(18.208.187.169) 您好!臺灣時間:2019/10/24 08:01
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
本論文永久網址: 
line
研究生:劉昱攢
研究生(外文):Yu-tsan Liu
論文名稱:以衰減全反射傅氏紅外線光譜技術研究親膜胜肽干擾脂質膜的機制
論文名稱(外文):The ATR-FTIR investigation on lipid-affinity peptide induced membrane perturbation
指導教授:指教授: 張定國, 蔡惠旭
學位類別:碩士
校院名稱:國立中央大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:90
中文關鍵詞:衰減全反射傅氏紅外線光譜穿膜蛋白蜂毒膜擾動次序係數融合胜肽
外文關鍵詞:transmembrane domainorder parameterfusion peptidemelittinATR-FTIR
相關次數:
  • 被引用被引用:0
  • 點閱點閱:198
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
蛋白質與脂質膜會發生多樣的交互作用,例如包膜病毒表面融合蛋白誘導的膜融合和蜂毒蛋白對細胞膜的消溶作用。其中,病毒表面的融合胜肽、胞外區與穿膜蛋白在病毒進入宿主細胞的過程中扮演了重要角色,蜂毒在脂質膜上的排列狀態與結構與其消溶的活性亦息息相關。本論文以衰減全反射霍氐紅外線光譜將上述幾種胜肽在脂質膜中的性質作一番探討。我們以化學合成方法將HIV和Influenza的融合胜肽、穿膜蛋白兩種胜肽合成出來,用E.coli表現胞外區蛋白,與脂質層混合後使用衰減全反射霍氏紅外線光譜偵測,我們發現融合蛋白的蛋白質二級結構以β結構為主,穿膜蛋白則以α螺旋為主。融合蛋白以傾斜角度約50度插入多層膜內部,穿膜蛋白則如同穿膜一樣以較為垂直的33度插入膜內,但依膜組成不同亦有不同角度出現;我們也發現脂質膜的分子排列均會隨融合胜肽或穿膜蛋白加入而更擾動,不過特別的是當HIV融合胜肽與穿膜胜肽等比例共存在脂質膜中時,脂質膜分子排列會趨於穩定,但Influenza沒有這種情況;而HIV和Influenza均可以在C=O吸收的地方,發現融合胜肽與穿膜胜肽在脂質膜中共存時造成的明顯去水作用。胞外區蛋白結構以α螺旋為主,以接近70度的角度,幾乎平躺在脂質膜上;以外加一段融合胜肽的胞外區蛋白比較,其對於脂質膜的擾動作用並不明顯。
蜂毒蛋白在負電性的脂質膜中,有比中性脂質膜更多的α螺旋結構,以ATR-FTIR和之前文獻推測,蜂毒蛋白應該是以4聚體頭尾相接的聚集方式,單體與空間Z軸呈50度斜躺的方式陷入脂質膜表面,形成四邊形的脂質膜孔洞。本論文利用ATR-FTIR技術驗證闡明,HIV與Influenza在後融合時期的脂質膜狀態有所不同,並驗證蜂毒在脂質膜上的排列模型,應該是由四聚體頭尾構成的四邊形孔洞,與前人所提的模型有部分差異。
Protein and lipid interaction is important for biology. For example, fusion protein of enveloped virus would mediate the viral membrane fusion, melittin has the hemolytic effect on cell membrane. Fusion peptide, ectodomain and transmembrane domain of viral fusion protein play pivotal roles in viral entry. Aggregation and the orientation of Melittin in membrane may determine its hemolytic activity. We synthesized HIV and Influenza fusion peptide and transmembrane domain by solid phase synthesis, and HIV ectodomain were expressed by E.coli system. Then ATR-FTIR was used to determine the properties of these lipid-affinity peptide and protein in lipid membrane. We found that fusion peptide has major β-sheet structure and transmembrane domain has major α-helix structure. Fusion peptide has tilt angle respect to membrane normal about 50°. Transmembrane domain has almost vertical angle (33°), but the angle may be different by changing the lipid composition.The lipid molecule will be more perturbation by adding the HIV and Influenza fusion peptide or transmembrane domain individually, however, the HIV fusion peptide and transmembrane domain will make lipid membrane molecule more order when they are together in lipid. We also found fusion peptide and transmembrane domain mixture will make membrane more dehydration than other peptide lonely. Finally, the HIV ectodomain can’t almost make any interaction on membrane comparing other lipid-affinity protein.
Melittin has more α-helix in electro-negative membrane, we thought that melittin should be 4-monomer combined by head to tail attach. The monomer was lying on membrane surface by 50° and formed a quadrangle pore. The new melittin model provided a clue to explain melittin pore-formation mechanism.
中文摘要-------------------------------------------------------------------------i
英文摘要-------------------------------------------------------------------------iii
致 謝----------------------------------------------------------------------------iv
章節目錄-------------------------------------------------------------------------v
圖目錄----------------------------------------------------------------------------vii
表目錄----------------------------------------------------------------------------ix
名詞縮寫及胺基酸序列對照表----------------------------------------------x
第 一 章 研 究 背 景
1-1病毒表面蛋白對細胞膜造成的融合作用---------------------1
1-2蜂毒蛋白對細胞膜造成的影響---------------------------------3
1-3 Melittin在脂質膜中的排列--------------------------------------6
1-4研究動機------------------------------------------------------------6
第 二 章 原理簡介
2-1傅氏轉換紅外線FTIR---------------------------------------------10
2-2 ATR-FTIR----------------------------------------------------------14
2-3 Polarized ATR-FTIR與生物分子的傾斜角-------------------15
第 三 章 實驗方法
3-1 Peptide樣品製備---------------------------------------------------33
3-2 蛋白質表現--------------------------------------------------------38
3-3 peptide與蛋白質定量---------------------------------------------39
3-4 SDS-PAGE----------------------------------------------------------40
3-5 SUV製備------------------------------------------------------------40
3-6 ATR-FTIR實驗操作----------------------------------------------41
第 四 章 實驗結果
4-1 1700~1600 cm-1 Amide I區間------------------------------------43
4-2 C=O 1730 cm-1振動區間-----------------------------------------44
4-3 3000~2800 cm-1區間CH2 symmetric stretching---------------45
4-4 Polarized ATR-FTIR 的amide I區間分析---------------------47
4-5 HIV與Influenza的穿膜蛋白SDS-PAGE 分析-------------48
4-6 結果總結-----------------------------------------------------------48
第 五 章 討 論------------------------------------------------------------76
參考文獻------------------------------------------------------------------------84
1. Weissenhorn,W., A.Hinz, and Y.Gaudin. (2007). Virus membrane fusion. FEBS Letters 581,2150-2155.
2. Nieva,J.L. and A.Agirre. (2003). Are fusion peptides a good model to study viral cell fusion? Biochimica et Biophysica Acta-Biomembranes 1614,104-115.
3. Castano,S. and B.Desbat. (2005). Structure and orientation study of fusion peptide FP23 of gp41 from HIV-1 alone or inserted into various lipid membrane models (mono-, bi- and multibi-layers) by FT-IR spectroscopies and Brewster angle microscopy. Biochimica et Biophysica Acta (BBA) - Biomembranes 1715,81-95.
4. Hunter, E., and Swanstrom, R. (1990). Retrovirus envelope glycoproteins. Curr. Top. Microbiol. Immunol. 157, 187-253.
5. Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J., and Hendrickson, W.A. (1998). Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393, 648-659.
6. Chan, D. C., and Kim, P. S. (1998). HIV entry and its inhibition. Cell 93, 681-684.
7. Chang,D.K. and S.F.Cheng. (2006). pH-dependence of intermediate steps of membrane fusion induced by the influenza fusion peptide. Biochemical Journal 396,557-563.
8. Tatulian,S.A. and L.K.Tamm. (2000). Secondary structure, orientation, oligomerization, and lipid interactions of the transmembrane domain of influenza hemagglutinin. Biochemistry 39,496-507.
9. Sackett,K. and Y.Shai. (2003). How structure correlates to function for membrane associated HIV-1 gp41 constructs corresponding to the N-terminal half of the ectodomain. Journal of Molecular Biology 333:47-58.
10. Haque,M.E., V.Koppaka, P.H.Axelsen, and B.R.Lentz. (2005). Properties and structures of the influenza and HIV fusion peptides on lipid membranes: Implications for a role in fusion. Biophysical Journal 89:3183-3194.
