研究生: |
朱冠宇 Kuan Yu Chu |
---|---|
論文名稱: |
溶菌酶展開的熱力學性質與模型的探討 A Study on the Model of the Unfolding of Lysozyme and its Thermodynamic Properties |
指導教授: |
林聖賢
Lin, Sheng-Hsien 林震煌 Lin, Cheng-Huang |
學位類別: |
碩士 Master |
系所名稱: |
化學系 Department of Chemistry |
論文出版年: | 2009 |
畢業學年度: | 97 |
語文別: | 中文 |
論文頁數: | 93 |
中文關鍵詞: | 溶菌酶 、蛋白質展開模型 、螢光光譜 、圓光二向光譜 |
英文關鍵詞: | Lysozyme, Two state model, Ising model, temperature induced unfolding, denaturant induced unfolding, Fluorescence spectrum, circular dichroism spectrum |
論文種類: | 學術論文 |
相關次數: | 點閱:223 下載:0 |
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蛋白質結構與其生理功能的完整性息息相關,處於正常折疊狀態下的蛋白質可以有效發揮其生理功能且十分穩定,但不正常的折疊會造成蛋白質失去其功能並且處於較不穩定的狀態,一些疾病如阿茲海默症與狂牛病等等,其起因皆是蛋白質的摺疊錯誤。為了了解蛋白質摺疊與展開的機制,並且對這些疾病的形成能有進一步地瞭解,甚至可以成為於該疾病的預防或治療,在本研究中,利用本實驗室所提出的Ising model來描述每個蛋白質的展開行為。在本實驗室所建構的模型裡,我們將蛋白質視為由許多個單元σi(unit)所組成,每一個單元可以是肽鍵、胺基酸、雙硫鍵或任何蛋白質摺疊的單位,而每個單元只有兩個狀態,分別為自然摺疊狀態或展開狀態,而不同的單元卻擁有相同的熱力學性質,則可以將這幾個單元視為相同一個摺疊子。在實驗上,我們選用溶菌酶當做研究對象,藉由加入變性劑或提高溫度,讓溶菌酶逐漸展開,並利用螢光光譜還有圓光二向光譜對溶菌酶做偵測,藉以得知細溶菌酶展開時局部結構的變化,我們發現Ising model可以成功地描述了在實驗上所得到的數據,結果顯示這二種光譜所偵測到的過渡曲線在不同的環境下會有所不同;並且可以將這二種光譜所測到的局部位置各視為數個單元,其中在螺旋結構局部位置(α-helix local site)可以視為三個單元所組成,而色氨酸局部位置(Trp local site)則可是為一個個別的單元,與傳統的兩態模型不同的是:藉由Ising model的擬合,不僅得知其局部位置的熱力學穩定度,而且還得知其局部位置與其他單元的作用力 J。並且藉由比較小角度散射的實驗,可以清楚明白的知道溶菌酶在展開時整個構型的改變,和光譜偵測的結果比較下,我們發現其展開的構型改變主要來自於螺旋結構局部位置展開的貢獻。
It is well knowing that the function of a protein is related to its psychology properties. Knowing the folding-unfolding processes of proteins is helpful in the search for possible remedies to a disease that’s caused by protein misfolding such as Alzheimer’s disease. In order to study the thermodynamic properties of the unfolding of proteins, lysozyme was chosen as the model protein. Equilibrium unfolding behaviors of lysozyme, induced by the presence of urea,GdHCl or changed in temperature, were examined via different spectroscopic techniques, such as a fluorescence spectrometer and circular dichroism spectrometer. The local structural changes can be detected by those spectrometers. The experimental data was fitted successfully to the modified Ising model, and obtained the thermodynamic parameters for the unfolding of lysozyme at different environments. for example, the temperature induced denature free energy for each local site. The results indicated that there are several unfolding grouping in α-helix local site and only a group in Trp local site . By comparing the spectroscopic data with the data from small angle X-ray scattering, we found that the native lysozyme is close to an ellipsoid envelope. This envelope elongated gradually at higher temperature. However, during the unfolding of the α-helix local site and the Trp local site, the semi-major axis of lysozyme elongated dramatically, whereas the semi-minor axis stayed roughly the same.
