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系統識別號 U0002-1308201216385000
中文論文名稱 利用CD與NMR研究Mastoparan-B衍生物的結構、活性與動力學行為
英文論文名稱 Structure, Dynamics and Activity of the Antimicrobial Peptide Mastoparan-B Analogue studied by CD and NMR
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 100
學期 2
出版年 101
研究生中文姓名 童偉誠
研究生英文姓名 Wei-Cheng Tung
學號 699160221
學位類別 碩士
語文別 中文
口試日期 2012-06-17
論文頁數 197頁
口試委員 指導教授-李長欣
委員-陳佩燁
委員-陳銘凱
中文關鍵字 核磁共振  弛緩實驗  動力學  擴散實驗  無模型法則  抗菌胜肽 
英文關鍵字 NMR  13C relaxation  Dynamics  Diffusion  Model-free  Antimicrobial peptides 
學科別分類 學科別自然科學化學
中文摘要 Mastoparan B (MP-B-NH2)是由14個胺基酸所組成的抗菌胜肽,從黑腹胡蜂(Vespa basalis)毒液中分離出來,其特色為含有多個正電性胺基酸殘基及C端修飾為NH2。在此,我們用asparagine及tyrosine分別取代MP-B-NH2序列的2號位置殘基(lysine)及9號位置殘基(tryptophan),合成出其衍生物N2Y9-MPB-NH2,並去探討它的結構、動力學行為與抗菌活性之間的關係。
從CD實驗中發現,N2Y9-MPB-NH2在水中為無序纏繞的構形,在30 % TFE溶液中及SDS微胞中形成α螺旋結構。NMR結構模擬結果為N2Y9-MPB-NH2在30 % TFE溶液中,溫度283 K及310 K時,分別在Lys4到Val13及Lys4到Lys12形成α螺旋結構;在SDS微胞中,溫度310 K時,則在Lys4到Val13出現α螺旋的結構。從Model-free分析N2Y9-MPB-NH2在30 % TFE溶液中的動力學行為發現,在溫度283 K及310 K時,其螺旋片段的動性皆較低。
NMR結構計算結果發現,N2Y9-MPB-NH2在30 % TFE溶液中和與SDS微胞結合時的構形有所差異,其Tyr9和Lys11殘基側鏈的位向改變了。我們推測N2Y9-MPB-NH2帶正電性的殘基Lys會先和SDS微胞陰電性的膜表面有靜電作用,與膜表面結合之後,再藉由Tyr9殘基的芳香環側鏈及Ile6殘基的疏水性側鏈插入膜內部的疏水性區域。而抗菌實驗結果發現,N2Y9-MPB-NH2的抗菌活性比MP-B-NH2低,這也許是因為用Tyr取代Trp9及Asn取代Lys2,同時降低了其疏水性及和膜的靜電作用力。這兩種作用力是調控MP-B-NH2及其衍生物,與細菌細胞膜的陰電性磷脂層結合時的親和力(affinity)之基礎。
英文摘要 Mastoparan B (MP-B) is an antimicrobial tetradecapeptide that was isolated from the hornet (Vespa basalis) venom with cationic character and the amidated C-terminus. We synthesized a MP-B analogue, N2Y9-MPB, mutated at position 2 and 9 with asparagine and tyrosine and investigated its antimicrobial activity, structure, and dynamics.
Spectra of circular dichroism (CD) indicated that N2Y9-MPB convert from a random coil conformation in water to an α-helical structure in TFE and SDS micelles. Structure calculated via NMR data indicated the induced helix involves residues from 4 to 13 and 4 to 12 in 30% TFE at 283 K and 310 K, respectively, and from 4 to 13 in SDS micelles. The diffusion studies suggested that the peptide may form larger oligomers in TFE as temperature decreased from 310 K to 283 K. In aqueous TFE, the model-free analysis of 13C relaxation data showed that the order parameters, S2, in the helical segment are more restricted at both 310 K and 283 K.
