Conformational Fine-Tuning of Pore-Forming Peptide Potency and Selectivity.
Conformational Fine-Tuning of Pore-Forming Peptide Potency and Selectivity.
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DOI：
10.1021/jacs.5b10595
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发表时间：
2015-12-30
影响因子：
15
通讯作者：
Wimley WC
中科院分区：
文献类型：
--
作者：
Krauson AJ;Hall OM;Fuselier T;Starr CG;Kauffman WB;Wimley WC
为了更好地理解控制膜透化肽的活性和选择性的序列-结构-功能关系，我们筛选了基于原型成孔剂蜂毒肽的肽库，用于功能丧失变体。这是通过测定文库成员在1：20的肽-脂质比下不能引起包埋的内容物从合成脂质囊泡中渗漏来实现的，所述肽-脂质比是蜂毒肽有效透化相同囊泡的浓度的10倍。令人惊讶的是，大约三分之一的文库成员在这些条件下是不活跃的。在阴性肽中，两个疏水残基变为甘氨酸的变化特别丰富。我们表明，功能丧失的活动可以完全重演的一个单一的残基变化的亮氨酸在位置16甘氨酸。与强效溶细胞蜂毒肽不同，功能丧失肽（包括单位点变体）对磷脂酰胆碱囊泡和多种类型的真核细胞基本上无活性。功能丧失是由于结合-折叠平衡从活性、结合、α-螺旋状态向非活性、未结合、无规卷曲状态的转变。因此，将阴离子脂质添加到合成脂质囊泡中恢复了“负”肽的结合、α-螺旋二级结构和有效活性。虽然对哺乳动物细胞无毒，但单位点变体具有有效的杀菌活性，与细菌膜的阴离子性质一致。结果表明，螺旋孔形成肽的构象微调是调节其活性和选择性的有效途径。
To better understand the sequence–structure–function relationships that control the activity and selectivity of membrane-permeabilizing peptides, we screened a peptide library, based on the archetypal pore-former melittin, for loss-of-function variants. This was accomplished by assaying library members for failure to cause leakage of entrapped contents from synthetic lipid vesicles at a peptide-to-lipid ratio of 1:20, 10-fold higher than the concentration at which melittin efficiently permeabilizes the same vesicles. Surprisingly, about one-third of the library members are inactive under these conditions. In the negative peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that loss-of-function activity can be completely recapitulated by a single-residue change of the leucine at position 16 to glycine. Unlike the potently cytolytic melittin, the loss-of-function peptides, including the single-site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Loss of function is shown to result from a shift in the binding–folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, α-helical secondary structure, and potent activity of the “negative” peptides. While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes. The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity.