利用 Cation-π 作用力誘導膠原蛋白異源三股螺旋形成及膠原蛋白胜肽自組裝之探討

簡易檢索 / 詳目顯示

回結果列表

研究生：	高堂畯 Kao, Tang-Chun
論文名稱：	利用 Cation-π 作用力誘導膠原蛋白異源三股螺旋形成及膠原蛋白胜肽自組裝之探討 Using Cation-π Interactions to Form Heterotrimeric Collagen Helices and Assemble Collagen-Related Peptides
指導教授：	洪嘉呈 Horng, Jia-Cherng
口試委員:	李賢明 Lee, Hsien-Ming 陳貴通 Tan, Kui-Thong
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2012
畢業學年度：	100
語文別：	中文
論文頁數：	122
中文關鍵詞：	膠原蛋白、異源三股螺旋、Cation-π 作用力、膠原蛋白胜肽自組裝
外文關鍵詞：	Collagen, Heterotrimeric helices, Cation-π Interactions, Assemble Collagen-Related Peptides
相關次數：	點閱：3 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

膠原蛋白是動物體內組織中含量最多的蛋白質，其特殊的構造是由包含了重複 X-Y-Gly 序列的三條左旋第二型聚脯胺酸胜肽鏈，沿著相同軸方向纏繞成的一右旋三股螺旋結構。第 Ⅰ 型膠原蛋白，為天然膠原蛋白含量最多的一種型態，其為異源三股螺旋結構，因此探討異源三股螺旋結構之膠原蛋白模擬胜肽能夠更接近於自然界中的情形。在這裡的實驗中我們利用 cation-π 作用力幫助合成的膠原蛋白模擬胜肽纏繞成異源三股螺旋結構，CD 及 NMR 測量結果顯示 (POGPRG)3(POG) & (POGFOG)3(POG)、 (POGPRG)3(POG) & (POGFOG)3(POG) & (POG)7 混合胜肽溶液可形成單一的異源三股螺旋結構，得到的 Tm 值分別為 26 ºC 及 27.5 ºC，且沒有伴隨各自成份的同源三股螺旋產生，所以利用設計過的胜肽序列，藉由不同胜肽鏈間的 cation-π 作用力能夠使我們得到穩定的膠原蛋白異源三股螺旋結構。
在先前實驗室研究我們了解在兩端的 cation-π 作用力能夠促進 RG(POG)10F 快速聚集成纖維結構，在這裡我們進一步合成 (POG)4(PRG)(FOG)(POG)4、(POG)3(PRG)(POG)2(FOG)(POG)3、(POG)2(PRG) (POG)4(FOG)(POG)2、RG(POG)3(PRG)(POG)2(FOG)(POG)3F，研究不同位置的 cation-π 作用力對其聚集過程的影響，turbidity 及 DLS 量測顯示股內的 cation-π 作用力並無明顯促進它們聚集成更大的超分子纖維結構，此結果也指出當將 (PRG) 和 (FOG) 序列崁入整個膠原蛋白胜肽的中間時，可能因為 arginine、phenylalanine 的置入使得結構的立體障礙變大而無法形成強的 cation-π 作用力，進而阻礙膠原蛋白三股螺旋間的堆疊。

Collagen is an important structural component of tissues in animals. It has an unique right-handed triple helix consisted of three left-handed polyproline II like chains which are composed of X-Y-Gly repeats in the sequence. TypeⅠcollagen, a heterotrimer, is the most abundant form, and thus using heterotrimeric helices can be more appropriate to mimic nature collagen. In this work, we used cation-π interactions to assist collagen-related peptides to fold into heterotrimers. CD and NMR measurements indicate that (POGPRG)3(POG) & (POGFOG)3(POG), (POGPRG)3(POG) & (POGFOG)3(POG) & (POG)7 mixed peptide solution could form a single heterotrimeric helices with Tm values of 26 and 27.5 ºC respectively, and no homotrimers were found. The results demonstrate that heterotrimers could be formed by interchain cation-π interactions.
We have previously shown that terminal cation-π interactions can promote RG(POG)10F to rapidly assemble into fibrils. Here we further synthesized (POG)4(PRG)(FOG)(POG)4, (POG)3(PRG)(POG)2(FOG)(POG)3, (POG)2(PRG) (POG)4(FOG)(POG)2, and RG(POG)3(PRG)(POG)2(FOG)(POG)3F to study the position dependent effects of cation-π interactions on their self-assembly process. Turbility and dynamic light scattering measurements showed that such designs and arrangements do not significantly promote the assembly process. The results imply that the PRG and FOG triplets in place of POG in the middle of a collagen related peptide may impose steric effects through the bulky side chains of arginine and phenylalanine residues, which prevent the formation of strong cation-π interactions and retard the packing between collagen triple helices.

第一章 緒論 ......................................12
1-1 膠原蛋白 (collagen) ......................... 12
1-2 膠原蛋白的結構 ............................... 13
1-2-1 甘胺酸 (Gly) 在膠原蛋白中的地位 ............. 14
1-2-2氫氧脯胺酸 (Hyp) 之重要性 .................... 17
1-2-3利用吡咯烷環 (pyrrolidine ring) 構形解釋 Hyp 穩定膠原蛋白之原因 ............................................ 18
1-3 常見影響蛋白質結構的作用力比較 ................. 20
1-4 Cation-π 作用力 (Cation-π interactions) ..... 21
1-4-1 水溶液環境下之cation-π 作用力 ............... 23
1-4-2 胺基陽離子型側鏈胺基酸和芳香環側鏈胺基酸 ...... 25
1-4-3 胺基型陽離子-芳香環作用力 .................... 27
1-5 膠原蛋白自組裝 ............................... 29
1-6異源三股螺旋結構之膠原蛋白與成骨不全症 ........... 37
1-7 研究動機 ..................................... 40
第二章實驗 ....................................... 41
2-1 實驗儀器 ..................................... 41
2-2 藥品 ........................................ 42
2-3 合成 Fmoc-Pro-Hyp-Gly-OH tripeptide ......... 46
2-3-1 Boc-(4R,2S)-hydroxyproline (1) 之合成 ..... 46
2-3-2 Boc-Hyp-Gly-OBn (2) 之合成 ................ 47
2-3-3 Fmoc-Pro-Hyp-Gly-OBn (3) 之合成 ........... 47
2-3-4 Fmoc-Pro-Hyp-Gly-OH (4) 之合成 ............ 48
2-4 固相胜肽合成法 (solid-phase peptide synthesis, SPPS) . 49
2-4-1酯化反應 (Esterification) .................. 52
2-4-2 去保護 (Deprotection) ..................... 52
2-4-3 活化 (Activation) ......................... 53
2-4-4 偶合 (Coupling) ........................... 54
2-4-5 切除 (Cleavage) ........................... 55
2-5 膠原蛋白模擬胜肽鏈之合成 ...................... 55
2-6 高壓液相層析儀 (HPLC) 純化胜肽鏈 .............. 56
2-7 圓二色光譜儀 (circular dichroism spectroscopy, CD) . 58
2-8 動態光散射 (dynamic light scattering, DLS) -粒徑量測 ............................................. 62
2-9 利用 cation-π 作用力形成異源三股螺旋胜肽實驗 ... 65
2-9-1 模擬胜肽溶液配製 ........................... 65
2-9-2 圓二色光譜儀 (CD) 量測 ..................... 65
2-9-3 核磁共振光譜 (NMR) 實驗 .................... 66
2-10 利用Cation-π 作用力促進膠原蛋白側向自組裝實驗 . 67
2-10-1 模擬胜肽溶液配製 .......................... 67
2-10-2 圓二色光譜儀 (CD) 量測 .................... 67
2-10-3 UV-VIS 光譜儀濁度 (turbidity) 量測實驗 .... 67
2-10-4 動態光散射量測實驗 (DLS) .................. 68
2-10-5 穿透式電子顯微鏡 (transmission electron microscopy, TEM) 影像實驗 ................................... 69
第三章 結果與討論 ................................ 70
3-1 模擬膠原蛋白異源三股螺旋胜肽 .................. 71
3-1-1 單個胺基酸取代之K、R、F、W、T7 混合探討 ..... 71
3-1-2 三個胺基酸取代之 K3、R3、F3、W3、Y3 混合探討 . 78
3-1-3 全部胺基酸取代之R7、F7探討 ................. 84
3-1-4 胜肽溶液預先加熱與沒有預先加熱之比較 ......... 86
3-1-5 R & F 以及 R3 & F3 依不同比例混合之探討 .... 90
3-1-6 T7 & R3 & F3依比例1：1：1混合之探討 ........ 93
3-1-7 K3/F3/T7、K3/W3/T7、K3/Y3/T7、R3/W3/T7、R3/Y3/T7 混合之探討 ........................................... 97
3-1-8 NMR HSQC光譜 ............................ 102
3-2利用 cation-π 作用力對膠原蛋白進行散狀式自組裝之探討 ............................................ 105
3-2-1 使用的膠原蛋白胜肽及胜肽溶液之製備 .......... 105
3-2-2 CD 光譜探討 .............................. 106
3-2-3 UV-Vis 光譜濁度 (turbidity) 實驗 .......... 107
3-2-4 動態光散射實驗 ............................ 109
3-2-5 穿透式電子顯微鏡 (TEM) 影像觀察 ............ 111
第四章 結論 ..................................... 117
第五章 參考文獻 (References) .................... 118
                                

1. (a) Chen, C. C.; Hsu,W.; Hwang, K. C.; Hwu, J. R.; Lin, C. C.; Horng, J. C.
Arch. Biochem. Biophys. 2011, 508, 46-53. (b) 陳佳青. 碩士論文, 國立清華大學, 2010.
2. (a) Chen, C. C.; Hsu,W.; Kao, T. C.; Horng, J. C. Biochemistry 2011, 50, 2381-2383. (b) 徐維. 碩士論文, 國立清華大學, 2011.
3. Brinckmann, J. Top. Curr. Chem. 2005, 247, 1-6.
4. Veit, G.; Kobbe, B.; Keene, D. R.; Paulsson, M.; Koch, M.; Wagener, R. J. Biol. Chem. 2006, 281, 3494-504.
5. (a) 黃彥富; 湯正明; 徐善慧. 科學發展期刊 2004, 380, 4; (b) 洪雅萍. 科學發展期刊 2004, 380, 6; (c) Lodish, H. F. Molecular cell biology. 4th ed.; W.H. Freeman: New York, 2000; p xxxvi, 1084, G-17, I-36 p.
6. Prockop, D. J.; Kivirriko, K. I. Ann. Rev. Biochem. 1995, 64, 403-34.
7. Prockop, D. J. Matrix Biol. 1998, 16, 519-28.
8. Prockop, D. J. Biochem. Soc. Trans. 1999, 27, 15-31.
9. (a) Long, C. G.; Li, M. H.; Baum, J.; Brodsky, B. J. Mol. Biol. 1992, 225, 1-4; (b) Kramer, R. Z.; Bella, J.; Mayville, P.; Brodsky, B.; Berman, H. M. Nat. Struct. Biol. 1999, 6, 454-7.
10. Rich, A.; Crick, F. H. J. Mol. Biol. 1961, 3, 483-506.
11. Okuyama, K.; Nagarajan, V.; Kamitori, S. J. Chem. Sci. 1999, 111, 19-34.
12. Okuyama, K. Connect. Tissue Res. 2008, 49, 299-310.
13. Bella, J.; Brodsky, B.; Berman, H. M. Structure 1995, 3, 893-906
14. Bella, J.; Eaton, M.; Brodsky, B.; Berman, H. M. Science 1994, 266, 75-81.
15. Shoulders, M. D.; Raines, R. T. Annu. Rev. Biochem. 2009, 78, 929-58.
16. Hinderaker, M. P.; Raines, R. T. Protein Sci. 2003, 12, 1188-94.
17. Shah, N.K.; Ramshaw, J.A.M.; Kirkpatrick, A.; Shah, C.; Brodsky, B. Biochemistry 1996, 35 ,10262-8.
18. Privalov, P.L. Adv. Protein Chem. 1982, 35,1-104.
19. Brodsky, B.; Ramshaw, J.A.M. Matrix Biol. 1997, 15, 545-54.
20. Persikov, A. V.; Ramshaw, J. A.; Kirkpatrick, A.; Brodsky, B. Biochemistry 2000, 39, 14960-7.
21. Ramshaw, J. A. M.; Shah, N. K.; Brodsky, B. J. Struct. Biol. 1998, 122, 86-91.
22. Yang, W.; Chan, V. C.; Kirkpatrick, A.; Ramshaw, J. A. M.; Brodsky, B. J.Biol. Chem. 1997, 272, 28837-40.
23. Dougherty, D. A. Science 1996, 271, 163-8.
24. Kearney, P. C.; Mizoue, L. S.; Kumpf, R. A.; Forman, J. E.; McCurdy, A.; Dougherty, D. A. J. Am. Chem. Soc. 1993, 115, 9907-19.
25. Mecozzi, S.; West, A. P., Jr.; Dougherty, D. A. Proc. Natl. Acad. Sci. USA 1996, 93, 10566-71.
26. Mecozzi, S.; West, A. P.; Dougherty, D. A. J. Am. Chem. Soc. 1996, 118, 2307-08.
27. Caldwell, J. W.; Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 4177-8.
28. Meot-Ner, M.; Deakyne, C. A. J. Am. Chem. Soc. 1985, 107, 474-9.
29. Sunner, J.; Nishizawa, K.; Kebarle, P. J. Phys. Chem. 1981, 85, 1814-20.
30. Guo, B. C.; Purnell, J. W.; Castleman Jr.; A. W. Chem. Phys. Lett. 1990, 168, 155-60.
31. Taft, R. W.; Anvia, F.; Gal, J.-F.; Walsh, S.; Capon, M.; Holmes, M. C.; Hosn, K.; Oloumi, G.; Vasanwala, R.; Yazdani, S. Pure Appl. Chem. 1990, 62, 17-23.
32. Ma, J. C.; Dougherty, D. A. Chem. Rev. 1997, 97, 1303-24.
33. Shepodd, T. J.; Petti, M. A.; Dougherty, D. A. J. Am. Chem. Soc. 1986, 108, 6085-87.
34. Petti, M. A.; Shepodd, T. J.; Barrans, J.; R. E.; Dougherty, D. A. J. Am. Chem. Soc. 1988, 110, 6825-40.
35. Dougherty, D. A.; Stauffer, D. A. Science 1990, 250, 1558-60.
36. Murayama, K.; Aoki, K. Chem. Commun. 1997, 119, 23.
37. Chipot, C.; Maigret, B.; Pearlman, D. A.; Kollman, P. A. J. Am. Chem. Soc. 1996, 118, 2998-3005.
38. Aoki, K.; Murayama, K.; Nishiyama, H. J. Chem. Soc., Chem. Commun. 1995, 2221-2.
39. Beene, D. L.; Brandt, G. S.; Zhong, W.; Zacharias, N. M.; Lester, H. A. Biochemistry 2002, 41, 10262-9.
40. Brocchieri, L.; Karlin, S. Proc. Natl. Acad. Sci. USA 1994, 91, 9297-301.
41. Karlin, S.; Zuker, M.; Brocchieri, L. J. Mol. Biol. 1994, 239, 227-48.
42. Burley, S. K.; Petsko, G. A. FEBS Lett. 1986, 203, 139-143.
43. Burley, S. K.; Petsko, G. A. Adv. Protein Chem. 1988, 39, 125-89.
Lett. 1986, 203, 139-43.
44. Singh, J.; Thornton, J. M. Mol. Biol. 1990, 211, 595-615.
45. Mitchell, J. B.; Nandi, C. L.; McDonald, I. K.; Thornton, J. M.; Price, S. L. J. Mol. Biol. 1994, 239, 315-31.
46. Boudon, S.; Wipff, G.; Maigret, B. J. Phys. Chem. 1990, 94, 6056-61.
47. Duffy, E. M.; Kowalczyk, P. J.; Jorgensen, W. L. J. Am. Chem. Soc. 1993, 115, 9271-5.
48. Nandi, C. L.; Singh, J.; Thornton, J. M. Protein Eng. 1993, 6, 247-59.
49. Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I. Science 1991, 253, 872-9.
50. Gribbon, C.; Channon, K. J.; Zhang, W.; Banwell, E. F.; Bromley, E. H. C.; Chaudhuri, J. B.; Oreffo, R. O. C.; Woolfson, D. N. Biochemistry 2008, 47, 10365-71.
51. Whitesides, G. M.; Boncheva, M. Proc. Natl. Acad. Sci. USA 2002, 99, 4769-74.
52. Kar, K.; Ibrar, S.; Nanda, V.; Getz, T. M.; Kunapuli, S. P.; Brodsky, B. Biochemistry 2009, 48, 7959-68.
53. Kar, K.; Amin, P.; Bryan, M. A.; Persikov, A. V.; Mohs, A.; Wang, Y. H.; Brodsky, B. J. Biol. Chem. 2006, 281, 33283-90.
54. Shoulders, M. D.; Raines, R. T. Annu Rev Biochem. 2009, 78, 929-58.
55. Przybyla, D. E.; Chmielewski, J. Biochemistry 2010, 49, 4411-9.
56. Kotch, F. W.; Raines, R. T. Proc. Natl. Acad. Sci. USA 2006, 103, 3028-33.
57. Werten, M. W.; Teles, H.; Moers, A. P.; Wolbert, E. J.; Sprakel, J.; Eggink, G.; de Wolf, F. A. Biomacromolecules 2009, 10, 1106-13.
58. Luo, J.; Tong, Y. W. ACS Nano 2011, 5 (10), 7739-47.
59. Waters, M. L., Curr. Opin. Chem. Biol. 2002, 6, 736–41.
60.黃祥鳴. 博士論文, 國立中央大學, 2002.
61. (a) Fallas, J. A.; Gauba, V.; Hartgerink, J. D. J. Biol. Chem. 2009, 284, 26851–9.
(b) Gauba, V.; Hartgerink, J. D. J. Am. Chem. Soc. 2007, 129, 2683–90.
(c) Gauba, V.; Hartgerink, J. D. J. Am. Chem. Soc. 2007, 129, 15034–41.
(d) Gauba, V.; Hartgerink, J. D. J. Am. Chem. Soc. 2008, 130, 7509–15.
(e) Venugopal, M. G.; Ramshaw, J. A.; Braswell, E.; Zhu, D.; Brodsky, B. Biochemistry 1994, 33, 7948–56.
62. Li, Y.; Mo, X.; Kim, D.; Yu, S. M. Biopolymers 2011, 95, 94-104.
63. (a) Krishna, O. D.; Kiick, K. L. Biomacromolecules 2009, 10, 2626–2631.
(b) Mechling, D. E.; Bachinger, H. P. J. Biol. Chem. 2000, 275, 14532–14536.
(c) Ottl, J.; Battistuta, R.; Pieper, M.; Tschesche, H.; Bode, W.; Kuhn, K.; Moroder, L. FEBS Lett. 1996, 398, 31–6.
64. (a) Shah, N. K.; Ramshaw, J. A.; Kirkpatrick, A.; Shah, C.; Brodsky, B. Biochemistry 1996, 35, 10262–8.
(b) Persikov, A. V.; Ramshaw, J. A.; Brodsky, B. J. Biol. Chem. 2005, 280, 19343–9.
(c) Persikov, A. V.; Ramshaw, J. A.; Kirkpatrick, A.; Brodsky, B. Biochemistry 2000, 39, 14960–7.
65. O’Leary, L. E. R.; Fallas, J. A.; Hartgerink, J. D. J. Am. Chem. Soc. 2011, 133, 5432-43.
66. Byers, P. H.; Cole, W. G. Connective Tissue and Its Heritable Disorders. Wiley-Liss, Inc. 2002, 385-430.
67. Marini, J. C.; Letocha, A. D. Disease of Bone and Mineral Metabolism. endotext.com. 2005.
68. Cabral, W. A.; Milgrom, S.; Letocha, A. D.; Moriarty, E.; Marini, J. C. J. Med. Genet.2006, 43, 685–90.
69. Bodian, D. L.; Madhan, B.; Brodsky, B.; Klein, T. E. Biochemistry 2008, 47, 5424-32.
70. Bretscher, L. E.; Jenkins, C. L.; Taylor, K. M.; DeRider, M. L.; Raines, R. T. J. Am. Chem. Soc. 2001, 123, 777-8.
71. Chorghade, M. S.; Mohapatra, D. K.; Sahoo, G.; Gurjar, M. K.; Mandlecha, M. V.; Bhoite, N.; Moghe, S.; Raines, R. T. J. Fluorine Chem. 2008, 129, 781-4.
72. Sreerama, N.; Woody, R. W. Methods Enzymol. 2004, 383, 318-51.
73. Greenfield, N. J. Nat. Protocols 2007, 1, 2527-35.
74. Fasman, G. D., Circular dichroism and the conformational analysis of biomolecules. Plenum Press 1996, ix, 738.
75. Brookhaven Instruction Manual for 90 plus.
76. Harding, S. E.; Jumel, K. Curr. Protoc. Protein Sci. 2001, 7, 7.
77. Kersteen, E. A.; Raines, R. T. Biopolymers 2001, 59, 24-8.
78. DeRider, M. L.; Wilkens, S. J.; Waddell, M. J.; Bretscher, L. E.; Weinhold, F.;
Raines, R. T.; and Markley, J. L. J. Am. Chem. Soc. 2002, 124, 2497–505.

全文公開日期本全文未授權公開 (校內網路)
全文公開日期本全文未授權公開 (校外網路)

簡易檢索 / 詳目顯示

相關論文