天然水解酶具有高度的催化效果以及受質選擇性，但容易受到環境影響而解摺疊失去催化活性，因此近年來陸續有許多研究參考天然酵素活性中心的胺基酸片段序列，並透過各式蛋白質結構作為人工水解酶骨架，但這些人工水解酶的催化效果仍不及天然水解酶。由於膠原蛋白是人體含量最為豐富的蛋白質，且本身為三股螺旋結構，因此本研究利用膠原蛋白模擬胜肽作為骨架並引入吡啶基團及組胺酸，觀察此胜肽作為人工水解酶的催化能力以及與金屬配位後自組裝能力。本實驗中使用圓二色光譜儀鑑定胜肽的結構及熱穩定性，透過穿透式電子顯微鏡觀察形成的自組裝結構，以及利用紫外光可見光光譜儀探討胜肽催化效率。從第一部分的實驗中得知引入吡啶並不會影響膠原蛋白胜肽的基本結構，而吡啶與組胺酸之間的距離也使得胜肽的催化效果有所不同，加入雙硫鍵後對於催化能力也有些許改變。第二部分實驗中透過加入金屬離子讓胜肽具有自組裝結構，而產生的結構對於不同的胜肽的催化效率也有所增減。透過膠原蛋白模擬胜肽作為骨架效仿天然水解酶的三維結構，雖整體結果仍不夠理想，但引入吡啶的研究方向能夠做為未來設計人工水解酶良好的參考。

Natural hydrolases have high catalytic activity and substrate specificity but they are easily affected to unfold and lose their activities by the environmental changes. In recent years, there are several studies referenced the amino acid sequences in the active sites of natural enzymes to design artificial hydrolases, though their catalytic efficiencies are still far below those of the natural ones. Collagen is the most abundant protein in the human body and has a unique triple helical structure. Therefore, in this study, we utilize collagen-mimetic peptides (CMPs) as the scaffolds to design artificial hydrolases and introduce pyridine moieties, histidine, and disulfides to evaluate the catalytic efficiency and self-assembly of these CMPs. We used circular dichroism (CD), transmission electron microscopy (TEM), and UV-Vis spectroscopy to characterize the structure, examine the morphology, and measure the catalytic efficiency of a series of CMPs. In the first part, all the designed CMPs were shown to form PPII structure and stable triple helices despite incorporating a pyridine moiety into the N-terminus of CMPs. The results show that the catalytic efficiency depends on the distance between pyridine and the side chain of histidine. Moreover, the catalytic efficiency is slightly affected by the linkage of disulfides. In the second part, we found the CMPs could be promoted to assemble into supramolecular structures by adding metal ions. The results also show that the large-scale structures do not benefit the catalytic efficiency. Although the catalytic efficiency of our designed CMPs in this study is not as good as that of natural enzymes, the introduced pyridine moiety does increase the activity and can be a good reference for the development of artificial hydrolases in the future.

