C端胺化與未胺化修飾的Mastoparan-B衍生物的結構、動力學與活性的關係

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Title:	C端胺化與未胺化修飾的Mastoparan-B衍生物的結構、動力學與活性的關係
Other Titles:	Relationship between structure, dynamics and activity of the analogues of Mastoparan-B with and without C-terminal carboxyamidation
Authors:	彭安邦;Peng, An-Pang
Contributors:	淡江大學化學學系碩士班李長欣;Li, Chang-Shin
Keywords:	胺化;NMR;13C relaxation;dynamics;diffusion;Antimicrobial peptides;Model-Free
Date:	2013
Issue Date:	2014-01-23 13:45:47 (UTC+8)
Abstract:	Mastoparan B (MP-B) 是從黑腹胡蜂(Vespa basalis)的毒液中所分離出來的，是由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。在水中的構形相當不穩定，但在TFE(三氟乙醇)水溶液中能夠形成穩定的兩親性結構。在此，我們將抗菌胜肽C端進行胺化的修飾，並探討MP-B-NH2和MP-B-COO-及其衍生物9F-MP-B-NH2和9F-MP-B-COO-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，胜肽在水中為無序纏繞的狀態，在30 % TFE溶液中形成α螺旋結構。由於C端胺化的原因，使得末端殘基的二次化學位移有明顯的不同，我們推測是由於胺化後的胜肽能夠提供額外的H鍵，進而使得靠近末端的殘基其環境產生改變。NMR結構模擬結果顯示MP-B-NH2及9F-MP-B-NH2在30 % TFE溶液中，溫度283 K及310 K時，都在Lys4到Val13形成α螺旋結構，表示283K到310K的溫差還不足以對MP-B-NH2產生影響；而MP-B-COO-及9F-MP-B-COO-在溫度283K時，會在Lys4到Lys11及Ser5到Lys12形成α螺旋結構，但在310K時光譜則無法判定。 Model-free分析13C的弛緩，其數據顯示胺化後的胜肽在溫度283 K時，其螺旋片段的動性都比未胺化的胜肽較低，因此我們推測由於C端自由基的影響，使得末端的胜肽分子結構不穩定，並影響了胜肽殘基與殘基之間的分子作用力，而造成去胺化的胜肽螺旋片段較短。從實驗結果發現抗菌胜肽C端胺化的修飾能夠使抗菌胜肽末端結構較為穩定，對於生物活性的影響也有非常大的改變。 Mastoparan B (MP-B) is an antimicrobial tetradecapeptide that was isolated from the hornet (Vespa basalis) venom with cationic character and amidated C-terminus.It forms a random coil in aqueous solution and adopts an amphiphilic α-helical conformation in trifluoroethanol(TFE). In this study, we investigate the structure, dynamic behavior and biological activity of Mastoparan-B and 9F-Mastoparan-B with and without C-terminal carboxyamidation. Spectra of circular dichroism (CD) indicated that our peptides will form random coil conformation in aqueous solution, and an α-helical conformation in 30%TFE. The C-terminal carboxyamidation contributes significant difference in amide proton secondary chemical shifts, we conclude that the C-terminal carboxyamidation can provide additional H-bond that changed the local environment of nearby residues in C-terminal. The NMR data of MP-B-NH2 and 9F-MP-B-NH2 indicated the induced helix involves residues from 4 to 13 in 30% TFE at 283K and 310K. It shows that there is no influence in structure from 283K to 310K, while MP-B-COO- and 9F-MP-B-COO- helices involve residues respectively from 4 to 11 and 5 to 12 in 30% TFE 283K, but the spectra at 310K can not phase. In the model-free analysis of 13C relaxation data showed that based on order parameter, S2, the helix segment of MP-B-NH2 are more restricted than that of MP-B-COO-. We proposed that the free carboxyl group at C-terminal result in structure instability, and might destabilize not only the hydrogen bond stretch of helical structure but the peptide as a whole. Antimicrobial peptides, the C-terminal carboxyaamidation of AMP can make the peptides become more stable, and has large influences in the biological activity.
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