The NMR data showed that the orientation of Tyr9 and Lys11 in the micelle-bound conformation is different from that in 30% TFE. We propose that the positively charged Lys residues in N2Y9-MPB may firstly develop the electrostatic interactions with the negatively charged surface of SDS micelle, then the aromatic side chain of Tyr9 and the aliphatic side chain of Ile6 insert into the hydrophobic interior of the mimicking membranes. As well, we observed that the antimicrobial activity of N2Y9-MPB is relatively lower than MP-B, it may result from the mutant of Trp9 with Tyr and the mutant of Lys2 with Asn, which descend both the hydrophobic and the electrostatic interaction of N2Y9-MPB with membrane. Both interactions are fundamental in modulating the binding affinities of MP-B and its analogous with the anionic phospholipid layer of membranes.
論文目次 目錄.................................................................................................I
表目錄......................................................................................................IV
圖目錄.....................................................................................................VII
縮寫表....................................................................................................XX
第一章 緒論
1.1 抗菌胜肽簡介.............................................................................1
1.2 Mastoparan家族...........................................................................2
1.3 Mastoparan B (MP-B) 介紹........................................................5
1.4 Mastoparan B在水溶液及類膜環境中的結構...........................6
1.5 Mastoparan B的結構與活性關係...............................................9
1.6 研究目的.....................................................................................11
第二章 實驗原理
2.1 固相胜肽合成...........................................................................12
2.2 圓二色旋光光譜儀原理...........................................................16
2.3 二維核磁共振...........................................................................21
2.3-1 COSY實驗.....................................................................23
2.3-2 TOCSY實驗...................................................................25
2.3-3 NOESY實驗...................................................................26
2.3-4 HSQC實驗.....................................................................29
2.4 DOSY實驗................................................................................30
2.5 化學位移指數...........................................................................36
2.6 結構計算...................................................................................39
2.7 無模型法則(Model-Free Approach)..........................................40

第三章 實驗材料與方法
3.1 實驗材料...................................................................................44
3.2 實驗方法...................................................................................49
3.2-1 胜肽合成........................................................................50
3.2-2 純化與分子量鑑定........................................................55
3.2-3 圓二色旋光光譜儀........................................................58
3.2-4 抗菌活性測試................................................................59
3.2-5 核磁共振實驗................................................................61
3.2-6 結構計算........................................................................65
3.2-7 無模型法則(model-free approach)計算........................69
3.2-8 黏度測量........................................................................72
第四章 實驗結果
4.1 圓二色旋光光譜.......................................................................73
4.2 抗菌活性...................................................................................75
4.3 NMR光譜判定..........................................................................78
4.4 化學位移指數( Chemical Shift Index, CSI )............................82
4.5 DOSY實驗 ................................................................................84
4.6 結構計算...................................................................................86
4.7 胜肽分子的動態行為...............................................................88
第五章 討 論........................................................................................173
第六章 結 論........................................................................................191
第七章 參考文獻..................................................................................193
參考文獻 (1) Syvitski, R. T.; Burton, I.; Mattatall, N. R.; Douglas, S. E.; Jakeman, D. L., Structural characterization of the antimicrobial peptide pleurocidin from winter flounder. Biochemistry 2005, 44 (19), 7282-7293.
(2) Peters, B. M.; Shirtliff, M. E.; Jabra-Rizk, M. A., Antimicrobial peptides: primeval molecules or future drugs? PLoS Pathog 2010, 6 (10), e1001067.
(3) Cabrera, M. P.; Alvares, D. S.; Leite, N. B.; Souza, B. M. d.; Palma, M. S.; Riske, K. A.; Neto, J. R., New insight into the mechanism of action of wasp mastoparan peptides: lytic activity and clustering observed with giant vesicles. Langmuir 2011, 27 (17), 10805-10813.
(4) Lin, C.-H.; Shyu, C.-L.; Kuo, Y.-M.; Tu, W.-C., A novel anti-microbial peptide isolated from the venom of Vespa affinis. 台灣昆蟲特刊 2006, 8, 33-41.
(5) Mendes, M. A.; Monson de Souza, B.; Delazari dos Santos, L.; Palma, M. S., Structural characterization of novel chemotactic and mastoparan peptides from the venom of the social wasp Agelaia pallipes pallipes by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2004, 18 (6), 636-642.
(6) Mendes, M. A.; de Souza, B. M.; Marques, M. R.; Palma, M. S., Structural and biological characterization of two novel peptides from the venom of the neotropical social wasp Agelaia pallipes pallipes. Toxicon 2004, 44 (1), 67-74.