摘要 II
ABSTRACT III
目錄 IV
圖目錄 VII
表目錄 IX
第一章 緒論 1
1-1 膠原蛋白 1
1-1-1 膠原蛋白結構 2
1-1-2 脯胺酸（Pro）與羥脯胺酸（Hyp）穩定膠原蛋白能力之比較 3
1-1-3 膠原蛋白置換胺基酸穩定性之探討 5
1-1-4 膠原蛋白之自組裝 6
1-2 酵素 7
1-2-1 水解反應 7
1-2-2 人工水解酶設計 8
1-3 雙硫鍵對蛋白質的影響 9
1-3-1 生物體內含有雙硫鍵之蛋白質 9
1-4 蛋白質胺基酸序列突變 10
1-5 研究動機 11
第二章 實驗部分 12
2-1 實驗儀器 12
2-2 實驗藥品 13
2-3 固相胜肽合成法（Solid Phase Peptide Synthesis, SPPS） 15
2-3-1 酯化反應（Esterification）/醯胺化反應（Amidation） 18
2-3-2 去保護（Deprotection） 18
2-3-3 活化（Activation） 19
2-3-4 耦合（Coupling） 20
2-3-5 切除（Cleavage） 20
2-4 圓二色光譜儀（Circular Dichrosim） 21
2-5 合成 Fmoc-Pro-Hyp-Gly-OH tripeptide 25
2-5-1 合成 Boc-Hyp-OH 25
2-5-2 合成 Boc-Hyp-Gly-OBn 26
2-5-3 合成 Fmoc-Pro-Hyp-Gly-OBn 27
2-5-4 合成 Fmoc-Pro-Hyp-Gly-OH 28
2-6 模擬胜肽之合成 29
2-6-1 模擬膠原蛋白系列之合成 30
2-6-2 胜肽的切除與純化 30
2-6-3 雙硫鍵膠原蛋白模擬胜肽合成 31
2-7 圓二色光譜實驗 31
2-7-1 Far-UV 光譜（Wavelength scan） 31
2-7-2 熱變性實驗（Thermal denaturation） 31
2-7-3 變溫實驗數據之處理 32
2-8 紫外光可見光光譜實驗 33
2-8-1 水解催化實驗 34
2-8-2 催化實驗數據之處理 34
2-9 穿透式電子顯微鏡觀察實驗 36
2-10 掃描式電子顯微鏡觀察實驗 36
第三章 實驗結果與討論 37
3-1 膠原蛋白模擬胜肽之設計 37
3-1-1 膠原蛋白模擬胜肽結構之鑑定 38
3-1-2 膠原蛋白模擬胜肽催化活性之探討 40
3-2 膠原蛋白模擬胜肽自組裝結構之探討 45
3-2-1 自組裝胜肽結構之鑑定 45
3-2-2 自組裝胜肽催化活性之探討 52
3-3 與過去人工水解酶研究文獻比較 56
第四章 結論 57
參考資料（REFERENCE） 58
附錄 62

1. Kar, K.; Ibrar, S.; Nanda, V.; Getz, T. M.; Kunapuli, S. P.; Brodsky, B., Aromatic Interactions Promote Self-Association of Collagen Triple-Helical Peptides to Higher-Order Structures. Biochemistry 2009, 48, 7959-7968.
2. Cowan, P. M.; McGavin, S.; North, A. C. T., The Polypeptide Chain Configuration of Collagen. Nature 1955, 176, 1062-1064.
3. Rich, A.; Crick, F. H. C., The Structure of Collagen. Nature 1955, 176, 915-916.
4. Rich, A.; Crick, F. H., The Molecular Structure of Collagen. J. Mol. Biol. 1961, 3, 483-506.
5. Bella, J.; Brodsky, B.; Berman, H. M., Hydration Structure of a Collagen Peptide. Structure 1995, 3, 893-906.
6. Bella, J.; Eaton, M.; Brodsky, B.; Berman, H. M., Crystal and Molecular Structure of a Collagen-Like Peptide at 1.9 Å Resolution. Science 1994, 266, 75-81.
7. Hinderaker, M. P.; Raines, R. T., An Electronic Effect on Protein Structure. Protein Sci. 2003, 12, 1188-1194.
8. Privalov, P. L., Stability of Proteins. Proteins Which Do Not Present a Single Cooperative System. Adv. Protein Chem. 1982, 35, 1-104.
9. Brodsky, B.; Ramshaw, J. A., The Collagen Triple-Helix Structure. Matrix Biol. 1997, 15, 545-554.
10. Sakakibara, S.; Inouye, K.; Shudo, K.; Kishida, Y.; Kobayashi, Y.; Prockop, D. J., Synthesis of (Pro-Hyp-Gly)n of Defined Molecular Weights Evidence for the Stabilization of Collagen Triple Helix by Hydroxypyroline. Biochim. Biophys. Acta 1973, 303, 198-202.
11. Shoulders, M. D.; Raines, R. T., Collagen Structure and Stability. Annu. Rev. Biochem. 2009, 78, 929-958.
12. Persikov, A. V.; Ramshaw, J. A. M.; Kirkpatrick, A.; Brodsky, B., Amino Acid Propensities for the Collagen Triple-Helix. Biochemistry 2000, 39, 14960-14967.