11. Jin,H., Leser G.P., Zhang J., and Lamb R.A. (1997). Influenza virus hemagglutinin and neuraminidase cytoplasmic tails control particle shape. Embo Journal 16:1236-1247.
12. Schwarz,G. and Beschiaschvili,G. (1989). Thermodynamic and kinetic studies on the association of melittin with a phospholipid bilayer. Biochim. Biophys. Acta, 979, 82-80.
13. Habermann,E. (1972). Bee and wasp venoms. Science, 177, 314-322.
14. Dempsey,C.E. (1990). The actions of melittin on membranes. Biochim. Biophys. Acta, 1031, 143-161.
15. Wade,D., Andreu,D., Mitchell,S.A., Silveira,A.M., Boman,A., Boman,H.G., and Terwilliger,T.C. and Eisenberg,E. (1982). Antibacterial peptides designed as analogs or hybrids of cecropins and melittin. J. Biol. Chem, 257, 6016-6022.
16. Schwarz,G., Zong,R. and Popescu,T. (1992). Kinetics of melittin induced pore formation in the membrane of lipid vesicles. Biochim. Biophys. Acta, 1110, 97-104.
17. Tosteson,M.T. and Tosteson,D.C. (1981). The sting. Melittin forms channels in lipid bilayers. Biophys. J., 36, 109-116.
18. Eytan,G.D. and Almary,T. (1983). Melittin-induced fusion of acidic liposomes. FEBS, 156, 29-32.
19. Sybille,R. (1996). Pore formation induced by the peptide melittin in different lipid vesicle membranes. Biophys. Chem., 58, 75-85.
20. Ladokhin,A.S., Selsted,M.E., and White,S.H. (1997). Sizing membrane pores in lipid vesicles by leakage of co-encapsulated markers: pore formation by melittin. Biophys. J., 72, 1762-1766.
21. Terwilliger,T.C., Weissman,L. and Eisenberg,D. (1982). The structure of melittin in the form I crystals and its implication for melittin''s lytic and surface activities. Biophys. J., 37, 353-361.
22. Bazzo,R.,Tappin,M.J.,Pastore,A.,Harvey,T.S.,Carver,J.A.,and Campbell,I.D.
(1988). The structure of melittin. A 1H-NMR study in methanol. Eur. J. Biochem., 173, 139-146.
23. Hermetter,A. and Lakowicz,J.R. (1986). The aggregation state of mellitin in lipid bilayers. An energy transfer study. J. Biol. Chem., 261, 8243-8248.
24. Frey,S. and Tamm,L. (1991). Orientation of melittin in phospholipid bilayers. A polarized attenuated total reflection infrared study. Biophys. J., 60, 922-930.
25. Lee,T.H., Mozsolits,H. and Aguilar,M.I. (2001). Measurement of the affinity of melittin for zwitterionic and anionic membranes using immobilized lipid biosensors. J.Peptide Res., 58, 464-476.
26. Bello,J., Bello,H.R. and Granados,E. (1982). Conformation and aggregation of melittin: dependence on pH and concentration. Biochemistry, 21, 461-465.
27. Gauldie,J., Hanson,J.M., Rumjanek,F.D., Shipolini,R.A. and Vernon,C.A. (1976). The peptide components of bee venom. Eur. J. Biochem., 61, 369-376.
28. Schubert,D., Pappert,G. and Boss,K. (1985). The nature of the stable noncovalent dimers of band 3 protein from erythrocyte membranes in solutions of Triton X-100. Biophys. J., 48, 327-329.
29. Ikura,T., Go,N., and Inagaki,F. (1991). Refined structure of melittin bound to perdeuterated dodecylphosphocholine micelles as studied by 2D-NMR and distance geometry calculation. Proteins, 9, 81-89.
30. Dempsey,C.E., and Butler,G.S. (1992). Helical structure and orientation of melittin in dispersed phospholipid membranes from amide exchange analysis in situ. Biochemistry, 31, 11973-11977.
31. Anderluh,G., M.D.Serra, G.Viero, G.Guella, P.Macek, and G.Menestrina. (2003). Pore Formation by Equinatoxin II, a Eukaryotic Protein Toxin, Occurs by Induction of Nonlamellar Lipid Structures. J Biol Chem 278:45216-45223.
32. 李遠鵬 (1975) 科儀產品新知. 72, 29~35.
