擁擠效應是體內細胞質主要能穩定蛋白質的原因，經由在溶液中的排除體積效應，使蛋白質在擁擠環境中能變得更加穩定。本研究中以兩種擁擠試劑Ficoll 70和Dextran 70做為體外模擬擁擠環境的高分子聚醣類，以HP36代表α螺旋；WW-domain代表β摺疊；膠原蛋白代表三股螺旋，分別觀察三種不同結構的胜肽在兩種擁擠環境中的變化，利用CD光譜量測在擁擠環境下結構的強度與藉由熱變性與化學變性的實驗結果來探討其穩定性的變化。
在大部分的體外擁擠環境模擬中，擁擠試劑通常為高分子，其穩定度的結果會因為Flory–Huggins solution effect造成穩定程度呈現階梯狀的上升，並且擁擠效應內擁擠試劑所具有的軟效應在排除體積效應較不明顯時，時常主導了整個環境，導致擁擠效應的結果在擁擠試劑濃度較低時反而使胜肽變得更加不穩定。直到擁擠試劑的濃度上升至ㄧ定比例，排除體積效應才逐漸與軟效應相互抵消，且溶液中的胜肽結構趨向穩定。在化學變性的過程中，觀察到胜肽結構在解摺疊狀態的壓縮，代表著在擁擠試劑與化學變性試劑兩者的交互作用下，將會使胜肽的解摺疊結構改變，隨著擁擠試劑濃度上升至30% 時，摺疊態與解摺疊態間的能階差將會下降。最後將擁擠試劑的形狀與胜肽的構形作為探討目標，發現在溶液中形狀相似、大小接近的分子將會導致更強且更平滑的排除體積效應。
藉由實驗結果歸納出以大分子模擬擁擠環境的過程中所會面對到的困難，提供出ㄧ個更為完備的方式去模擬擁擠環境，在未來選用擁擠試劑與決定溶液中擁擠試劑的重量百分濃度時，將可以針對其溶液中的作用力做出更全面的考量，期望此結果可對於蛋白質舒適的擁擠環境所具有的作用力引發更多的探討，進而更加了解蛋白質結構在擁擠環境中對於環境的容忍力提升的原因，對於穩定蛋白質提供另一種簡單且不具破壞性的途徑。

The proteins in cells can be stabilized by the macromolecular crowding effects in cytoplasm due to excluded volumn effects. Here we used two different carbohydrate polymers, semi-rigid spherical Ficoll 70 and rod-like Dextran 70, to mimic the enviroment in cytoplasm. In our study, three different peptides, HP36 for an α-helix; WW-domain for a β-sheet; collagen for a triple helix, were chosen to study the crowding effects on their conformational stability in vitro.
Using polymers to be a crowder will have Flory–Huggins solution effect, and thus the peptide will be significantly stabilized as the concentration of crowders increases to approximately 15%. In this case, the crowding effect is not obvious at first and the soft effect may actually destabilize the peptide at low concentrations of crowders. After increasing the concentration of crowders, the crowding effect can gradually compensate the destabilization due to the soft effect and make the peptide more stable. In chemical denaturation, the interactions including crowders and denaturants are involved in the unfolded state, which will make the unfolded state more compact. As the crowder concentration increases up to 30%, the free energy gap between the folded state and the unfolded state will decrease. Furthermore, we will discuss the relationship between peptide conformations and crowder shapes. Our finding indicates that the crowding effect will be more significant if the shape and size of crowders are similar to those of peptides.
To sum up the results, our study attempted to provide a more accurate method to mimic a crowding enviroment. Hopefully, the results can initiate more attention to the interactions between solvents and proteins, and provide a simple physical way to stabilize protein.

目錄
摘要 I
Abstract II
謝誌 III
目錄 IV
圖目錄 VII
表目錄 VIIII
第一章、緒論 1
1.1 雞絨毛蛋白 (Villin) headpiece, HP36 1
1.2 Human Pin1 WW-Domain 3
1.3 膠原蛋白 Collagen 4
1.4 擁擠效應(Crowding Effect) 6
1.4.1 排除體積效應 7
1.4.2 聚蔗糖(Ficoll)與葡聚醣(Dextran) 9
1.4.3 擁擠試劑所具有的軟效應(soft effect) 9
1.5 弗洛里－哈金斯溶液理論(Flory-Huggins solution theory)12
1.6 固相胜肽合成法(Solid Phase Peptide Synthesis) 14
1.6.1 酯化（esterification） /醯胺化反應(amidation) 15
1.6.2 去保護 (deprotection) 15
1.6.3 活化 (activation) 16
1.6.4 耦合 (coupling) 16
1.6.5 切除 (cleavage) 17
1.7 圓二色光譜儀(Circular Dichroism, CD) 18
1.8 研究動機 21
第二章、實驗步驟 22
2.1 實驗步驟 22
2.2 實驗儀器 23
2.3 實驗藥品 24
2.4 胜肽之合成、純化與鑑定 26
2.4.1 固相胜肽合成法 26
2.4.2 純化與鑑定 27
2.5 光譜測量 28
2.5.1 胜肽濃度測定 28
2.5.2 CD光譜測量 28
2.6 變性實驗之數據分析 31
2.6.1 熱變性曲線分析 31
2.6.2 化學變性曲線分析 33
第三章、結果與討論 36
3.1 擁擠效應對HP36上α-helix的影響 36
3.1.1 擁擠效應對於HP36結構的影響 36
3.1.2 擁擠效應對於HP-36熱誘導變性的影響 38
3.1.3 擁擠效應對於HP-36化學誘導變性的影響 41
3.2 擁擠效應對WW-domain上β-sheet的影響 43
3.2.1 擁擠效應對於WW-domain結構的影響 43
3.2.2 擁擠效應對於WW-domain熱誘導變性的影響 45
3.2.3 擁擠效應對於WW-domain化學誘導變性的影響 48
3.3 擁擠效應對不同長短的膠原蛋白三股螺旋的影響 50
3.3.1 擁擠效應對於膠原蛋白三股螺旋結構的影響 50
3.3.2 擁擠效應對於膠原蛋白三股螺旋熱誘導變性的影響52
3.4 討論 55
第四章、結論 57
參考文獻 58

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