研究生: |
林駿達 Lin, Chun Ta |
---|---|
論文名稱: |
探討高自組裝性之短胜肽序列對膠原蛋白模擬胜肽摺疊和自組裝性質影響 The Effects of High Self-Assembled Oligopeptides on the Folding and Self-Assembly of Collagen Mimetic Peptides |
指導教授: |
洪嘉呈
Horng, Jia Cherng |
口試委員: |
江昀緯
Chiang, Yun Wei 許馨云 Hsu, Hsin Yun |
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: | 2015 |
畢業學年度: | 103 |
語文別: | 中文 |
論文頁數: | 98 |
中文關鍵詞: | 膠原蛋白 、細胞毒性實驗 、自組裝 、三股螺旋 |
外文關鍵詞: | Collagen, Cytotoxicity experiment, Self-assemble, Triple helix |
相關次數: | 點閱:4 下載:0 |
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膠原蛋白是哺乳動物中含量最多的蛋白質,目前已廣泛用在生醫材料上。為了增加其結構穩定度和應用性,我們設計並製備膠原蛋白模擬胜肽。主要的構想是將具有高自組裝性質的胺基酸序列當成基底,並接上膠原蛋白序列,合成新的膠原蛋白模擬胜肽,目的是利用高自組裝性質的胺基酸序列來幫助原始的膠原蛋白序列形成更穩定且高階的結構,並探討其抑制其它蛋白質聚集的可能性。
研究工作分為兩部分,第一部分是接續研究之前本實驗室所設計和製備的胜肽,這些胜肽是由膠原蛋白序列與貝它類澱粉蛋白序列這兩條序列片段所組成的新胜肽鏈。之前的光譜量測顯示,這些合成的新胜肽中,有一些能夠抑制類澱粉蛋白Ab聚集,有一些在TEM下可以觀察到有別於典型膠原蛋白和類澱粉蛋白這兩種蛋白所形成的纖維。故在第一部分,旨在探討這些胜肽鏈是否對於生物神經細胞會產生毒性,鑑定其是否可以成為新的材料或者是具有潛力的抑制藥物。因這些含Ab序列的膠原蛋白胜肽均具細胞毒性,因此在第二部分,我們選用CILFWG 這段胺基酸序列來做研究,將其接於膠原蛋白相關序列(POG)n之胜肽鏈,共合成五段胜肽: CILFWG、CILFWG(POG)7 、(POG)7CILFWG、OG(POG)4CILFWG和 (POG)7。利用可見光紫外光分光光譜儀、TEM、CD來探討新胜肽鏈的結構特性。
我們由CILFWG(POG)7這段胜肽的實驗結果發現此一具高自組裝性的短序列胜肽除了能夠穩定膠原蛋白摺疊,亦能幫助其自組裝形成大型結構。我們認為CILFWG(POG)7能夠繼續進行其他生物性實驗,來進一步探討其成為新的生物材料之可能性。
Collagen, the most abundant protein in mammals, has been widely used in biomedical materials. In order to increase the structural stability of collagen, we designed and synthesized the collagen mimetic peptides (CMPs) in which an oligopeptide with a high self-assembly propensity was attached. By this design, we expected that the oligopeptide could stabilize the collagen triple helix and assist their self-assembly into higher order structures.
Since some of the CMPs containing Ab(16-22) sequence showed the ability to inhibit the aggregation of A protein in our previous studies, in the first part of this study, we further investigated their cytotoxicity by MTT assay. The results indicate that these CMPs are actually toxic to a Neuro cell N2a.
Therefore, in the second part, we chose another oligopeptide CILFWG as an attachment to CMPs. From this design, we synthesized a series of CMPs : CILFWG(POG)7 , (POG)7CILFWG, and OG(POG)4CILFWG. The collagen peptide (POG)7 and CILFWG peptide were also synthesized for comparison. We used UV-VIS spectroscopy, TEM, and CD to characterize these peptides. The results showed that the oligopeptide could stabilize the collagen triple helices and assist their self-assembly into higher order structures. In particular, this oligopeptide has a more pronounced effect on CILFWG(POG)7 than any other peptide studied. Thus, this oligopeptide could be potentially useful in designing stable collagen assemblies and related biomaterials.
1. 張峻銘.(2014) 膠原蛋白模擬胜肽對貝它類澱粉蛋白Ab(16-22)聚集探討,碩士學位論文,清華大學化學研究所.
