跳到主要內容

臺灣博碩士論文加值系統

(3.231.230.177) 您好!臺灣時間:2021/07/28 23:24
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
: 
twitterline
研究生:高亦芬
研究生(外文):Yi-Fen Kao
論文名稱:奈瑟氏腦膜炎雙球菌之膜蛋白Ag473不同長度重複串聯序列(EAVTEAK)的結構特性分析
論文名稱(外文):Structural Basis of Neisseria meningitidis Ag473 Proteins, the Membrane-associated Proteins with Different Tandem Repeats (EAVTEAK)
指導教授:楊秋英
指導教授(外文):Chiou-Ying Yang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:86
中文關鍵詞:奈瑟氏腦膜炎雙球菌核磁共振蛋白
外文關鍵詞:Neisseria meningitidisNMRprotein
相關次數:
  • 被引用被引用:0
  • 點閱點閱:122
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
腦膜炎雙球菌(Neisseria meninggitidis)膜上新發現的脂蛋白Ag473,擁有七個胺基酸的重複串聯序列(EAVTEAK),其相關研究仍很少。目前證據顯示當感染宿主細胞時此蛋白會大量表現,加上此蛋白在目前所有檢驗過的腦膜炎雙球菌上皆存在,顯示出Ag473蛋白極具潛力開發成疫苗。本研究著重以生物物理方式了解此蛋白結構上的訊息並嘗試比較不同長短的串聯序列功用為何。為排除載體本身表現的殘基、訊息序列及Lipobox motif,我們重新構築一個及三個串聯序列的Ag473-1及Ag473-3於pET30a表現系統,大量表現蛋白並純化取得。根據序列組成可知蛋白質內帶電性胺基酸多、疏水性胺基酸少,蛋白質電泳分析結果分子量位置也大於預期結果。圓二色光譜(circular dichroism)顯示,蛋白在pH 7.0 生理環境之二級結構呈現結構鬆散(random coil)的型態,判斷是因為生理狀態下疏水核心無法形成,胺基酸間彼此電荷排斥,鬆散的結構與水結合較多使得水合半徑較大,顯示Ag473為非序化蛋白(disordered protein)。兩蛋白收集到的核磁共振(nuclear magnitude resonance ) HSQC圖譜相似且可以重疊,顯示蛋白在重複串聯序列以外的部分立體結構應相似。加入有機溶劑(MeOH,EtOH,TFE)的實驗中,所有溶劑皆能在一定濃度以上誘導蛋白產生α-螺旋,且Ag473-1及Ag473-3誘導出的二級結構類似;但在加入SDS給予微胞(micelle)環境,誘導出α-螺旋時,Ag473-3比 Ag473-1明顯提早形成螺旋之二級結構。以 Ag473-3蛋白為進一步研究目標,用核磁共振光譜觀測SDS加入蛋白溶液後的圖譜變化,配合部分骨架判讀完成的Ag473-3結果及判讀完成的Ag473-1’結果,可知蛋白質以C端E95-E107 12個左右的殘基與SDS作結合,其餘N端殘基暴露於水溶液中。配合螺旋輪(helical wheel)預測出重複串聯序列為兩性螺旋結構(amphipatic helix),我們提出Ag473蛋白本身為非序化蛋白,透過給予類似細胞膜的環境會誘導其形成延展的螺旋式結構(extended helical structure),是以C端和膜作結合,其餘序列也在模擬膜環境中誘導成螺旋結構貼覆於膜表面。此模型的正確性還必須要做更高解析度的實驗才得以驗證此模型的正確性。整體而言,此結果有助於提升有效疫苗的製備,透過加入微脂粒(liposome)或微胞環境(micelle),能誘導出具有構型的Ag473蛋白,提升免疫辨識力。
Ag473 protein, which has heptads tandem repeats (EAVTEAK), was recently identified as a lipoprotein on the outer membrane of Neisseria meninggitidis. Not many studies on this protein have been done. However, prelimilary results revealed that the protein is upregulated during infection and the proteins can be found on outer membrane of every NM that examined. All these results suggest Ag473 is a potential vaccine candidate. Our study focuses on finding out the structural basis about Ag473 protein of four types of tandem repeats and what role the heptads tandem repeats will play of in the whole protein structure. I re-constructed Ag473-1 and Ag473-3 into pET30a expression system to get rid off redundant vector-expressed residues, signal peptide and Lipobox motif. After expressing and purification process, non-labeled form and labeled form of proteins were obtained. Further analysis of the composition of protein sequence reveals low content of hydrophobic residues and high proportion of particular polar and charged amino acids. Circular dichroism (CD) spectra show that the secondary structure of both Ag473-1 and Ag473-3 are in random coil under physiology environment (pH 7.0). A possible explanation is unbalance in the competition between the charge repulsion driving unfolding and hydrophobic interactions driving folding. When running protein electrophoresis, the molecular weights of proteins on SDS-PAGE seem practically larger than original estimation. It also gives a