13. Whitesides, G. M.; Boncheva, M., Beyond Molecules: Self-Assembly of Mesoscopic and Macroscopic Components. Proc. Nat. Acad. Sci. U.S.A. 2002, 99, 4769.
14. Selkoe, D. J., Cell Biology of Protein Misfolding: The Examples of Alzheimer's and Parkinson's Diseases. Nat. Cell Biol. 2004, 6, 1054-1061.
15. Stefani, M.; Dobson, C. M., Protein Aggregation and Aggregate Toxicity: New Insights into Protein Folding, Misfolding Diseases and Biological Evolution. J. Mol. Med. 2003, 81, 678-699.
16. Bruice, T. C.; Schmir, G. L., Imidazole Catalysis. I. The Catalysis of the Hydrolysis of Phenyl Acetates by Imidazole. J. Am. Chem. Soc. 1957, 79, 1663-1667.
17. Jencks Wp Fau - Carriuolo, J.; Carriuolo, J., Imidazole Catalysis. II. Acyl Transfer and the Reactions of Acetyl Imidazole with Water and Oxygen Anions. J. Biol. Chem. 1959, 234, 1272–1279.
18. Bezer, S.; Matsumoto, M.; Lodewyk, M. W.; Lee, S. J.; Tantillo, D. J.; Gagné, M. R.; Waters, M. L., Identification and Optimization of Short Helical Peptides with Novel Reactive Functionality as Catalysts for Acyl Transfer by Reactive Tagging. Org. Biomol. Chem. 2014, 12, 1488-1494.
19. Matsumoto, M.; Lee, S. J.; Waters, M. L.; Gagné, M. R., A Catalyst Selection Protocol That Identifies Biomimetic Motifs from Β-Hairpin Libraries. J. Am. Chem. Soc. 2014, 136, 15817-15820.
20. Burton, A. J.; Thomson, A. R.; Dawson, W. M.; Brady, R. L.; Woolfson, D. N., Installing Hydrolytic Activity into a Completely De Novo Protein Framework. Nat. Chem. 2016, 8, 837-844.
21. Wang, P. S. P.; Nguyen, J. B.; Schepartz, A., Design and High-Resolution Structure of a β3-Peptide Bundle Catalyst. J. Am. Chem. Soc. 2014, 136, 6810-6813.
22. Gulseren, G.; Khalily, M. A.; Tekinay, A. B.; Guler, M. O., Catalytic Supramolecular Self-Assembled Peptide Nanostructures for Ester Hydrolysis. J. Mater. Chem. 2016, 4, 4605-4611.
23. Rufo, C. M.; Moroz, Y. S.; Moroz, O. V.; Stöhr, J.; Smith, T. A.; Hu, X.; DeGrado, W. F.; Korendovych, I. V., Short Peptides Self-Assemble to Produce Catalytic Amyloids. Nat. Chem. 2014, 6, 303-309.
24. Al-Garawi, Z. S.; McIntosh, B. A.; Neill-Hall, D.; Hatimy, A. A.; Sweet, S. M.; Bagley, M. C.; Serpell, L. C., The Amyloid Architecture Provides a Scaffold for Enzyme-Like Catalysts. Nanoscale 2017, 9, 10773-10783.
25. Stickle, D. F.; Presta, L. G.; Dill, K. A.; Rose, G. D., Hydrogen Bonding in Globular Proteins. J. Mol. Biol. 1992, 226, 1143-1159.
26. Privalov, P. L.; Gill, S. J., Stability of Protein Structure and Hydrophobic Interaction. Adv. Protein Chem. 1988, 39, 191-234.
27. Burley, S. K.; Petsko, G. A., Amino-Aromatic Interactions in Proteins. FEBS Lett. 1986, 203, 139-143.
28. Betz, S. F., Disulfide Bonds and the Stability of Globular Proteins. Protein Sci. 1993, 2, 1551-1558.
29. Beeby, M.; O'Connor, B. D.; Ryttersgaard, C.; Boutz, D. R.; Perry, L. J.; Yeates, T. O., The Genomics of Disulfide Bonding and Protein Stabilization in Thermophiles. PLOS Biology 2005, 3, e309.
