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    Please use this identifier to cite or link to this item: http://tkuir.lib.tku.edu.tw:8080/dspace/handle/987654321/87403

    Title: 芳香環對抗菌胜肽Mastoparan-B及其衍生物的結構與活性的影響 : NMR及CD的研究
    Other Titles: Aromatic effects on the structure and activity of the antibacterial peptide Mastoparan-B and its mutant analogous studied by CD and NMR
    Authors: 林夆羲;Lin, Feng-Shi
    Contributors: 淡江大學化學學系碩士班
    李長欣;Lee, Chang-Shin
    Keywords: 芳香環;抗菌胜肽;生物結構;生物活性;核磁共振;MPB-NH2;9F-MPB-NH2;9Y-MPB-NH2;aromatic ring;NOEs;CD;NMR
    Date: 2012
    Issue Date: 2013-04-13 11:03:20 (UTC+8)
    Abstract: Mastoparan B自黑腹虎頭蜂蜂毒液中分離出,由十四個胺基酸(LKLKSIVSWAKKVL-NH2)所組成,MPB-NH2在水中的構形相當不穩定,而在TFE(三氟乙醇)水溶液中會形成穩定的雙性二級結構(α螺旋)。
    將MPB-NH2的Trp置換成Tyr,利用固相胜肽合成法(Solid Phase Peptide Synthesis)合成9Y-MPB-NH2,經由高效能液相層析儀(HPLC)純化、MOLDI-TOF確認分子量,再以圓二色光譜儀(CD)和二維核磁共振(2D NMR)光譜:TOCSY、NOESY、[1H,13C]-HSQC光譜判定得到化學位移與NOE資訊﹔弛緩實驗(relaxation)測得9Y-MPB-NH2內部運動的參數T1、T2與NOE ﹔利用DOSY的變溫實驗,測量胜肽分子所產生的聚集現象導致擴散運動的改變,用以估計胜肽在溶液中的分子量,與理論分子量相比較的結果,推測胜肽分子與溶劑分子相互作用下的胜肽聚集行為 。
    經由Xplor計算軟體模擬出9Y-MPB-NH2的二級結構,得知其283K 30%TFE環境下之螺旋結構位於K4-V13;310 K 30%TFE環境下之螺旋結構位於S5-V13。利用無模型法則(Model-free approach)進行分子動態行為計算,計算出9Y-MPB-NH2各殘基的次序參數(order parameter, S2)與全關聯時間(Overall correlation time, τm)。
    另外,我們利用大腸桿菌(E. coli)對MPB-NH2、9F-MPB-NH2和9Y-MPB-NH2做抗菌活性測定,並透過影響抗菌胜肽活性之結構因素來了解為何置換不同芳香環胺基酸會對抗菌活性有所影響。
    Mastoparan-B (MPB-NH2) is an antibacterial cationic tetradecapeptide, isolated from the venom of black-bellied hornet (Vespa basalis) with a primary structure
    (LKLKSIVSWAKKVL -CONH2) and amide C-terminus. It forms a random coil in aqueous solution and adopts an ampliphlic α-helical conformation in trifluoroethanol(TFE). A previous study showed that Alanine (Ala) in substitution for Trp9 cause a collapse of the helix. The aromatic ring of the tryptophan in MPB-NH2 is important in the helical stabilization. Recognizing this, we attempt to uncover how an aromatic residue affect the structure and the biological activity of MPB-NH2 and its analogous.
    In this study, we replaced Trp9 by phenylalanine and tyrosine in the primary structure of MPB-NH2. Results of CD and NMR indicated no significant change in the helical structure in 30% trifluoroethanol solution.
    However, the experiments of antibacterial activity showed that MPB-NH2 has stronger antibacterial activity than [Phe9]- MPB-NH2and [Tyr9]- MPB-NH2. It is likely that the indole ring of tryptophan residue is more effective for membrane perturbation than benzene ring of phenylalanine and the phenol of tyrosine.
    Also, a number of NOEs have been found between aromatic residue and other residues.
    Here, we proposed that some of these NOEs may imply the fundamental interaction for structure stabilization and activity.
    Appears in Collections:[化學學系暨研究所] 學位論文

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