hint to a extended hydrodynamic dimension of the protein due to the loosely folded compaction. Therefore, Ag473 protein is a disordered protein. When 2D Nuclear Magnitude resonance (NMR) spectra of the two proteins are compared, most of the signals on the HSQC spectra can overlay very well, which mean the two proteins possess the same conformation except the region of tandem repeats. In a quest to simulate the effect of membrane fields on Ag473, addition of mixtures of water with organic solvent (methanol, ethanol, triflouroethonal) was performed. Both proteins were induced to have high helical content with similar CD absorbing tendency in the three different kinds of alcohol. In addition, Micelle-forming detergent created by sodium dodecyl sulfate (SDS) induces helical structure of Ag473-3 earlier than Ag473-1. To gain more detailed structural information about the interaction between Ag473-3 and SDS micelle, changes on the SDS-added HSQC spectra were then assigned by combining assignments obtained from Ag473-1’and Ag473-3. The results indicatethat these missing resonances in SDS-added spectra correspond to the last 12 residues of the protein (E95-E107). Here, we provided a detailed model for the structure of Ag473-3 added with SDS, based on the data of this study and helical wheel representation about tandem repeat: Ag473, a disordered protein, incorporates its C terminal residues into SDS micelle. In the meantime the rest part of protein form extended helical structure intermittent association/dissociation with the micelle surface by contacting with hydrophobic part of inducing helix. High resolution structural data need to be presented to test this model of lipid-associated Ag473. Overall, these results will be helpful for the preparation of effective Ag473 vaccine by inducing a stabilizing conformation.
目次
摘要 v
Abstract vi
壹、前言 1
貳、材料與方法 7
參、結果與討論 17
一、Ag473蛋白質胺基酸組成 17
二、蛋白質表現載體之構築 17
三、Ag473-1’重組蛋白之大量表現與純化 17
四、Ag473-1’蛋白質在結構上的分析 18
(一) 不同pH值和不同溫度的結構變化(圓二色光譜) 18
(二) Ag743-1’蛋白質之核磁共振骨架光譜判讀 18
五、不同變體之Ag473蛋白質結構預測 19
六、不同變體之Ag473蛋白質之表現與純化 19
七、不同變體的Ag473蛋白質在結構上的分析及比較 20
(一) 不同變體的Ag473蛋白質在不同pH值的結構變化(圓二色光譜) 20
(二) 不同變體之Ag473蛋白質HSQC的比較 20
八、溶劑和蛋白交互作用 21
(一) 醇類水溶液模擬細胞膜環境 21
(二) 形成微胞(micelle)的介面活性劑和蛋白的交互作用 22
九、溫度對蛋白質的摺疊影響 23
十、Ag743-3蛋白質之核磁共振圖譜分析 24
(一) Ag473-3蛋白核磁共振骨架光譜判讀 24
(二) HSQC上SDS和蛋白質交互作用的影響 24
肆、圖表 27
伍、參考文獻 67
陸、縮寫表 73
柒、附錄 75
許志安 (2003) B型奈瑟氏腦膜炎球菌單株抗體之製備及其抗原表位分析。國立中興大學分子生物研究所碩士論文。
劉伊倫 (2006) 腦膜炎雙球菌表面抗原 Ag473 之功能及免疫特性分析。國立中興大學分子生物研究所碩士論文。
Achtman, M., Kusecek, B., Morelli, G., Eickmann, K., Wang, J.F., Crowe, B., Wall, R.A., Hassan-King, M., Moore, P.S., and Zollinger, W. (1992). A comparison of the variable antigens expressed by clone IV-1 and subgroup III of Neisseria meningitidis serogroup A. J Infect Dis 165, 53-68.
Akey, D.L., Malashkevich, V.N., and Kim, P.S. (2001). Buried polar residues in coiled-coil interfaces. Biochemistry 40, 6352-6360.
Batra-Safferling, R., Abarca-Heidemann, K., Korschen, H.G., Tziatzios, C., Stoldt, M., Budyak, I., Willbold, D., Schwalbe, H., Klein-Seetharaman, J., and Kaupp, U.B. (2006). Glutamic acid-rich proteins of rod photoreceptors are natively unfolded. J Biol Chem 281, 1449-1460.
