膠原蛋白為動物體含量最多的蛋白質，主要分布於骨骼、皮膚與細胞組織中，其基底結構為三條具備聚脯胺酸第二型結構( PPII )的胜肽鏈以右旋方式纏繞而成的三股螺旋，其中每條胜肽鏈再以Xaa-Yaa-Gly之方式組成。穩定三股螺旋結構的作用力於許多文獻皆曾探討，其中靜電交互作用也不乏被設計於序列中以增強結構的穩定效果，因此本研究探討此作用力對三股螺旋結構穩定性之影響，並針對末端點靜電作用力對結構影響進行一系列的討論。
研究實驗中，我們以Gly-Gly-(Pro-Hyp-Gly)7-Gly-Gly作為原生型胜肽，並將末端的胺基酸分別置換為Lys與Glu以分析靜電交互作用對結構的影響。我們發現當碳端Gly置換為Lys時，藉由其側鏈上帶正電荷之胺基與末端羧基所產生的靜電吸引力可使結構提升穩定性，相反地，氮端Glu的置換則較無顯著的影響；當Lys與Glu分別置換在氮端以及碳端時，則會因為電荷間的排斥力效應使得結構穩定性下降。此外，我們也將胜肽溶入高濃度的NaCl溶液中，藉由離子的遮蔽效應再次證實靜電交互作用力的存在。
接著，我們進一步將帶電荷胺基酸之側鏈長度縮短，以觀察側鏈長度對於作用力大小的影響。實驗結果發現僅有同區域存在羧基之胜肽產生靜電排斥現象，其餘胜肽皆無此效應，因此我們推測當側鏈縮短時，作用距離存在限制性，因而帶電荷官能基的作用範圍成為重要的影響因子。

Collagen, the primary protein in mammals, is abundant among bones, skins and connective tissues. The single strand of native collagen adopts the polyproline II ( PPII ) structure and consists of the repeating tripeptide Xaa-Yaa-Gly. Three PPII strands then form the collagen basic structure, generally known as triple helices. From the previous studies, various interactions have been utilized to improve the stability of a collagen triple helix. Here we focused on the study of electrostatic interactions on collagen triple helices.
In this study, we explored the contribution of terminal charge charge interactions to the triple helix stability. We designed Gly Gly-(Pro-Hyp-Gly)7-Gly-Gly as a parent peptide and incorporated Lys or Glu into the terminus to examine the effects of terminal electrostatic interactions on the triple helix stability. We found that incorporating Lys into the C-terminus of a peptide generates the electrostatic attractions between its side chain and the terminal carboxylic group to stabilize the triple helix. While installing Glu into the N-terminus of a peptide only introduces a relatively small stabilization effect. In contrast, incorporating Lys into the N-terminus or Glu to the C-terminus destabilizes the triple helix because of the charge-charge repulsions. Such effects were further confirmed by the experiments with concentrated salts present, which the electrostatic interactions are shielded by the salts in solution.
In addition, we also analyzed the relationship between the length of the side chains and the electrostatic interactions. Surprisingly, the results revealed that only Asp induced larger charge-charge repulsions to further destabilize the triple helix. The results suggest that the size of the charged functional groups may play a critical role affecting the interactions.

中文摘要 I
Abstract II
謝誌 III
目錄 IV
圖目錄 VII
表目錄 X
第一章 緒論 1
1.1 蛋白質結構簡介 1
1.2 膠原蛋白 1
1.2.1 膠原蛋白結構 2
1.2.2 膠原蛋白作用力 3
1.2.3 同源三股螺旋( Homotrimer ) 4
1.3 分子間的交互作用 4
1.3.1 共價鍵作用力 5
1.3.2 非共價鍵作用力 5
1.3.3 靜電交互作用( Electrostatic interaction ) 6
1.4 研究動機 9
第二章 實驗部分 10
2.1 實驗儀器 10
2.2 實驗藥品 11
2.3 固相胜肽合成法( Solid-phase peptide synthesis, SPPS )24 13
2.3.1 酯化反應( Esterrification )或醯胺化反應( Amidation ) 16
2.3.2 去保護( Deprotection ) 16
2.3.3 活化( Activation ) 17
2.3.4 耦合( Coupling ) 17
2.3.5 切除( Cleavage ) 17
2.4 圓二色光譜儀 ( Circular Dichroism Spectroscopy, CD )25 18
2.5 微量差式掃描量熱儀( Differential Scanning Calorimetry, DSC ) 21
2.6 Fmoc-Pro-Hyp-Gly-OH之合成 22
2.6.1 Boc-(2S,4R)-Hyp-OH之合成 22
2.6.2 Boc-Hyp-Gly-OBn之合成 23
2.6.3 Fmoc- Pro-Hyp-Gly- OBn之合成 23
2.6.4 Fmoc-Pro-Hyp-Gly-OH之合成 24
2.7 胜肽的合成、純化與鑑定 25
2.7.1 預置胺基酸樹脂 26
2.7.2 自動胜肽合成儀 27
2.7.3 微波胜肽合成儀 27
2.7.4 胜肽的切除、純化與鑑定 28
2.8 緩衝溶液配製 28
2.8.1 PB buffer ( pH 7.0 ) 28
2.8.2 CH3COOH ( pH 3.0 ) 28
2.8.3 PB buffer ( pH 11.0 ) 29
2.8.4 NaCl(aq) ( pH 7.0 ) 29
2.9 圓二色光譜實驗 29
2.9.1 Far-UV CD光譜( Wavelength scan ) 29
2.9.2 變溫CD光譜( Thermal denaturation ) 29
2.9.3 微示差掃描熱卡分析儀 30
2.9.4 變溫CD實驗數據處理 30
2.10 熱力學實驗數據處理 31
第三章 結果與討論 33
第一部分 GG(POG)7GG序列衍生物之探討 33
3.1 胜肽序列之設計 33
3.2 CD光譜探討 33
3.3 碳端置換GG(POG)7GX之胜肽探討 34
3.4 氮端置換YG(POG)7GG之胜肽探討 38
3.5 碳端與氮端同時置換YG(POG)7GX之胜肽探討 41
3.6 NaCl離子溶液對胜肽穩定度之探討 43
3.7 pH值變化與胜肽結構穩定性之探討 45
第二部分 胺基酸側鏈長度與靜電作用力之影響探討 52
3.1 胜肽序列之設計 52
3.2 Far-UV CD光譜 52
3.3 變溫CD光譜 53
3.4 碳端與氮端置換之胜肽側鏈作用力探討 53
3.5 pH值變化與胜肽結構穩定性之探討 55
3.6 熱力學性質之探討 57
第四章 結論 58
參考文獻 60
附錄 63

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