膠原蛋白（collagen）是哺乳動物中含量最多的蛋白質，由於其生物相容性與生物降解性，現今已廣泛應用於生醫材料與藥物等方面，為增加其應用性，尋找簡單、快速且有效的製備方法來促使膠原蛋白模擬胜肽自組裝成高階或超分子結構已成為許多研究上的努力目標。本研究中，利用組胺酸（Histidine, His）置換至膠原蛋白模擬胜肽中，藉由組胺酸側鏈的咪唑（imidazole）與金屬離子之配位作用來促進膠原蛋白模擬胜肽自組裝。我們將基底胜肽長度縮短，由 (Pro-Hyp-Gly)9 改為 (Pro-Hyp-Gly)7 與 (Pro-Hyp-Gly)5 並於中間置換組胺酸，合成胜肽鏈：GG(POG)3(PHG)(POG)3GG 與 G(POG)2(PHG)(POG)2Y。探討胜肽長度改變與加入不同的金屬離子對於自組裝效率與結構之影響。實驗結果顯示，胜肽長度改變並未影響胜肽纏繞形成第二型聚脯胺酸鏈（polyproline II, PPII），但會降低三股螺旋的穩定性，此外，較短的胜肽鏈在自組裝上需要較長時間的延遲期且雖然以 (Pro-Hyp-Gly)5 為基底的膠原蛋白模擬胜肽並無法形成穩定的三股螺旋結構，但是仍可以藉由金屬離子的誘導形成較高階的結構；同時，我們亦發現自組裝前期是否給予適當地攪拌對於自組裝之高階結構有不同以往之形狀，如可呈現尺寸較一致且微厚片蓮花般形態且此結構為特定金屬離子（鋅離子）誘導時才可形成。在低溫（4 °C）與高溫（80 °C）的實驗中，我們發現製備溫度亦會影響自組裝結構。雖然目前仍對於我們的膠原蛋白模擬胜肽自組裝過程未有明確的解釋，但發現其組裝結構有特殊形態，希望藉此進一步設計不同的膠原蛋白模擬胜肽發展不同的自組裝方法和組裝結構。

Collagen is the most abundant protein in mammals and has been widely used in biomedical materials. Searching for a quick and easy way to effectively assemble short collagen-mimetic peptides (CMPs) into higher-order or supramolecular structures has been an emerging research topic in biomaterials. In this study, we incorporated histidine (His) into CMPs to promote their self-assembly into higher-order structures via His-metal coordination bonds. We used the 21-mer peptide (Pro-Hyp-Gly)7 and 15-mer peptide (Pro-Hyp-Gly)5, which are shorter than the 27-mer peptide (Pro-Hyp-Gly)9, as the parent peptides and prepared the peptides: GG(POG)3(PHG)(POG)3GG and G(POG)2(PHG)(POG)2Y. Our aim was to investigate the impacts of the change in peptide length or differences in metal ions on the self-assembly of CMPs. The results showed that the change in peptide length did not affect its polyproline II (PPII) structure but decreased the thermal stability of the triple-helical structure and the assembly process became relatively slow. Besides, we also found that stirring in the beginning of the self-assembly process could assist the CMPs in becoming size-consistent lotus leaf-like structures, which were different from the commonly found higher-order structural shapes, and they were induced by binding with a specific metal ion, Zn(II). The effects of incubation temperature on the assembled structures were also examined, and the results showed that the assemblies prepared at 4 °C had a different morphology from that of the assemblies prepared at 80 °C. We were not able to clarify the self-assembly process of collagen, but we did find the special large-scale construction. Our finding may be useful for the future development of collagen-related materials.