1. Dobson C. M. ; Philos. Trans. R. Soc. B. 2001 , 356 , 133
2. Shiu Y. J. ; Yeh Y. L. ; Liang K. K. ; Hayashi M. ; Mo Y. ; Yan Y. ; Lin S. H. ; J.Chin.Chem. Soc. 2004 , 51 , 1161.
3. Varhac R. ; Antalik M. ; Bano M. ; J Biol Inorg Chem. 2004 , 9 , 12
4. Latypov R. F. ; Cheng H. ; Roder N.A. ; J.Mol.Biol. 2006 , 357 , 1009.
5. Maity H. ; Maity M. ; Krishna M. M. G. ; Mayne L. ; Englander S. W. ; Proc. Natl. Acad. Sci. USA. 2005 , 102 , 4741
6. Fleming A. ; Proc Roy Soc Ser B 1992 93 306
7. Blake C.C. ; Koenig D.F. ; Mair G.A. ; North A.C. ; Phillips D.C. ; Sarma V.R. ; Nature 2001 , 206 , 757
8. JOHNSON L.N. ; PHILLIPS D. C. ; Nature 1995 , 206 , 761
9. Kirby A.J. Nature structural biology 2001 , 8 , 737-739
10. Privalov P. L. ; Khechinashvili N. N. ; J. Mol. Biol. 1974 , 86 , 665.
11. Chen L.L. ; Hodgson K.O. ; Doniach S. ; J.Mol.Biol. 1996 , 261 , 658
12. Sasahar K. ; Demura M. ; Nitta K. ; PROTEINS: Structure, Function, and Genetics 2002 , 49 , 472
13. Sasahar K. ; Demura M. ; Nitta K. ; Biochemistry 2000 , 39 , 6475
14. Ibarra-Molero B. ; Sanchez-Ruiz J.M. ; Biochemistry 1997 , 36 , 9616
15. Yamamoto T. ; Fukui N. ; Hori A. ; Matsui Y. ; Journal of Molecular Structure 2006 , 782 , 60
16. Krishna M. M. G. ; Maity H. ; Rumbley J. N. ; Lin Y. ; Englander S. W. ; J. Mol. Biol. 2006 , 359 , 1410.
17. Bakk A. ; Hoye J. S. ; Physica A. 2003 , 323 , 504.
18. Liang K. K. ; Hayashi M. ; Shiu Y. J. ; Mo Y. ; Shao J. ; Yan Y. ; Lin S. H. Phys. Chem. Chem. Phys. 2003 , 5 , 5300.
19. Shiu Y. J. ; Jeng U. S. ; Su C. ; Huang Y. S. ; Hayashi M. ; Liang K. K. ; Yeh Y. L. ; Lin S. H. ; Journal of Applied Crystallography, 2007 , 40 , s195.
20. Xu Q. ; Keiderling T. A.; Protein Science, 2004 , 13 , 2949
21. Zhong L. ; Johnson W. C. ; Pro. Natl. Acad. Sci. 1992 , 89 , 4462.
22. Waterhous A. V. ; Johnson W. C. ; Biochemistry. 1994 , 33 , 2121.
23. Alison R. ; Bengt N. ; Circular Dichroism and Linear Dichroism
24. Proctor V.A. ; Cunning F.E. ; CRC critical review in food science and Nutrition 1998 , 26 , 359
25. Privalov P. L. ; FEBS Letter 1974 , 40 , s140.
26. Eftink M.R. ; Ghirron C.A. ; Biochemistry 1976 , 15 , 672.
27. Huang Y.S. ; Jeng U.S. ; Shiu Y.J. ; Lai Y.H. ; Sun Y.S. ; Journal of Applied Crystallography, 2007 , 40 , s165
28. Glatter O. ; Kratky O. ; Small angle X-ray scattering Ch.1 and Ch. 2. 1983, London Academic Press
29. Leninger A. Principles of biochemistry 4th edition
30. Eaton W.A. ; Munoz V. ; Thompson P.A. ; Henry R. ; Hofrichter J. ; Acc.Chem.Res ; 1998, 31, 745
31. Huang R. ; Setnicka V. ; Etienne M. A. ; Kim J. ; Kubelka J. ; Hammer R. P. ; Keiderling T.A. J.Am.Chem.Soc. 2007, 129, 13592
32. Guo J. ; Harn N. ; Robbin A. ; Dougherty R. ; Middaugh C. R. Biochemistry 2006, 45, 8686
33. Munoz V. ; Thompson P.A. ; Hofrichter J. ; Eaton W.A. Nature 1997, 390, 196
34. Hauser K. ; Krejtschi C. ; Huang R. ; Wu L. ; Keiderling T.A. J.Am.Chem.Soc. 2008, 130, 2984
35. Setnicka V. ; Huang R. ; Thomas L. ; Etienne M.A. ; Kubelka J. ; Hammer R.P. ; Keiderling T.A. ; J.Am.Chem.Soc. 2005, 127, 4992
36. Matagne A. ; Dobson C.M. ; CMLS 1998, 54, 363
37. Arial S. ; Hirai M. ; Biophysical journal, 1999, 76, 2129
38. Yang W.Y. ; Gruebele M. ; J.Am.Chem.Soc., 2004, 126, 7785
39. Takekiyo T. ; Wu L. ; Yoshimura Y. ; Shimizu A. ; Keiderling T.A. ; Biochemistry., 2009, 48, 1543
40. Zhao C. ; Poavarapu P.L. ; Das C. ; Balaram P. ; J.Am.Chem.Soc., 2000, 122, 8228
41. Hilario J. ; Kubelka J. ; Keiderling T.A. ; J.Am.Chem.Soc., 2003, 125, 7562
42. Du D. ; Zhu Y. ; Huang C.Y. ; Gai F. ;PNAS, 2004, 101, 15915
43. Bour P. ; Keiderling T.A. ; J. Phys. Chem. B, 2005, 109, 23678
44. Hammed M. ; Ahmad B. ; Fazili K.J. ; Andrabi K. ;Khan R.H. ; J. Biochem, 2007, 141, 573
45. Chowdhry B. ; Leharne S. ; Journal of chemical education, 1997, 74, 236