Bell, S., Klein, C., Muller, L., Hansen, S., and Buchner, J. (2002). p53 contains large unstructured regions in its native state. J Mol Biol 322, 917-927.
Callaghan, M.J., Buckee, C., McCarthy, N.D., Ibarz Pavon, A.B., Jolley, K., Faust, S., Gray, S.J., Kaczmarski, E.B., Levin, M., Kroll, J.S., et al. (2008). The Opa protein repertoires of disease-causing and carried meningococci. J Clin Microbiol.
Christodoulides, M., Brooks, J.L., Rattue, E., and Heckels, J.E. (1998). Immunization with recombinant class 1 outer-membrane protein from Neisseria meningitidis: influence of liposomes and adjuvants on antibody avidity, recognition of native protein and the induction of a bactericidal immune response against meningococci. Microbiology 144 ( Pt 11), 3027-3037.
Cole, N.B., Murphy, D.D., Grider, T., Rueter, S., Brasaemle, D., and Nussbaum, R.L. (2002). Lipid droplet binding and oligomerization properties of the Parkinson''s disease protein alpha-synuclein. J Biol Chem 277, 6344-6352.
Comanducci, M., Bambini, S., Brunelli, B., Adu-Bobie, J., Arico, B., Capecchi, B., Giuliani, M.M., Masignani, V., Santini, L., Savino, S., et al. (2002). NadA, a novel vaccine candidate of Neisseria meningitidis. J Exp Med 195, 1445-1454.
de Jonge, M.I., Bos, M.P., Hamstra, H.J., Jiskoot, W., van Ulsen, P., Tommassen, J., van Alphen, L., and van der Ley, P. (2002). Conformational analysis of opacity proteins from Neisseria meningitidis. Eur J Biochem 269, 5215-5223.
Dunker, A.K., Lawson, J.D., Brown, C.J., Williams, R.M., Romero, P., Oh, J.S., Oldfield, C.J., Campen, A.M., Ratliff, C.M., Hipps, K.W., et al. (2001). Intrinsically disordered protein. J Mol Graph Model 19, 26-59.
Dyson, H.J., and Wright, P.E. (1998). Equilibrium NMR studies of unfolded and partially folded proteins. Nat Struct Biol 5 Suppl, 499-503.
Eliezer, D., Kutluay, E., Bussell, R., Jr., and Browne, G. (2001). Conformational properties of alpha-synuclein in its free and lipid-associated states. J Mol Biol 307, 1061-1073.
Findlow, J., Lowe, A., Deane, S., Balmer, P., van den Dobbelsteen, G., Dawson, M., Andrews, N., and Borrow, R. (2005). Effect of sequence variation in meningococcal PorA outer membrane protein on the effectiveness of a hexavalent PorA outer membrane vesicle vaccine in toddlers and school children. Vaccine 23, 2623-2627.
Finne, J., Leinonen, M., and Makela, P.H. (1983). Antigenic similarities between brain components and bacteria causing meningitis. Implications for vaccine development and pathogenesis. Lancet 2, 355-357.
Fioroni, M., Diaz, M.D., Burger, K., and Berger, S. (2002). Solvation phenomena of a tetrapeptide in water/trifluoroethanol and water/ethanol mixtures: a diffusion NMR, intermolecular NOE, and molecular dynamics study. J Am Chem Soc 124, 7737-7744.
Fletcher, L.D., Bernfield, L., Barniak, V., Farley, J.E., Howell, A., Knauf, M., Ooi, P., Smith, R.P., Weise, P., Wetherell, M., et al. (2004). Vaccine potential of the Neisseria meningitidis 2086 lipoprotein. Infect Immun 72, 2088-2100.
Frasch, C.E. (1987). Prospects for the prevention of meningococcal disease: special reference to group B. Vaccine 5, 3-4.
Geddie, M.L., O''Loughlin, T.L., Woods, K.K., and Matsumura, I. (2005). Rational design of p53, an intrinsically unstructured protein, for the fabrication of novel molecular sensors. J Biol Chem 280, 35641-35646.
Girard, M.P., Preziosi, M.P., Aguado, M.T., and Kieny, M.P. (2006). A review of vaccine research and development: meningococcal disease. Vaccine 24, 4692-4700.
Granoff, D.M., Moe, G.R., Giuliani, M.M., Adu-Bobie, J., Santini, L., Brunelli, B., Piccinetti, F., Zuno-Mitchell, P., Lee, S.S., Neri, P., et al. (2001). A novel mimetic antigen eliciting protective antibody to Neisseria meningitidis. J Immunol 167, 6487-6496.
Hamada, D., Kato, T., Ikegami, T., Suzuki, K.N., Hayashi, M., Murooka, Y., Honda, T., and Yanagihara, I. (2005). EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein. FEBS J 272, 756-768.