(7) Lin, C.-H.; Tzen, J. T. C.; Shyu, C.-L.; Yang, M. J.; Tu, W.-C., Structural and biological characterization of mastoparans in the venom of Vespa species in Taiwan. Peptides 2011, 32 (10), 2027-2036.
(8) Argiolas, A.; Pisano, J. J., Facilitation of phospholipase A2 activity by mastoparans, a new class of mast cell degranulating peptides from wasp venom. J. Biol. Chem. 1983, 258 (22), 13697-13702.
(9) Mendes, M. A.; de Souza, B. M.; Palma, M. S., Structural and biological characterization of three novel mastoparan peptides from the venom of the neotropical social wasp Protopolybia exigua (Saussure). Toxicon 2005, 45 (1), 101-106.
(10) Murata, K.; Shinada, T.; Ohfune, Y.; Hisada, M.; Yasuda, A.; Naoki, H.; Nakajima, T., Novel mastoparan and protonectin analogs isolated from a solitary wasp, Orancistrocerus drewseni drewseni. Amino Acids 2009, 37 (2), 389-394.
(11) Ho, C.-L.; Lin, Y.-L.; Chen, W.-C.; Yu, H.-M.; Wang, K.-T.; Hwang, L.-L.; Chen, C.-T., Immunogenicity of mastoparan B, a cationic tetradecapeptide isolated from the hornet (Vespa basaus) venom, and its structural requirements. Toxicon 1995, 33 (11), 1443-1451.
(12) Yu, K.; Kim, Y.; Kang, S.; Park, N.; Shin, J., Relationship between the tertiary structures of mastoparan B and its analogs and their lytic activities studied by NMR spectroscopy. J. Pept. Res. 2000, 55 (1), 51-62.
(13) Chuang, C.-C.; Huang, W.-C.; Yu, H.-M.; Wang, K.-T.; Wu, S.-H., Conformation of Vespa basalis mastoparan-B in trifluoroethanol-containing aqueous solution. Biochim. Biophys. Acta 1996, 1292 (1), 1-8.
(14) Yeaman, M. R.; Yount, N. Y., Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev. 2003, 55 (1), 27-55.
(15) Yu, K.; Kang, S.; Kim, S. D.; Ryu, P. D.; Kim, Y., Interactions between mastoparan B and the membrane studied by 1H NMR spectroscopy. J. Biomol. Struct. Dyn. 2001, 18 (4), 595-606.
(16) Ho, C. L.; Lin, Y. L.; Chen, W. C.; Hwang, L. L.; Yu, H. M.; Wang, K. T., Structural requirements for the edema-inducing and hemolytic activities of mastoparan B isolated from the hornet (Vespa basalis) venom. Toxicon 1996, 34 (9), 1027-1035.
(17) Mierke, D. F.; Dürr, H.; Kessler, H.; Jung, G., Neuropeptide Y. Optimized solid-phase synthesis and conformational analysis in trifluoroethanol. Eur. J. Biochem. 1992, 206 (1), 39-48.
(18) Sabatino, G.; Chelli, M.; Brandi, A.; Papini, A. M., Analytical methods for solid phase peptide synthesis. Curr. Org. Chem. 2004, 8, 291-301.
(19) Nelson, D. L.; Cox, M. M., Lehninger principles of biochemistry. 4th ed.; W. H. Freeman: 2005; p 104-105.
(20) Berova, N.; Nakanishi, K.; Woody, R. W., Circular dichroism: principles and applications. Wiley-VCH: New York, 2000.
(21) Kelly, S. M.; Jess, T. J.; Price, N. C., How to study proteins by circular dichroism. Biochim. Biophys. Acta 2005, 1751 (2), 119-139.
(22) Engel, M.; Williams, R. W.; Erickson, B. W., Designed coiled-coil proteins: synthesis and spectroscopy of two 78-residue .alpha.-helical dimers. Biochemistry 1991, 30 (13), 3161-3169.
(23) 余靖; 李長欣, 核磁共振儀專輯(二). 行政院國科會精密儀器發展中心: 1988; p 111-127.
