苦荞α-螺旋发夹抗菌肽的分子改造抑制植物真菌活性

本研究对来源于苦荞的α-螺旋发夹抗菌肽FtAMP抗真菌机制与结构之间的关系进行了研究。首先人工合成了FtAMP分子中N-端和C-端的α-螺旋(FtAMP-N和FtAMP-C），探究两个α-螺旋究竟是哪个螺旋在起抗菌作用。然后以FtAMP为模板，α-螺旋区电荷和两亲性特征为变化要素，利用螺旋轮投影和特定氨基酸残基替换的方法，对其进行初步分子改造，并通过对多肽结构和活性比较，探讨FtAMP结构-功能的关系。研究表明，FtAMP-N和FtAMP-C都显示出良好的抗菌活性。根据螺旋轮投影方法分析螺旋的两亲性特征，并以FtAMP氨基酸序列为模板，分别表达4个FtAMP突变体(FtAMP-E12A、FtAMP-E12A/E9K、FtAMP-E12A/E9A和FtAMP-E12A/E9K/T24E）。圆二色光谱分析显示，4个多肽都可正确折叠成α-螺旋结构，在208 nm和222 nm处有典型的双负峰，表明氨基酸的改变及其表达过程中并未改变多肽的二级结构。抗真菌活性分析显示，与FtAMP相比，4种突变体对植物真菌的抑制作用均有一定增强。特别是FtAMP-E12A/E9K突变体，其抗真菌作用增强约1倍，同时诱导溶血活性并不显著，选择特异性提高近2倍。该研究也进一步表明，α-螺旋发夹抗菌肽发挥抗真菌效应主要与其螺旋结构有关，而与其抑制剂的活性位点没有关系，为该类抗菌肽结构和功能的关系提供了一定的参考。

Molecular Modification and Antifungal Activities of α-Helix Hairpins in Antimicrobial Peptides from Tartary Buckwheat

The relationship between antifungal mechanisms and structures of the antimicrobial peptide FtAMP from tartary buckwheat was studied. First, two helices at the N-terminal and C-terminal of FtAMP (FtAMP-N and FtAMP-C) were synthesized artificially to investigate the antimicrobial activity site. Then we used FtAMP as a template, the charge and amphiphilic characteristics at α-helical regions as change elements, helical wheel projection and specific amino acid residue substitution as main methods on the molecular modification, and compared the secondary structure and activity of peptides. The relationship between the structure and function of FtAMP were investigated. The results showed that both FtAMP-N and FtAMP-C had good antifungal activities. According to helix projection results, four FtAMP mutants (FtAMP-E12A, FtAMP-E12A/E9K, FtAMP-E12A/E9A and FtAMP-E12A/E9K/T24E) were expressed using FtAMP amino acid sequence as the template. Circular dichroism analysis showed that the secondary structures of the four peptides were folded into α-helical structures with typical double negative peaks at 208 nm and 222 nm, indicating that the amino acid mutation and protein expression did not change the spatial structure of the parent peptide. The antifungal activity analysis showed that the inhibitory effect of the mutants on the fungus was enhanced compared with FtAMP. In particular, the effectiveness of FtAMP-E12A/E9K was improved approximately two-fold compared to FtAMP, and the induction of hemolytic activity was not significant at the same time. The specificity of selection was improved approximately two-fold compared to FtAMP. This study also showed that the antifungal effect of α-helix hairpin antimicrobial peptides was mainly related to its helix structure, but not to the active sites of inhibitor. The results provided a reference for the relationship between the structure and function of this kind of antimicrobial peptide.