Harrison, L.H. (2006). Prospects for vaccine prevention of meningococcal infection. Clin Microbiol Rev 19, 142-164.
Hsu, C.A., Lin, W.R., Li, J.C., Liu, Y.L., Tseng, Y.T., Chang, C.M., Lee, Y.S., and Yang, C.Y. (2008). Immunoproteomic identification of the hypothetical protein NMB1468 as a novel lipoprotein ubiquitous in Neisseria meningitidis with vaccine potential. Proteomics 8, 2115-2125.
Iakoucheva, L.M., Brown, C.J., Lawson, J.D., Obradovic, Z., and Dunker, A.K. (2002). Intrinsic disorder in cell-signaling and cancer-associated proteins. J Mol Biol 323, 573-584.
Jo, E., McLaurin, J., Yip, C.M., St George-Hyslop, P., and Fraser, P.E. (2000). alpha-Synuclein membrane interactions and lipid specificity. J Biol Chem 275, 34328-34334.
Jones, C. (2005). Vaccines based on the cell surface carbohydrates of pathogenic bacteria. An Acad Bras Cienc 77, 293-324.
Kelly, S.M., Jess, T.J., and Price, N.C. (2005). How to study proteins by circular dichroism. Biochim Biophys Acta 1751, 119-139.
Klugman, K.P., Gotschlich, E.C., and Blake, M.S. (1989). Sequence of the structural gene (rmpM) for the class 4 outer membrane protein of Neisseria meningitidis, homology of the protein to gonococcal protein III and Escherichia coli OmpA, and construction of meningococcal strains that lack class 4 protein. Infect Immun 57, 2066-2071.
Lee, H., Mok, K.H., Muhandiram, R., Park, K.H., Suk, J.E., Kim, D.H., Chang, J., Sung, Y.C., Choi, K.Y., and Han, K.H. (2000). Local structural elements in the mostly unstructured transcriptional activation domain of human p53. J Biol Chem 275, 29426-29432.
Lee, S., Mesleh, M.F., and Opella, S.J. (2003). Structure and dynamics of a membrane protein in micelles from three solution NMR experiments. J Biomol NMR 26, 327-334.
Libich, D.S., and Harauz, G. (2008). Solution NMR and CD spectroscopy of an intrinsically disordered, peripheral membrane protein: evaluation of aqueous and membrane-mimetic solvent conditions for studying the conformational adaptability of the 18.5 kDa isoform of myelin basic protein (MBP). Eur Biophys J.
Linhart, B., and Valenta, R. (2005). Molecular design of allergy vaccines. Curr Opin Immunol 17, 646-655.
Moe, G.R., Tan, S., and Granoff, D.M. (1999). Differences in surface expression of NspA among Neisseria meningitidis group B strains. Infect Immun 67, 5664-5675.
Munishkina, L.A., Phelan, C., Uversky, V.N., and Fink, A.L. (2003). Conformational behavior and aggregation of alpha-synuclein in organic solvents: modeling the effects of membranes. Biochemistry 42, 2720-2730.
Nakakido, M., Tanaka, Y., and Tsumoto, K. (2007). The N-terminal domain of elastin-binding protein of Staphylococcus aureus changes its secondary structure in a membrane-mimetic environment. J Biochem 142, 131-134.
Otzen, D.E., Sehgal, P., and Nesgaard, L.W. (2007). Alternative membrane protein conformations in alcohols. Biochemistry 46, 4348-4359.
Perham, M., Liao, J., and Wittung-Stafshede, P. (2006). Differential effects of alcohols on conformational switchovers in alpha-helical and beta-sheet protein models. Biochemistry 45, 7740-7749.
Perrett, K.P., and Pollard, A.J. (2005). Towards an improved serogroup B Neisseria meningitidis vaccine. Expert Opin Biol Ther 5, 1611-1625.
Pervushin, K., Vamvaca, K., Vogeli, B., and Hilvert, D. (2007). Structure and dynamics of a molten globular enzyme. Nat Struct Mol Biol 14, 1202-1206.
Pizza, M., Scarlato, V., Masignani, V., Giuliani, M.M., Arico, B., Comanducci, M., Jennings, G.T., Baldi, L., Bartolini, E., Capecchi, B., et al. (2000). Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287, 1816-1820.
Polverini, E., Fasano, A., Zito, F., Riccio, P., and Cavatorta, P. (1999). Conformation of bovine myelin basic protein purified with bound lipids. Eur Biophys J 28, 351-355.