2. Bella, J., Brodsky, B., and Berman, H. M. Hydration structure of a collagen peptide. Structure 1995;3:893-906
3. Selkoe, D. J. Cell biology of protein misfolding : the example of Alzheimer's and Parkinson's diseases. Nat Cell Bio 2004;6:1054-61
4. Stefani, M., and Dobson, C. Protein aggregation and aggregate toxicity : new insights into protein folding, misfolding diseases and biological evolution. J Mol Med 2003;81:678-99
5. Truant, R., Atwal, R. S., Desmond, C., Munsie, L., and Tran, T. Huntington's disease : revisiting the aggregation hypothesis in polyglutamine neurodegenerative diseases. FEBS J 2008;275:4252-62
6. Höppener, J. W. M., Ahrén, B., and Lips, C.J.M. Islet amyloid and type 2 diabetes mellitus. N Engl J Med 2000;343:411-9
7. Irvine, G., Elagnaf, OM., Shankar, GM., Walsh, DM. Protein aggregation in the brain : the molecular basis for Alzheimer's and Parkinson's diseases. Mol Med 2008;14:451-64
8. Tjernberg, L. O., Callaway, D. J. E., Tjernberg, A., Hahne, S., Lilliehöök, C., Terenius, L., Thyberg, J., and Nordstedt, C. A molecular model of Alzheimer amyloid b-Peptide fibril formation. J Biol Chem 1999;274:12619-25
9. Tjernberg, L. O., Näslund, J., Lindqvist, F., Johansson, J., Karlström, A. R., Thyberg, J., Terenius, L., and Nordstedt, C. Arrest of amyloid fibril formation by a pentapeptide ligand. J Biol Chem 1996;271:8545-8
10. Balbach, J. J., Ishii, Y., Antzutkin, O. N., Leapman, R. D., Rizzo, N. W.,Dyda, F., Reed, J. , and Tykyo, R. Amyloid fibril formation by Ab16-22, a seven-residue fragment of the Alzheimer's b-amyloid peptide, and structural characterization by solid state NMR. Biochemistry 2000;39:13748-59
11. Wang, Q., Liang, G., Zhang, M., Zhao, J., Patel, K., Yu, X., Zhao, C., Ding, B., Zhang, G., Zhou, F., Zheng, J. De novo design of self-assembled hexapeptides as b-Amyloid (Ab) peptide inhibitors. ACS Chem Neurosci 2014;5:972-81
12. 徐維.(2011) 利用組胺酸-金屬配位鍵與 cation-π 作用力來進行膠原蛋白自組裝之探討,碩士學位論文,清華大學化學研究所.
13. Cowan, P. M., Stewart, M., North, A. C. T. The polypeptide chain configuration of collagen. Nature 1995;176:1062-4
14. Rich, A., and Crick, F. H. C. The structure of collagen. Nature 1955;176:915
15. Rich, A., and Crick, F. H. C. The molecular structure of collagen. J Mol Biol 1961;3:483-4
16. 陳伯翰、劉中行. 科學發展期刊 2004;380:4-35
17. 黃彥覆、湯正明、徐善慧. 科學發展期刊 2003;362:44-7
18. Sakakibara, S., Inouye, K.,Shudo, K., Kishida, Y., Kobayashi, Y., and Prockop, D.J. Sythesis of (Pro-Hyp-Gly)n of defined molecular weights evidence for the stabilization of collagen triple helix by hydroxyproline. Biochim Biophys Acta 1973;303:198-202
19. Bella, J., Eaton, M., Brodsky, B.,and Berman, H. Crystals and molecular structure of a collagen-like peptide at 1.9 Å resolution. Science 1994;266:75-81
20. Hinderaker, M. P., and Raines, R. T. An electronic effect on protein structure Protein Sci 2003;12:1188-94
21. Brodsky, B., and Ramshaw, J. A. M. The collagen triple-helix structure. Matrix Biol 1997;15:545-54
22. Primalov, P. L. Stability of proteins : proteins which do not present a single cooperative system. Protein Chem 1982;35:1-104
23. Whitesides, G. M., Boncheva, M. Beyond molecules : self-assembly of mesoscopic and macroscopic components. Proc Natl Acad Sci USA 2002;99:4769-74
24. Przybyla, D. E., Chmielewski, J. Higher-order assembly of collagen peptides into nano- and microscale materials. Biochemistry 2010;49:4411-9
25. Kotch, F. W., Raines, R. T. Self-assembly of synthetic collagen triple helices. Proc Natl Acad Sci USA 2006;103:3028-33
26. Werten, M. W., Teles, H., Moers, A. P., Wolbert, E. J., Sprakel, J., Eggink, G.,de Wolf, F. A. Precission gels from collagen-inspired triblock copolymers. Biomacromolecules 2009;10:1106-13
27. Pederson, S. L., Tofteng, A. P., Malik, L., and Jenson, K. J. Microwave heating in solid-phase peptides synthesis. Chem Soc Rev 2012;41:1826-44
28. Merrifield, R. B. Solid phase peptide synthesis. I. The synthesis of a tetrapeptides. J Am Chem Soc 1963;85:2149-54
29. 張湘戎.(2003) 體抗素胜肽分子內雙硫鍵建構之研究,碩士學位論文,中原大學化學研究所.
30. Berova, N., Nakanishi, K., and Woody, R. Circular dichroism : principles and applications ,Wiley, Hoboken. 2000
31. Whitmore, L., and Wallace, B. A. Protein secondary structure analyses from circular dichroism spectroscopy : methods and reference databases. Biopolymers 2008;89:392-400
32. 陳力俊、張立、梁鉅銘、林文台、楊哲人、鄭晃忠.(2004) 材料電子顯微鏡學,行政院國家科學委員會精密儀器發展中心
33. Bretcher, L. E., Jenkins, C. L., Taylor, K. M., Derider, M. L., and Raines, R. T. Conformational stability of collagen relies on a stereoelectronic effect. J Am Chem Soc 2001;123:777-8
34. Leonard, D. W., and Meek, K. M. Refractive indice of the collagen fibrils and extrafibrillar material of the corneal stroma. Biophys J 1997;72:1382-7
35. 陳佳青.(2009) Cation-π 作用力對膠原蛋白穩定性及自組裝影響之探討,碩士學位論文,清華大學化學研究所.
36. Brookhaven instruction manual for 90 plus.
37. Chen, Y. -S., Chen, C.-C., and Horng, J.-C. Thermodynamic and kinetic consequences of substituting glycine at different position in a Pro-Hyp-Gly repeat collagen model peptide. Biopolymers(pept Sci) 2011;96:60-8
38. Ackerman, M. S., Bhate, M., Shenoy, N., Beck, K., Ramshaw, J. A. M., and Brodsky, B. Sequence dependence of the folding of collagen-like peptides : single amino acids affect the rate of triple-helix nucleation. J Biol Chem 1999;274:2713-21