................................................................41
2.10.2 1H, 1H-TOCSY和1H, 1H-NOESY光譜...............................................................42
第三章、結果與討論....................................................................................................43
第一部分 (POG)6(PYG)3系列胜肽之探討........................................................................43
3.1 胜肽序列之設計....................................................................................................43
3.2 CD光譜探討.........................................................................................................44
3.2.1 Far-UV CD光譜.................................................................................................44
3.2.2 變溫實驗...........................................................................................................45
3.3 由2D-NMR驗證異源三股螺旋的形成.......................................................................47
3.3.1 1H,15N-HSQC光譜...........................................................................................47
3.3.2 1H,1H-TOCSY及1H,1H-NOESY光譜..................................................................50
3.4 熱力學性質之探討.................................................................................................51
3.5 胜肽摺疊速率.......................................................................................................53
3.6 三股螺旋在不同離子強度與pH值下穩定性之變化.......................................................55
3.6.1 離子強度對三股螺旋穩定性之影響........................................................................55
3.6.2 pH值對三股螺旋穩定性之影響.............................................................................56
第二部分 (POG)6(PYG)3系列胜肽中Sar置換之探討..........................................................59
3.7 置換位置之設計....................................................................................................59
3.8 CD光譜探討.........................................................................................................59
3.8.1 Far-UV CD光譜.................................................................................................59
3.8.2 變溫實驗...........................................................................................................60
3.9由2D-NMR驗證異源三股螺旋的形成........................................................................63
3.9.1 1H,15N-HSQC光譜...........................................................................................64
3.9.2 1H,1H-TOCSY及1H,1H-NOESY光譜...................................................................66
3.10 熱力學性質之探討...............................................................................................67
3.11 胜肽摺疊速率.....................................................................................................69
第四章、結論.............................................................................................................71
參考文獻....................................................................................................................74
附錄..........................................................................................................................79

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