[[alternative]]Structure, dynamics and activity of the antimicrobial peptide Mastoparan-B analogue studied by CD and NMR

碩士[[abstract]]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。[[abstract]]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.[[tableofcontents]]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[[note]]學號: 699160221, 學年度: 10