摘要 I
Abstract II
謝誌 III
目錄 IV
圖目錄 VI
表目錄 XI
第一章 緒論 1
1-1 膠原蛋白簡介 1
1-2 膠原蛋白的結構 2
1-2-1 脯胺酸和羥脯胺酸穩定膠原蛋白能力比較 3
1-2-2 膠原蛋白模擬胜肽單一置換穩定性探討 5
1-2-3 膠原蛋白的自組裝 6
1-3 生物系統中的金屬 10
1-3-1 生物系統中的重要元素 10
1-3-2 生物系統中的配位基 13
1-3-3 金屬誘發膠原蛋白自組裝 14
1-3-4 金屬離子對酵素的作用 15
1-4 研究動機 17
第二章 實驗部分 21
2-1 實驗儀器 21
2-2 實驗藥品 22
2-3 合成 Fmoc-Pro-Hyp-Gly-OH tripeptide 25
2-3-1 合成 Boc-Hyp-OH 25
2-3-2 合成 Boc-Hyp-Gly-OBn 25
2-3-3 合成 Fmoc-Pro-Hyp-Gly-OBn 26
2-3-4 合成 Fmoc-Pro-Hyp-Gly-OH 27
2-4 合成膠原蛋白模擬胜肽鏈 28
2-4-1 固相胜肽合成法 - 自動胜肽合成儀 28
2-4-2 固相胜肽合成法 - 手動合成 29
2-4-3 Cleavage 29
2-4-4 高效能液相層析儀純化 29
2-5 配製 pH 7.4 磷酸鹽緩衝溶液與配製樣品 29
2-6 圓二色光譜儀光譜量測與資料處理 30
2-6-1 Far-UV CD光譜量測（Wavelength scans） 32
2-6-2 變溫 CD 光譜量測（Thermal denaturation） 32
2-6-3 變溫實驗數據處理 33
2-7 掃描式電子顯微鏡量測 34
2-8 穿透式電子顯微鏡量測 34
2-9 組胺酸-金屬離子配為作用誘導之結構進行催化水解 34
2-10 催化水解活性實驗之數據處理 35
第三章 實驗結果與討論 37
3-1 Far-UV CD光譜 37
3-2 變溫 CD 光譜 39
3-3 穿透式電子顯微鏡影像實驗 41
3-4 掃描式電子顯微鏡影像實驗 43
3-4-1 探討攪拌、金屬濃度與溫度對 PHGT3 自組裝結構之影響 43
3-4-2 探討形成三股螺旋與否、金屬離子種類對自組裝結構之影響 47
3-4-3 PHGT3、PHGT2 自組裝結構構形整理 53
3-5 組胺酸-金屬配位作用誘導對酯類水解催化效率 58
第四章 結論與未來研究 61
參考文獻 62
附錄 67

[1] Brodsky, B.; Persikov, A. V. Molecular structure of the collagen triple helix. Adv. Protein Chem. 2005, 70, 301-339
[2] Friess, W. Collagen-biomaterial for drug delivery. Eur. J. Pharm. Biopharm. 1998, 45, 113-136
[3] Prockop, D. J.; Kivirriko, K. I. Collagens: molecular biology, diseases, and potentials for therapy. Annu. Rev. Biochem. 1995, 64, 403-434
[4] Lutolf, M. P.; Hubbell, J. A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotechnol. 2005, 23, 47-55
[5] Malafaya, P. B.; Silva, G. A.; Reis, R. L. Natural-origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications. Adv. Drug Delivery Rev. 2007, 59, 207-233
[6] Lee, C. H.; Singla, A.; Lee, Y. Biomedical applications of collagen. Int. J. Pharm. 2001, 221, 1-22
[7] Ramachandran, G. N.; Kartha, G. Structure of collagen. Nature 1955, 176, 593-595
[8] Rich, A.; Crick, F. H. C. The structure of collagen. Nature 1955, 176, 915-916
[9] Cowan, P. W.; McGavin, S.; North, A. C. T. The polypeptide chain configuration of collagen. Nature 1955, 176, 1062-1064
[10] Rich, A.; Crick, F. H. C. The molecular structure of collagen. J. Mol. Biol. 1961, 3, 483-506
[11] Okuyama, K.; Nagarajan, V.; Kamitori, S. 7/2-Helical model for collagen — evidence from model peptides. Sciences 1999, 111, 19-34
[12] Okuyama, K. Revisiting the molecular structure of collagen. Connect Tissue Res. 2008, 49, 299-310
[13] Bella, J.; Brodsky, B.; Berman, H. M. Hydration structure of a collagen peptide. Structure 1995, 3, 893-906
[14] Hinderaker, M. P.; Raines, R. T. An electronic effect on protein structure. Protein Sci. 2003, 12, 1188-1194
[15] Shoulders, M. D.; Satyshur, K. A.; Forest, K. T.; Raines, R. T. Stereoelectronic and steric effects in side chains preorganize a protein main chain. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 559-564
[16] Privalov, P. L. Stability of proteins. proteins which do not present a single cooperative system. Adv. Protein Chem. 1982, 35, 1-104
[17] Brodsky, B.; Ramshaw, J. A. The collagen triple-helix structure. Matrix Biol. 1997, 15, 545-554
[18] 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
[19] Shoulders, M. D.; Raines, R. T. Collagen structure and stability. Annu. Rev. Biochem. 2009, 78, 929-958
[20] Persikov, A. V.; Ramshaw, J. A.; Kirkpatrick, A.; Brodsky, B. Amino acid propensities for the collagen triple-helix. Biochemistry 2000, 39, 14960-14967
[21] Whitesides, G. M.; Boncheva, M. Beyond molecules: self-assembly of mesoscopic and macroscopic components. Proc. Natl. Acad. Sci. USA 2002, 99, 4769-4774
[22] Stefani, M.; Dobson, C. M. Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J. Mol. Med. (Berl.) 2003, 81, 678-699
[23] Selkoe, D. J. Cell biology of protein misfolding: the examples of Alzheimer's and Parkinson's diseases. Nat. Cell Biol. 2004, 6, 1054-1061
[24] Sakaguchi, M.; Hori, H.; Hattori, S.; Irie, S.; Imai, A.; Yanagida, M.; Miyazawa, H.; Toda, M.; Inouye, S. IgE reactivity to α1 and α2 chains of bovine type 1 collagen in children with bovine gelatin allergy. J. Allergy Clin. Immunol. 1999, 104, 695-699
[25] Lynn, A. K.; Yannas, I. V.; Bonfield, W. Antigenicity and immunogenicity of collagen. J. Biomed. Mater. Res., Part B 2004, 71, 343-354
[26] Przybyla, D. E.; Chmielewski, J. Higher-order assembly of collagen peptides into nano- and microscale materials. Biochemistry 2010, 49, 4411-4419
[27] Koide, T.; Homma, D. L.; Asada, S.; Kitagawa, K. Self-complementary peptides for the formation of collagen-like triple helical supramolecules. Bioorg. Med. Chem. Lett. 2005, 15, 5230-5233
[28] Kotch, F. W.; Raines, R. T. Self-assembly of synthetic collagen triple helices. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3028-3033
[29] Yamazaki, C. M.; Asada, S.; Kitagawa, K.; Koide, T. Artificial collagen gels via self‐assembly of de novo designed peptides. J. Pept. Sci. 2008, 14, 186-186
[30] Yamazaki, C. M.; Asada, S.; Kitagawa, K.; Koide, T. Artificial collagen gels via self-assembly of de novo designed peptides. Biopolymers 2008, 90, 816-823
[31] Rele, S.; Song, Y.; Apkarian, R. P.; Qu, Z.; Conticello, V. P.; Chaikof, E. L. D-periodic collagen-mimetic microfibers. J. Am. Chem. Soc. 2007, 129, 14780-14787
[32] Paramonov, S. E.; Gauba, V.; Hartgerink, J. D. Synthesis of collagen-like peptide polymers by native chemical ligation. Macromolecules 2005, 38, 7555-7561
[33] Cejas, M. A.; Kinney, W. A.; Chen, C.; Leo, G. C.; Tounge, B. A.; Vinter, J. G.; Joshi, P. P.; Maryanoff, B. E. Collagen-related peptides:  self-assembly of short, single strands into a functional biomaterial of micrometer scale. J. Am. Chem. Soc. 2007, 129, 2202-2203
[34] Cejas, M. A.; Kinney, W. A.; Chen, C.; Vinter, J. G.; Harold, R.; Almond, J.; Balss, K. M.; Maryanoff, C. A.; Schmidt, U.; Breslav, M.; Mahan, A.; Lacy, E.; Maryanoff, B. E. Thrombogenic collagen-mimetic peptides: Self-assembly of triple helix-based fibrils driven by hydrophobic interactions. Proc. Natl. Acad. Sci. U.S.A. 2008, 24, 8513-8518
[35] 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
[36] Przybyla, D. E.; Chmielewski, J. Metal-triggered radial self-assembly of collagen peptide fibers. J. Am. Chem. Soc. 2008, 130, 12610-12611
[37] Pires, M. M.; Chmielewski, J. Self-assembly of collagen peptides into microflorettes via metal coordination. J. Am. Chem. Soc. 2009, 131, 2706-2712
[38] Pires, M. M.; Przybyla, D. E.; Chmielewski, J. A metal-collagen peptide framework for three‐dimensional cell culture. Angew. Chem. Int. Ed. 2009, 48, 7813-7817
[39] Przybyla, D. E.; Chmielewski, J. Metal-triggered collagen peptide disk formation. J. Am. Chem. Soc. 2010, 132, 7866-7867
[40] Brodsky, B.; Thiagarajan, G.; Madhan, B.; Kar, K. Triple‐helical peptides: An approach to collagen conformation, stability, and self‐association. Biopolymers 2008, 89, 345-353
[41] Hwang, E. S.; Thiagarajan, G.; Parmar, A. S.; Brodsky, B. Interruptions in the collagen repeating tripeptide pattern can promote supramolecular association. Protein Sci. 2010, 19, 1053-1064
[42] Yu, S. M.; Li, Y.; Kim, D. Collagen mimetic peptides: progress towards functional applications. Soft Matter 2011, 7, 7927-7938
[43] Werten, M. W. T.; Teles, H.; Moers, A. P. H. A.; Wolbert, E. J. H.; Sprakel, J.; Eggink, G.; de Wolf, F. A. Precision gels from collagen-inspired triblock copolymers. Biomacromolecules 2009, 10, 1106-1113
[44] Chen, C. C.; Hsu, W.; Kao, T. C.; Horng, J. C. Self-assembly of short collagen-related peptides into fibrils via cation−π interactions. Biochemistry 2011, 50, 2381-2383
[45] Hsu, W.; Chen, Y. L.; Horng, J. C. Promoting self-assembly of collagen-related peptides into various higher-order structures by metal-histidine coordination. Langmuir 2012, 28, 3194-3199
[46] Crichton, R. Biological inorganic chemistry: an introduction, 1st edition, 2008
[47] Aggeli, A.; Bell, M.; Boden, N.; Keen, J. N.; Knowles, P. F.; McLeish, T. C. B.; Pitkeathly, M.; Radford, S. E. Responsive gels formed by the spontaneous self-assembly of peptides into polymeric β-sheet tapes. Nature 1997, 386, 259-262
[48] Altunbas, A.; Lee, S. J.; Rajasekaran, S. A.; Schneider, J. P.; Pochan, D. J. Encapsulation of curcumin in self-assembling peptide hydrogels as injectable drug delivery vehicles. Biomaterials 2011, 32, 5906-5914
[49] Korendovych, I. V.; DeGrado, W. F. Catalytic efficiency of designed catalytic proteins. Curr. Opin. Struct. Biol. 2014, 27, 113-121
[50] Rufo, C. M.; Moroz, Y. S.; Moroz, O. V.; Stohr, 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
[51] Wang, M.; Lv, Y.; Liu, X.; Qi, W.; Su, R.; He, Z. Enhancing the activity of peptide-based artificial hydrolase with catalytic Ser/His/Asp triad and molecular imprinting. ACS Appl. Mater. Interfaces 2016, 8, 14133-14141
[52] Wong, Y. M.; Masunaga, H.; Chuah, J. A.; Sudesh, K.; Numata, K. Enzyme-mimic peptide assembly to achieve amidolytic activity. Biomacromolecules 2016, 17, 3375-3385
[53] Zhang, C.; Xue, X.; Luo, Q.; Li, Y.; Yang, K.; Zhuang, X.; Jiang, Y.; Zhang, J.; Liu, J.; Zou, G.; Liang, X. J. Self-assembled peptide nanofibers designed as biological enzymes for catalyzing ester hydrolysis. ACS Nano 2014, 8, 11715-11723
[54] Pires, M. M.; Ernenwein, D.; Chmielewski, J. Selective decoration and release of his-tagged proteins from metal-assembled collagen peptide microflorettes. Biomacromolecules 2011, 12, 2429-2433
[55] Gleaton, J.; Chmielewski, J. Thermally controlled collagen peptide cages for biopolymer delivery. ACS Biomater. Sci. Eng. 2015, 1, 1002-1008
[56] 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
[57] Bretscher, L. E.; Jenkins, C. L.; Taylor, K. M.; DeRider, M. L.; Raines, R. T. Conformational stability of collagen relies on a stereoelectronic effect. J. Am. Chem. Soc. 2001, 123, 777-778
[58] Leonard, D.W.; Meek, K. M. Refractive indices of the collagen fibrils and extrafibrillar material of the corneal stroma. Biophys. J. 1997, 72, 1382-1387
[59] 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
[60] Chan, W. C. Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Practical Approach Series) , 1st edition 2000
[61] Greenfield, N. J. 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
[62] Chen, Y. S.; Chen, C. C.; Horng, J. C. Thermodynamic and kinetic consequences of substituting glycine at different positions in a Pro-Hyp-Gly repeat collagen model peptide. Biopolymers 2011, 96, 60-68
[63] Michaelis, L.; Menten, M. L. Kinetics of invertase action. Biochem. Z. 1913, 49, 333-369
[64] Berova, V.; Nakanishi, K.; Woody, R. W. Circular Dichroism: Principles and Applications, 2nd edition 2000