Wiedman Gregory, Fuselier Taylor, He Jing, Searson Peter C, Hristova Kalina, Wimley William C
Department of Biochemistry and Molecular Biology, Tulane University School of Medicine , New Orleans, Louisiana 70112, United States.
J Am Chem Soc. 2014 Mar 26;136(12):4724-31. doi: 10.1021/ja500462s. Epub 2014 Mar 13.
Peptides that self-assemble, at low concentration, into bilayer-spanning pores which allow the passage of macromolecules would be beneficial in multiple areas of biotechnology. However, there are few, if any, natural or designed peptides that have this property. Here we show that the 26-residue peptide "MelP5", a synthetically evolved gain-of-function variant of the bee venom lytic peptide melittin identified in a high-throughput screen for small molecule leakage, enables the passage of macromolecules across bilayers under conditions where melittin and other pore-forming peptides do not. In surface-supported bilayers, MelP5 forms unusually high conductance, equilibrium pores at peptide:lipid ratios as low as 1:25000. The increase in bilayer conductance due to MelP5 is dramatically higher, per peptide, than the increase due to the parent sequence of melittin or other peptide pore formers. Here we also develop two novel assays for macromolecule leakage from vesicles, and we use them to characterize MelP5 pores in bilayers. We show that MelP5 allows the passage of macromolecules across vesicle membranes at peptide:lipid ratios as low as 1:500, and under conditions where neither osmotic lysis nor gross vesicle destabilization occur. The macromolecule-sized, equilibrium pores formed by MelP5 are unique as neither melittin nor other pore-forming peptides release macromolecules significantly under the same conditions. MelP5 thus appears to belong to a novel functional class of peptide that could form the foundation of multiple potential biotechnological applications.
在低浓度下能自组装形成跨双层孔从而允许大分子通过的肽，在生物技术的多个领域都将是有益的。然而，几乎没有天然的或设计的肽具有这种特性。在这里我们表明，26个氨基酸残基的肽“MelP5”，是在小分子泄漏的高通量筛选中鉴定出的蜂毒溶解肽蜂毒素的合成进化功能获得变体，在蜂毒素和其他成孔肽不能使大分子通过双层的条件下，它能使大分子通过双层。在表面支持的双层中，MelP5在肽与脂质比低至1:25000时形成异常高电导的平衡孔。就每个肽而言，MelP5导致的双层电导增加比蜂毒素的亲本序列或其他肽成孔剂导致的增加显著更高。在这里我们还开发了两种用于检测大分子从囊泡泄漏的新方法，并使用它们来表征双层中的MelP5孔。我们表明，MelP5在肽与脂质比低至1:500时，且在既不发生渗透裂解也不发生囊泡总体失稳的条件下，能使大分子通过囊泡膜。MelP5形成的大分子尺寸的平衡孔是独特的，因为在相同条件下蜂毒素和其他成孔肽都不会显著释放大分子。因此，MelP5似乎属于一类新型功能性肽，可为多种潜在生物技术应用奠定基础。
