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| 題名: | 利用NMR探討Mastoparan-B衍生物的結構、活性與動力學行為 |
| 其他題名: | Characterization of structural, dynamic and functional relationships of a mutant analogous from mastoparan-B by NMR |
| 作者: | 張馨如;Chang, Hsin-Ru |
| 貢獻者: | 淡江大學化學學系碩士班
李長欣;Lee, Chang-Shin |
| 關鍵詞: | 黑腹胡蜂;蜂毒液;抗菌胜肽;MP-B-9F;2-D NMR;Relaxation;S2 |
| 日期: | 2011 |
| 上傳時間: | 2011-12-28 18:11:39 (UTC+8) |
| 摘要: | 黑腹胡蜂為目前台灣最危險、最具攻擊性的胡蜂之一，其蜂毒液中富含多種具生物活性的分子，經研究結果顯示極具醫藥價值。黑腹胡蜂的蜂毒液中，最主要的一種成分即為mastoparan-B，簡稱MP-B。MP-B是一種由14個胺基酸組成的小分子胜肽(LKLKSIVSWAKKVL-NH2)，其功能包括A2、C磷脂酸酶的活化、抗菌作用、溶血作用、組織胺的釋放、活化G蛋白等，在水溶液中會形成兩性α螺旋結構，使其可以和細胞膜結合，形成穿孔作用。
MP-B-9F，將MP-B的W9制換成Phe的小分子胜肽，利用固相胜肽合成法(Solid Phase Peptide Synthesis)合成MP-B-9F，經由高效能液相層析儀(HPLC)純化，質譜儀(ESI-MASS)確認分子量，利用大腸桿菌(E. coli)對MP-B和MP-B-9F做抗菌活性測定，再以圓二色光譜儀(CD)和二維核磁共振(2-D NMR)的光譜：TOCSY、NOESY、[1H,13C]-HSQC光譜判定得到化學位移與限制條件，弛緩實驗(relaxation)測得MP-B-9F內部運動的參數T1、T2與NOE，DOSY實驗測得MP-B-9F擴散係數，計算出流體力學半徑，可知MP-B-9F為寡聚體結構。
經由Xplor-nih計算軟體模擬出MP-B-9F的二級結構，得知其在283K 30%TFE環境下之螺旋結構位於S5-S8。由模擬焠熄實驗之後得到的結果推疊出MP-B-9F主幹RMSD為0.227。
利用無模型法則(Model-free approach)與光譜密度函數(Spectral density function)進行分子動態行為計算，計算出MP-B-9F與MP-B各殘基的次序參數(order parameter, S2)與全關連時間(Overall correlation time, τm)。
綜合比較以上實驗結果，發現MP-B以Phe取代Trp9之後可能導致溶血活性下降的原因與結構的變化有極大的關係。
In Taiwan, the black-bellied hornet (vespa basalis) is certainly the most dangerous and most aggressive one in all kinds of vaspine wesp. Mastoparan-B (MP-B), a main component of the venom of black-bellied hornet, is a potent anti-bacteria peptide consisting of 14 amino acid residues (LKLKSIVSWAKKVL-NH2). Previous study had shown that hemolytic activity of MP-B is almost abolished as tryptophan at position 9 is mutated by phenylalanine.
MP-B-9F, a mutant analogous at position 9 was mutated by phenylalanine, was synthesized by Solid Phase Peptide Synthesis (SPPS), purified by HPLC and checked the molecular weight of peptide by ESI-MASS. The structure has been characterized by CD and 2D-NMR spectroscopy. As comparing with MP-B, the α-helix content in MP-B-9F is less at 283K but is more stable as temperature increased. The structure was simulated by Xplor-nih, and shows that residues 5-8 adopt a helical conformation in 283K, 30% TFE/water mixture.
The 13C relaxation parameters of MP-B and MP-B-9F were determined in 30% TFE/water mixture. The relaxation parameters, T1, T2, and NOE were analyzed using model-free approach. We calculated the order parameters, S2 and overall correlation times, τm of both peptides. |
| 顯示於類別: | [化學學系暨研究所] 學位論文
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