30. Rigobello, M. P.; Donella-Deana, A.; Cesaro, L.; Bindoli, A., Distribution of Protein Disulphide Isomerase in Rat Liver Mitochondria. Biochem. J 2001, 356, 567-570.
31. Starman, B. J.; Eyre D Fau - Charbonneau, H.; Charbonneau H Fau - Harrylock, M.; Harrylock M Fau - Weis, M. A.; Weis Ma Fau - Weiss, L.; Weiss L Fau - Graham, J. M., Jr.; Graham Jm Jr Fau - Byers, P. H.; Byers, P. H., Osteogenesis Imperfecta. The Position of Substitution for Glycine by Cysteine in the Triple Helical Domain of the Pro Alpha 1(I) Chains of Type I Collagen Determines the Clinical Phenotype. J. Clin. Invest. 1989, 84, 1206–1214.
32. Gajko-Galicka, A., Mutations in Type I Collagen Genes Resulting in Osteogenesis Imperfecta in Humans. Acta Biochim. Pol. 2002, 49, 433-441.
33. Shapiro, J. R.; Stover, M. L.; Burn, V. E.; McKinstry, M. B.; Burshell, A. L.; Chipman, S. D.; Rowe, D. W., An Osteopenic Nonfracture Syndrome with Features of Mild Osteogenesis Imperfecta Associated with the Substitution of a Cysteine for Glycine at Triple Helix Position 43 in the Pro Alpha 1(I) Chain of Type I Collagen. J. Clin. Invest. 1992, 89, 567-573.
34. Narcisi, P.; J.Richards, A.; Ferguson, S. D.; Pope, F. M., A Family with Ehlers — Danlos Syndrome Type III/Articular Hypermobility Syndrome Has a Glycine 637 to Serine Substitution in Type III Collagen. Hum. Mol. Genet. 1994, 3, 1617-1620.
35. Prockop, D. J.; Kivirikko, K. I., Collagens: Molecular Biology, Diseases, and Potentials for Therapy. Annu. Rev. Biochem. 1995, 64, 403-434.
36. Hung, P.-Y.; Chen, Y.-H.; Huang, K.-Y.; Yu, C.-C.; Horng, J.-C., Design of Polyproline-Based Catalysts for Ester Hydrolysis. ACS Omega 2017, 2, 5574-5581.
37. Ting, Y.-H.; Chen, H.-J.; Cheng, W.-J.; Horng, J.-C., Zinc(II)–Histidine Induced Collagen Peptide Assemblies: Morphology Modulation and Hydrolytic Catalysis Evaluation. Biomacromolecules 2018, 19, 2629-2637.
38. 張彤瑋. 膠原蛋白模擬胜肽與其自組裝結構對酯類水解反應的催化效率探討. 國立清華大學, 新竹市, 2019.
39. Yamagami, M.; Sawada, T. A.-O.; Fujita, M. A.-O., Synthetic β-Barrel by Metal-Induced Folding and Assembly. J. Am. Chem. Soc. 2018, 140, 8644–8647.
40. Merrifield, B., Solid Phase Synthesis. Science 1986, 232, 341-347.
41. Berova, N.; Nakanishi, K.; Woody, R., Circular Dichroism. Principles and Applications 2nd Edition. 2000.
42. Greenfield, N., Using Circular Dichroism Collected as a Function of Temperature to Determine the Thermodynamics of Protein Unfolding and Binding Interactions. Nat. Protoc. 2006, 1, 2527-2535.
43. Sundberg, R. J.; Martin, R. B., Interactions of Histidine and Other Imidazole Derivatives with Transition Metal Ions in Chemical and Biological Systems. Chem. Rev. 1974, 74, 471-517.
44. Krężel, A.; Maret, W., The Biological Inorganic Chemistry of Zinc Ions. Arch. Biochem. Biophys. 2016, 611, 3-19.
45. Zastrow, M. L.; Pecoraro, V. L., Influence of Active Site Location on Catalytic Activity in De Novo-Designed Zinc Metalloenzymes. J. Am. Chem. Soc. 2013, 135, 5895-5903.