Povey, J.F., Smales, C.M., Hassard, S.J., and Howard, M.J. (2007). Comparison of the effects of 2,2,2-trifluoroethanol on peptide and protein structure and function. J Struct Biol 157, 329-338.
Raussens, V., Slupsky, C.M., Sykes, B.D., and Ryan, R.O. (2003). Lipid-bound structure of an apolipoprotein E-derived peptide. J Biol Chem 278, 25998-26006.
Rispoli, P., Carzino, R., Svaldo-Lanero, T., Relini, A., Cavalleri, O., Fasano, A., Liuzzi, G.M., Carlone, G., Riccio, P., Gliozzi, A., et al. (2007). A thermodynamic and structural study of myelin basic protein in lipid membrane models. Biophys J 93, 1999-2010.
Romero, P., Obradovic, Z., Kissinger, C.R., Villafranca, J.E., Garner, E., Guilliot, S., and Dunker, A.K. (1998). Thousands of proteins likely to have long disordered regions. Pac Symp Biocomput, 437-448.
Romero, P., Obradovic, Z., Li, X., Garner, E.C., Brown, C.J., and Dunker, A.K. (2001). Sequence complexity of disordered protein. Proteins 42, 38-48.
Ruan, K.H., Li, D., Ji, J., Lin, Y.Z., and Gao, X. (1998). Structural characterization and topology of the second potential membrane anchor region in the thromboxane A2 synthase amino-terminal domain. Biochemistry 37, 822-830.
Sanchez, S., Abel, A., Arenas, J., Criado, M.T., and Ferreiros, C.M. (2006). Cross-linking analysis of antigenic outer membrane protein complexes of Neisseria meningitidis. Res Microbiol 157, 136-142.
Singh, V.K., Pacheco, I., Uversky, V.N., Smith, S.P., MacLeod, R.J., and Jia, Z. (2008). Intrinsically disordered human C/EBP homologous protein regulates biological activity of colon cancer cells during calcium stress. J Mol Biol 380, 313-326.
Taha, M.K., and Alonso, J.M. (2008). Molecular epidemiology of infectious diseases: the example of meningococcal disease. Res Microbiol 159, 62-66.
Tettelin, H., Saunders, N.J., Heidelberg, J., Jeffries, A.C., Nelson, K.E., Eisen, J.A., Ketchum, K.A., Hood, D.W., Peden, J.F., Dodson, R.J., et al. (2000). Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 287, 1809-1815.
Theriault, Y., Pochapsky, T.C., Dalvit, C., Chiu, M.L., Sligar, S.G., and Wright, P.E. (1994). 1H and 15N resonance assignments and secondary structure of the carbon monoxide complex of sperm whale myoglobin. J Biomol NMR 4, 491-504.
Uversky, V.N. (2003). Protein folding revisited. A polypeptide chain at the folding-misfolding-nonfolding cross-roads: which way to go? Cell Mol Life Sci 60, 1852-1871.
Uversky, V.N., Gillespie, J.R., and Fink, A.L. (2000). Why are "natively unfolded" proteins unstructured under physiologic conditions? Proteins 41, 415-427.
Uversky, V.N., Oldfield, C.J., and Dunker, A.K. (2005). Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling. J Mol Recognit 18, 343-384.
Verheul, A.F., Snippe, H., and Poolman, J.T. (1993). Meningococcal lipopolysaccharides: virulence factor and potential vaccine component. Microbiol Rev 57, 34-49.
Virji, M., Makepeace, K., Ferguson, D.J., Achtman, M., and Moxon, E.R. (1993). Meningococcal Opa and Opc proteins: their role in colonization and invasion of human epithelial and endothelial cells. Mol Microbiol 10, 499-510.
Vucetic, S., Brown, C.J., Dunker, A.K., and Obradovic, Z. (2003). Flavors of protein disorder. Proteins 52, 573-584.
Watson, R.M., Woody, R.W., Lewis, R.V., Bohle, D.S., Andreotti, A.H., Ray, B., and Miller, K.W. (2001). Conformational changes in pediocin AcH upon vesicle binding and approximation of the membrane-bound structure in detergent micelles. Biochemistry 40, 14037-14046.
Yang, C.Y., Lee, Y.S., Huang, L.S., Kuo, Y.L., Liu, Y.L., and Lu, C.H. (2006). Antigenic diversity of Neisseria meningitidis isolated in Taiwan between 1995 and 2002. Scand J Infect Dis 38, 273-280.
Zollinger, W.D., and Mandrell, R.E. (1977). Outer-membrane protein and lipopolysaccharide serotyping of Neisseria meningitidis by inhibition of a solid-phase radioimmunoassay. Infect Immun 18, 424-433.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top