(24) Cavanagh, J.; Fairbrother, W. J.; Arthur, G. P. I.; Skelton, N. J., Protein NMR spectroscopy: principles and practice. Academic Press: 1996.
(25) Bax, A.; Davis, D. G., Assignment of complex 1H NMR spectra via two-dimensional homonuclear Hartmann-Hahn spectroscopy. J. Am. Chem. Soc. 1985, 107, 2820-2821.
(26) Ösapay, K.; Case, D. A., A new analysis of proton chemical shifts in proteins. J. Am. Chem. Soc. 1991, 113 (25), 9436-9444.
(27) Ösapay, K.; Case, D. A., Analysis of proton chemical shifts in regular secondary structure of proteins. J. Biomol. NMR 1994, 4 (2), 215-230.
(28) Bax, A.; Ikura, M.; Kay, L. E.; Torchia, D. A.; Tschudin, R., Comparison of different modes of two-dimensional reverse-correlation NMR for the study of proteins. J. Magn. Reson. 1990, 86 (2), 304-318.
(29) Yao, S.; Howlett, G. J.; Norton, R. S., Peptide self-association in aqueous trifluoroethanol monitored by pulsed field gradient NMR diffusion measurements. J. Biomol. NMR 2000, 16 (2), 109-119.
(30) Dingley, A. J.; Mackay, J. P.; Shaw, G. L.; Hambly, B. D.; King, G. F., Measuring macromolecular diffusion using heteronuclear multiple-quantum pulsed-field-gradient NMR. J. Biomol. NMR 1997, 10 (1), 1-8.
(31) Price, W. S., Pulsed-field gradient nuclear magnetic resonance as a tool for studying translational diffusion: part 1. Basic theory. Concepts Magn. Reson. 1997, 9 (5), 299-336.
(32) Wishart, D. S.; Sykes, B. D.; Richards, F. M., The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry 1992, 31 (6), 1647-1651.
(33) Wishart, D. S.; Sykes, B. D., The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J. Biomol. NMR 1994, 4 (2), 171-180.
(34) Wishart, D. S.; Sykes, B. D.; Richards, F. M., Relationship between nuclear magnetic resonance chemical shift and protein secondary structure. J. Mol. Biol. 1991, 222 (2), 311-333.
(35) Schwieters, C. D.; Kuszewski, J. J.; Clore, G. M., Using Xplor-NIH for NMR molecular structure determination. Prog. Nucl. Magn. Reson. Spectrosc. 2006, 48, 47-62.
(36) Wüthrich, K., NMR of proteins and nucleic acids. Wiley-Interscience: 1986.
(37) Esposito, L.; De Simone, A.; Zagari, A.; Vitagliano, L., Correlation between ω and ψ dihedral angles in protein structures. J. Mol. Biol. 2005, 347 (3), 483-487.
(38) Koerdel, J.; Skelton, N. J.; Akke, M.; Palmer, A. G.; Chazin, W. J., Backbone dynamics of calcium-loaded calbindin D9k studied by two-
dimensional proton-detected nitrogen-15 NMR spectroscopy. Biochemistry 1992, 31 (20), 4856-4866.
(39) Lipari, G.; Szabo, A., Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. J. Am. Chem. Soc. 1982, 104 (17), 4546-4559.
(40) Ellis, J. P.; Bakke, C. K.; Kirchdoerfer, R. N.; Jungbauer, L. M.; Cavagnero, S., Chain dynamics of nascent polypeptides emerging from the ribosome. ACS Chem. Biol. 2008, 3 (9), 555-566.
(41) 黃太煌, 利用核磁共振光譜學探討蛋白質的結構、動性及功能. 物理雙月刊 2001, 24 (3), 403-412.
(42) Rule, G. S.; Hitchens, T. K., Fundamentals of protein NMR spectroscopy. Springer Netherlands: 2005; p 449-473.
(43) Novabiochem® 2004/5 Catalog. Merck.
(44) Fioroni, M.; Diaz, M. D.; Burger, K.; Berger, S., 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. 2002, 124 (26), 7737-7744.
(45) Li, Y.; Han, X.; Tamm, L. K., Thermodynamics of fusion peptide−membrane interactions. Biochemistry 2003, 42 (23), 7245-7251.
