Testing the limits of rational design by engineering pH sensitivity into membrane-active peptides.
Testing the limits of rational design by engineering pH sensitivity into membrane-active peptides.
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DOI：
10.1016/j.bbamem.2014.12.023
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发表时间：
2015-04
期刊：
影响因子：
--
通讯作者：
Hristova K
中科院分区：
文献类型：
--
作者：
Wiedman G;Wimley WC;Hristova K
在这项工作中，我们试图合理设计膜活性肽，这些肽由低 pH 值触发，在脂质双层中形成大分子尺寸的孔。此类肽可在生物技术和纳米医学中具有广泛的用途，作为癌症治疗剂或促进内体释放大分子的药物递送载体。我们的合理设计方法是将不依赖 pH 的肽 MelP5 的特性与两种 pH 依赖的膜活性肽 pHlip 和 GALA 的特性相结合，MelP5 形成大孔，允许大分子通过。我们通过使用 pHlip 和 GALA 上酸性残基的分布作为指导，将酸性氨基酸插入 MelP5 的两亲性螺旋中，创建了两个杂合序列 MelP5_Δ4 和 MelP5_Δ6。我们证明新的肽与脂质双层结合并以 pH 依赖性方式获得二级结构。这些肽还以 pH 依赖性方式破坏双层的稳定性，使得脂质囊泡仅在低 pH 下释放小分子 ANTS/DPX。因此，我们成功设计了 pH 触发的成孔肽。然而，在任何条件下都没有观察到大分子释放。因此，我们通过在其序列中引入pH敏感性，废除了MelP5独特的大分子穿孔特性。我们得出结论，pHlip、GALA 和 MelP5 的特性是相加的，但只是部分如此。我们认为，这种可加性的缺乏是新型膜活性肽合理设计的限制，而高通量的发现方法对于该领域的持续进展至关重要。
In this work, we sought to rationally design membrane active peptides that are triggered by low pH to form macromolecular-sized pores in lipid bilayers. Such peptides could have broad utility in biotechnology and in nanomedicine as cancer therapeutics or drug delivery vehicles that promote release of macromolecules from endosomes. Our approach to rational design was to combine the properties of a pH-independent peptide, MelP5, which forms large pores allowing passage of macromolecules, with the properties of two pH-dependent membrane active peptides, pHlip and GALA. We created two hybrid sequences, MelP5_Δ4 and MelP5_Δ6 by using the distribution of acidic residues on pHlip and GALA as a guide to insert acidic amino acids into the amphipathic helix of MelP5. We show that the new peptides bind to lipid bilayers and acquire secondary structure in a pH-dependent manner. The peptides also destabilize bilayers in a pH-dependent manner, such that lipid vesicles release the small molecules ANTS/DPX at low pH only. Thus, we were successful in designing pH-triggered pore-forming peptides. However, no macro-molecular release was observed under any conditions. Therefore, we abolished the unique macromolecular poration properties of MelP5 by introducing pH-sensitivity into its sequence. We conclude that the properties of pHlip, GALA and MelP5 are additive, but only partially so. We propose that this lack of additivity is a limitation in the rational design of novel membrane active peptides, and that high-throughput approaches to discovery will be critical for continued progress in the field.
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