33. Tamm L.K. and Tatulian S.A. (1997) Infrared spectroscopy of proteins and peptides in lipid bilayers. Quart. Rev.Biophy. 30, 365-429.
34. Casal, H. L. and Mantsch, H. H. (1984) Polymorphic phase behaviour of phospholipids membranes studied by infrared spectroscopy. Biochim. Biophys. Acta. 779, 381-401.
35. Mantsch, H.H. and McElhaney, R. N. (1991) Phospholipid phase transitions in model and biological membranes as studied by infrared spectroscopy. Chem. Phys. Lipid 57, 213-226.
36. Casal,H. L. and McElhaney, R. N. (1990) Quantitive determination of hydrocarbon chain conformational order in bilayers of saturated phosphatidyl-cholines of various chain lengths by Fourier transform infrared spectroscopy. Biochemistry 29, 5423-5427.
37. Goni, F. M. and Arrondo, J. L. R. (1986) A study of phospholipid phosphate groups in model membranes by Fourier transform infrared spectroscopy. Faraday Discuss. Chem. Soc. 81, 117-126.
38. Gomez-Fernandez, J. C. and Villalain, J. (1998) The use of FT-IR for quantitative studies of the apparent pKa of lipid carboxyl groups and the dehydration degree of the phosphate group of phospholipid. Chem. Phys. Lipid 96, 41-52.
39. Krimm, S. and Bandekar, J. (1986) Vibrational spectroscopy and conformation of peptides, polypeptides, and protein. Adv. Protein Chem. 38, 181-365.
40. Surewicz, W. K. Mantsch, H. H. and Chapman, D. (1993) Determination of protein sccondary structure by Fourier transform infrared spectroscopy: a critical assessment. Biochemistry 32, 389-394.
41. Arrondo, J. L. R., Blanco, F. J. Serrano, L. and Goni, F. M. (1996) Infrared evidence of a β-hairpin peptide structure insolution. FEBS letters 384, 35-37.
42. Chehin, R. Iloro, I., Marcos, M. J., Villar, E., Shnyrov, V. L. and Arrondo, J. L. R. (1999) Thermal and pH-induced conformational changesof a β-sheet protein monitored by infrared spectroscopy. Biochemistry 38, 1525-1530.
43. de Jongh, H. H. J., Goormaghtigh, E. and Ruysschaert, J.-M. (1997) Amide-protein exchange of water-soluble proteins of different structural classes studied at the submolecular level by infrared spectroscopy. Biochemistry 36, 13603-13610.
44. Raussens, V., Narayanaswami, V., Gooemaghtigh, E., Ryan, R. O. and Ruysschaert, J.-M. (1996) Hydrogen/Deuterium exchange kinetics of apolipophorin-III in lipid-free and phospholipid-bound states. J. Biol. Chem. 271, 23089-23095.
45. Griebenow, K. and Kilibanov, A. M. (1995) Lyophilization-induced reversible changes in the secondary structure of proteins. Proc. Natl. Acad. Sci. USA. 92, 10969-10976.
46. Rahmelow, K., Hubner, W. and Ackermann, T. (1998) Infrared absorbances of protein side chains. Analy. Biochem. 257, 1-11.
47. Longas, M. and bBreitweiser, K. L. (1991) Sulfate composition of glycosamino-glycans determined by infrared spectroscopy. Analy. Biochem. 192, 193-196.
48. Lijour, Y. Gentric, E. Deslandes, E. and Guezennec, J. (1994) Estimation of the sulfate content of hydrothermal vent baterial polysaccharides by Fourier transform infrared spectroscopy. Analy. Biochem. 220, 244-248.
49. Grant, D., Long, W. F., Moffat, C. F. and Williamson, F. B. (1991) Infared spectroscopy of heparins suggests that the region 750-950cm-1 is sensitive to change in iduronate residue ring conformation. Biochem. J. 275, 193-197.
50. Cheng, H., Sukal, S., Callender, R. and Leyh, T. (2001) r-Phosphate protonation and pH-dependent unfolding of the Ras-GTP.Mg2+ complex. J. Biol. Chem. 276, 9931-9935.