(46) Rautenbach, M.; Gerstner, G. D.; Vlok, N. M.; Kulenkampff, J.; Westerhoff, H. V., Analyses of dose–response curves to compare the antimicrobial activity of model cationic α-helical peptides highlights the necessity for a minimum of two activity parameters. Anal. Biochem. 2006, 350 (1), 81-90.
(47) Hsu, S.-T. D.; Varnai, P.; Bugaut, A.; Reszka, A. P.; Neidle, S.; Balasubramanian, S., A G-rich sequence within the c-kit oncogene promoter forms a parallel G-quadruplex having asymmetric G-tetrad dynamics. J. Am. Chem. Soc. 2009, 131 (37), 13399-13409.
(48) Bader, R.; Bettio, A.; Beck-Sickinger, A. G.; Zerbe, O., Structure and dynamics of micelle-bound Neuropeptide Y: comparison with unligated NPY and implications for receptor selection. J. Mol. Biol. 2001, 305 (2), 307-329.
(49) Vostrikov, V. V.; Gu, H.; Ingólfsson, H. I.; Hinton, J. F.; Andersen, O. S.; Roux, B. t.; Koeppe, R. E., Gramicidin A backbone and side chain dynamics evaluated by molecular dynamics simulations and nuclear magnetic resonance experiments. II: nuclear magnetic resonance experiments. J. Phys. Chem. B 2011, 115 (22), 7427-7432.
(50) Reddy, T.; Rainey, J. K., Interpretation of biomolecular NMR spin relaxation parameters. Biochem. Cell Biol. 2010, 88 (2), 131-142.
(51) Clore, G. M.; Driscoll, P. C.; Wingfield, P. T.; Gronenborn, A. M., Analysis of the backbone dynamics of interleukin-1 beta using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy. Biochemistry. 1990, 29 (32), 7387-7401.
(52) Copié, V.; J.A.Battles; Schwab, J. M.; Torchia, D. A., Secondary structure of beta-hydroxydecanoyl thiol ester dehydrase, a 39-kDa protein, derived from H alpha, C alpha, C beta and CO signal assignments and the chemical shift index: comparison with the crystal structure. J. Biomol. NMR 1996, 7 (4), 335-340.
(53) Kay, L. E.; Torchia, D. A.; Bax, A., Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry. 1989, 28 (23), 8972-8979.
(54) Cheng, J. W.; Lepre, C. A.; Chambers, S. P.; Fulghum, J. R.; Thomson, J. A.; Moore, J. M., Nitrogen-15 NMR relaxation studies of the FK506 binding protein: backbone dynamics of the uncomplexed receptor. Biochemistry 1993, 32 (35), 9000-9010.
(55) Palmer, A. G.; Williams, J.; McDermott, A., Nuclear magnetic resonance studies of biopolymer dynamics. J. Phys. Chem. 1996, 100 (31), 13293-13310.
(56) Yan, C.; Digate, R. J.; Guiles, R. D., NMR studies of the structure and dynamics of peptide E, an endogenous opioid peptide that binds with high affinity to multiple opioid receptor subtypes. Biopolymers 1999, 49 (1), 55-70.
(57) Song, X.-j.; Flynn, P. F.; Sharp, K. A.; Wand, A. J., Temperature dependence of fast dynamics in proteins. Biophys. J. 2007, 92 (6), L43-L45.
(58) Lee, A. L.; Sharp, K. A.; Kranz, J. K.; Song, X.-J.; Wand, A. J., Temperature dependence of the internal dynamics of a calmodulin−peptide complex. Biochemistry 2002, 41 (46), 13814-13825.
(59) Kemple, M. D.; Buckley, P.; Yuan, P.; Prendergast, F. G., Main chain and side chain dynamics of peptides in liquid solution from 13C NMR:  melittin as a model peptide. Biochemistry 1997, 36 (7), 1678-1688.
(60) Wang, G.; Treleaven, W. D.; Cushley, R. J., Conformation of human serum apolipoprotein A-I(166-185) in the presence of sodium dodecyl sulfate or dodecylphosphocholine by 1H-NMR and CD. Evidence for specific peptide-SDS interactions. Biochim. Biophys. Acta 1996, 1301 (3), 174-184.
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