臺灣博碩士論文加值系統

蛋白質折疊是生命科學領域中一個基本且重要的問題。台灣眼鏡蛇毒蛋白分子三（CTX III）是一個全部由β-摺板結構組成的小蛋白質，共有60個胺基酸，五個β-摺板。本論文對這個蛋白質分子做了16條的再折疊的分子模擬動力學模擬軌跡，並且試著由這些模擬軌跡來探討這個CTX III蛋白質再折疊的路徑。模擬結果發現，在16條模擬軌跡中，有5條成功地折成NMR類似結構，在大部分的模擬軌跡中，β345都會先形成，β12由於屬於擾動性較大的結構，較不易形成。經由構形簇分析得到的過渡態、中間態結構中氫氫對的距離與實驗上由NOE訊號推得之CTX III部份展開結構有良好的一致性；此外，NMR結構的氫鍵中氫的保護係數與再折疊軌跡中的氫鍵的形成比率在定性上亦有不錯的相關度；在此我們亦描述了一條成功軌跡的再折疊路徑。根據模擬結果，本論文建議，雙硫鍵對折疊核心結構具有重要的穩定性，折疊核心之疏水效應可能具有引導β12之氫鍵形成的效果。另外，我們亦將CTX III之β34股單獨切下做β-髮夾的再折疊模擬並探討其再折疊路徑。歸納10條模擬軌跡發現，同一種序列之β-髮夾可能循兩種不同的折疊路徑折疊，這兩種再折疊路徑分別為上拉鍊模型以及下拉鍊模型。模擬結果指出，前者是因為第一對位於轉彎結構附近形成的氫鍵所引導；後者是位於尾部的幾對氫鍵一起形成所致，顯示這些氫鍵形成的合作效應。我們報告討論這兩種折疊路徑的特徵以及氫鍵形成順序的異同。

Protein folding is one of the most fundamental and important problems in life science. Taiwan Cobra Cardiotoxin III（CTX III）is a small all-β-sheet protein of 60 amino acids and 5 β-sheets. Molecular dynamics simulations of CTX III were carried out to examine the refolding pathway(s) of this protein. In our simulations, 5 of 16 trajectories successfully refolded into NMR-like structure. Experimentally, it was observed thatβ-sheet of strand III, IV and V are formed earlier than β-sheet of strand I and II. The simulations gave the same result. From the refolding trajectories, transition states and intermediate states were identified using a conformational-cluster analysis. The calculated transition state structure showed good consistency with the experimental partially unfolded structure based on NOE data. It also noted that some calculated H-bond formation percentage correlate qualitatively well with experimental H-bond donor protection factors. The result of a refolding trajectory, which folded close to NMR structure, suggests that the disulfide bonds play an important role in CTX III folding, and the hydrophobic effect in head region may guide the hydrogen bond formation of β-strand I and II in CTX III. In addition to CTX III simulations, we cut residue 20-39 from CTX III and simulate 10 trajectories for this peptide to examine if this peptide form a β-hairpin. The simulations show that the H-bonds between two strands can form either in zip-up or zip-down direction. Formation of H-bonds showed cooperative effect. We report and discuss the physical basis of these simulated results.

目錄
第一章、緒論………………………………………...1-1
1-1、 台灣眼鏡蛇心臟毒蛋白III ( CTX III )簡介…………………………. 1-1
1-2、 β-髮夾的折疊問題與折疊模型介紹……………………………….. 1-3
1-2.1、 β-髮夾的折疊問題………………………………………………1-3
1-2.2、 β-hairpin的折疊模型…………………………………………..1-4
1-3、 蛋白質折疊問題與折疊理論簡述……………………………………1-5
1-3.1、 蛋白質折疊問題………………………………………………….1-5
1-3.2、 影響蛋白質的作用力…………………………………………….1-6
1-3.3、 蛋白質折疊模型………………………………………………….1-7
1-4、 利用分子動力學模擬方法輔助了解蛋白質折疊問題……………..1-10
1-5、 本論文的研究目標…………………………………………………..1-11
第二章、方法………………………………………..2-1
2-1、 模擬方法………………………………………………………………2-1
2-1.1、 升溫程序………………………………………………………….2-2
2-1.2、 GB/SA內涵水合模型……………………………………………2-3
2-1、 分析方法………………………………………………………………2-7
2-2.1、 氫鍵分析………………………………………………………….2-7
2-2.2、 RMSD分析………………………………………………………2-7
2-2.3、 構形簇分析……………………………………………………….2-8
2-2.4、 SASA值分析…………………………………………………….2-9
第三章、結果與討論………………………………..3-1
第一部份、 CTX III再折疊的分子動力學模擬…………………...3-2
3-1、 CTX III再折疊的模擬結果…………………………………………..3-2
3-2、 CTX III再折疊模擬軌跡的RMSD值分析與氫鍵分析……………3-4
3-2.1、 CTX III再折疊模擬軌跡的RMSD值分析…………………….3-4
3-2.2、 CTX III起始結構RMSD值對折疊軌跡的影響………………3-21
3-2.3、 再折疊軌跡的氫鍵分析與NMR結果比較……………………3-22
3-3、 CTX III再折疊所經過的過渡態、中間態與折疊態……………….3-35
3-3.1、 過渡態與中間態( Transition state and intermediate state )..3-35
3-3.2、 CTX III再折疊模擬的折疊態 ( Folded state )……………….3-43
3-4、 CTX III再折疊模擬的疏水效應與靜電作用力分析……………….3-46
3-4.1、 疏水效應與靜電作用力分析…………………………………...3-46
3-4.2、 Asn 55的SASA值降低與β12形成的關係………………...3-56
3-5、 CTX III再折疊模擬與實驗結果對照……………………………….3-60
3-5.1、 與H/D交換實驗中氫保護係數結果對照……………………..3-60
3-5.2、 與部份展開 ( partially unfolded )結構對照…………………..3-64
3-6、 CTX III的再折疊路徑描述………………………………………….3-70
第二部份、 β-髮夾再折疊的分子動力學模擬…………………..3-73
3-7、 β-髮夾再折疊的模擬結果………………………………………….3-73
3-8、 β-髮夾再折疊模擬的RMSD值分析與氫鍵分析………………...3-76
3-8.1、 RMSD值分析……………………………………………….….3-76
3-8.2、 氫鍵分析………………………………………………………...3-86
3-9、 β-髮夾再折疊的路徑……………………………………………….3-90
3-9.1、 上拉鍊模型 ( Zip-up model )………………………………….3-90
3-9.2、 下拉鍊模型 ( Zip-down model )………………………………3-92
3-10、 β-髮夾兩種再折疊路徑比較與折疊機制的探討……………...3-93
第四章、結論………………………………………..4-1
第五章、參考資料…………………………………..5-1

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