51. El-Mabdaoui, L., Neault, f. F. and Tajmir-Riabi, H. A. (1997) Carbohydrate-nucleotide interaction. The effects of mono- and disaccharides on the solution structure of AMP, dAMP, ATP, GMP, dGTP, and GTP studied by FTIR difference spectroscopy. J. Inorg. Biochem. 65, 123-131
52. Jamin, N., Dumas, P., Moncuit, J., Fridman, W.H., Teillaud, J.L., Carr, G.L. and Williams, G.P. (1998) Highly resolved chemical imaging of living cells by using synchrotron infrared microspectrometry. Proc. Natl. Acad. Sci. U. S. A. 95, 4837-4840.
53. Wetzel, D. L. and LeVine, S. M. (1999) Imaging molecular chemistry with infrared microscopy. Science 285, 1224-1225.
54. Dong, A., Huang, P. and Caughey, W.S. (1990) Protein secondary structures in water from second-derivative amide I infrared spectra. Biochemistry 29, 3303-3308.
55. Susi, H. and Byler, D. M. (1986) Resolution-enhanced Fourier transform infrared spectroscopy of enzymes. Methods Enzymol. 130, 290-311.
56. James, D. I., Maddams, W. F. and Tooke, P. B. (1987) The use of Fourier deconvolution in infrared spectroscopy. Part I: studies with synthetic single-peak system. Appl. Spectrosc. 41, 1362-1370.
57. Moffatt, D. J. and Mantsch, H. H. (1992) Fourier resolution enhancement of infrared spectral data. Methods Enzymol. 210, 192-200.
58. Goormaghtigh, E., Raussens, V. and Ruysschaert, J.-M. (1999) Attenuated total reflection infrared spectroscopy of proteins and lipids in biological membranes. Biochim. Biophys. Acta. 1422, 105-185.
59. Harrick N. J. (1979) Internal reflection spectroscopy. Ossining, New York.
60. Tamm, L.K. and Tatulian, S.A. (1993) Orientation of functional and nonfunctional PTS permease signal sequences in lipid bilayers. A polarized attenuated total reflection infrared study. Biochemistry 32, 7720– 7726.
61. Oberg, K.A. and Fink, A.L. (1998) A new attenuated total reflectance Fourier transform infrared spectroscopy method for the study of proteins in solution. Anal. Biochem. 256, 92-106.
62. Marsh, D., Muller, M. and Schmitt, F.J. (2000) Orientation of the infrared transition moments for an alpha-helix. Biophys J. 78, 2499-510.
63. Marsh, D. (1997) Dichroic ratios in polarized Fourier transform infrared for nonaxial symmetry of beta-sheet structures. Biophys. J. 72, 2710-2718.
64. Lafrance, C.-P., Nabet, A., Prud’homme, R. E. and Pezolet, M. (1995) On the relationship between the order parameter and the shape of orientation distributions. Can. J. Chem. 73, 1497-1505.
65. Picard, F., Buffeteau, T., Desbat, B., Auger, M. and Pezolet, M. (1999) Quantitative orientation measurements in thin lipid films by attenuated total reflection infrared spectroscopy. Biophys. J. 76, 539-551.
66. Ter-Minassian-Saraga, L., Okamura, E., Umemura, J. and Takenaka, T. (1988) Fourier transform infrared-attenuated total reflection spectroscopy of hydration of dimyristoylphosphatidylcholine multibilayers. Biochim. Biophys. Acta. 946, 417-423.
67. Okamura, E., Umemura, J. and Takenaka, T. (1985) Fourier transform-infrared-attenuated total reflection spectra of dipalmitoylphosphatidylcholine monomolecular films. Biochim. Biophys. Acta. 812, 139-146.
68. Pace,C.N., F.Vajdos, L.Fee, G.Grimsley, and T.Gray. (1995). How to Measure and Predict the Molar Absorption-Coefficient of A Protein. Protein Science 4,2411-2423.
69. Okamura, E., Umemura, J. and Takenaka, T. (1986) Orientation of gramicidin D incorporated into phospholipid multibilayers: a Fourier transform infrared-attenuated total reflection spectroscopic study. Biochim. Biophys. Acta. 856, 68-75.
70. 黃維寧,眼鏡蛇心臟毒素在細胞膜形成孔洞之機制研究,國立清華大學,博士論文,民國92年
71. 陳振瑞,具生物活性寡胜肽之生化性質研究:HIV抑制物之機制及蜂毒與胞膜之作用,國立中央大學,碩士論文,民國92年
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊
 
系統版面圖檔 系統版面圖檔