抗菌胜肽Mastoparan-B第9號位置色胺酸殘基對結構、動力學和活性的影響

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题名:	抗菌胜肽Mastoparan-B第9號位置色胺酸殘基對結構、動力學和活性的影響
其它题名:	Effects of the ninth position of residue, tryptophan, on the structure, dynamics and activity of an antimicrobial peptide, mastoparan-B
作者:	朱學桂;Zhu, Xue-Gui
贡献者:	淡江大學化學學系碩士班李長欣;Lee, Chang-Shin
关键词:	抗菌胜肽;NMR;13C relaxation;dynamics;diffusion;Antimicrobial peptides;Model-Free
日期:	2014
上传时间:	2015-05-04 09:48:30 (UTC+8)
摘要:	Mastoparan B（MP-B）是從黑腹胡蜂(Vespa basalis)毒液中分離出來，由14個胺基酸所組成的抗菌胜肽，其特色為含有多個正電性胺基酸殘基。在水中的構型為無序殘捲，但在TFE水溶液中能夠形成穩定的兩親性螺旋結構。在先前的文獻表明，MP-B上的第9號位置的芳香環殘基對於細胞膜相互作用與裂解是有影響的。在本研究，我們將9號位置的Tryptophan用非芳香環殘基Leucine置換(MP-BL9)，從之前研究發現9號位置芳香環殘基與13號位置Valine的γCH3有很強的疏水作用，因此將Valine用短鏈的Alanine取代(MP-BA13)，來探討芳香環側鏈和第13號位置的作用對結構及活性的影響。 CD實驗結果顯示，這些胜肽在30 % TFE溶液中形成α螺旋結構，螺旋長度沒有明顯不同，NMR結構模擬結果其螺旋範圍分別為，MPB : K4到V13、MPB-A13 : K4到A13和MPB-L9 : K4到V13。利用DOSY實驗分析分子的聚集行為，發現MP-B比MP-BA13、MP-BL9更容易形成寡聚體。再用弛緩實驗進行分子動態行為估算，螺旋片段的範圍動性較低，而MPB-L9在9號位置後的動性較高，推測是芳香環測鏈的影響，MPB-A13因其疏水表面積的影響，造成其Ala13動性較低。並利用大腸桿菌(E. coli)對MP-B、MP-BA13、MP-BL9做抗菌活性測試，發現MP-B抗菌活性優於其他其它類似物，這可能是MP-B其螺旋範圍具有較大的疏水表面積或是其芳香環與其它側鏈間作用的影響，造成它比其它類似物具有更好的疏水性和靜電作用力。 Mastoparan B (MP-B), a tetradecapeptide toxin isolated from the venom of the hornet(Vespa basalis), is an amphiphilic α-helical peptide with a primary structure of Leu-Lys-Leu-Lys-Ser-Ile-Val-Ser-Trp-Ala-Lys-Lys-Val-Leu-NH2. It forms a random coil in aqueous solution and adopts an ampliphlic α-helical conformation in trifluoroethanol (TFE). A previous study showed that the ninth position of aromatic residue, Tryptophan, is important for the helical conformation. Recognizing this fact, we attempt to answer the question how the structure and the biological activity of MP-B are affected by the aromatic residue in this study. In this work, we replaced Trp9 by Leu9, or Leu13 by Ala13 in the primary structure of MP-B. Results of CD indicated no significant change in the helical structures in 30% TFE solution. The NMR data of MP-B, MP-BA13, MP-BL9 indicated the induced helix involves residues from 4 to 13 in 30% TFE at 310K. The diffusion studies suggested that MP-B is more easier to form oligomers than MP-BA13, MP-BL9. in the model-free analysis of 13C relaxation data showed that the order parameter, S2, in the helix segment of these peptides are more restricted. The S2 of MP-BL9 is motional flexibile, which may be less interaction existed between aromatic residue and other residues. The experiments of antibacterial activity showed that MP-B has strongest activity among all the peptides. It is likely that the tryptophan residue, which has larger hydrophobic area and more NOEs, is more effective in both hydrophobic and electrostatic interactions than the other aromatic and Leucine residues. Both the interactions are essential in the binding affinities of MP-B with an anionic phospholipid layer of membranes.
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