Peptides that self-assemble into nanometer-sized pores in lipid bilayers could have utility in a variety of biotechnological and clinical applications if we can understand their physical chemical properties and learn to control their membrane selectivity. To empower such control, we have used synthetic molecular evolution to identify the pHD peptides, a family of peptides that assemble into macromolecule-sized pores in membranes at low peptide concentration, but only at pH < ∼6. Further advancements will also require better selectivity for specific membranes. Here we determine the effect of anionic headgroups and bilayer thickness on the mechanism of action of the pHD peptides by measuring binding, secondary structure, and macromolecular poration. The peptide pHD15 partitions and folds equally well into zwitterionic and anionic membranes but is less potent at pore-formation in phosphatidylserine-containing membranes. The peptide also binds and folds similarly in membranes of various thicknesses, but its ability to release macromolecules changes dramatically. It causes potent macromolecular poration in vesicles made from PC with 14 carbon acyl chains, but macromolecular poration decreases sharply with increasing bilayer thickness, and does not occur at any peptide concentration in fluid bilayers made from PC lipids with 20-carbon acyl chains. The effects of headgroup and bilayer thickness on macromolecular poration cannot be accounted for by the amount of peptide bound but instead reflect an inherent selectivity of the peptide for inserting into the membrane-spanning pore state. Molecular dynamics simulations suggest that the effect of thickness is due to hydrophobic match/mismatch between the membrane spanning peptide and the bilayer hydrocarbon. This remarkable degree of selectivity based on headgroup and especially bilayer thickness is unusual and suggests ways that pore-forming peptides with exquisite selectivity for specific membranes can be designed or evolved.

中文翻译：

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具有强成分依赖性膜选择性的 pH 触发成孔肽

如果我们能够了解它们的物理化学性质并学会控制它们的膜选择性，那么在脂质双层中自组装成纳米尺寸孔的肽可以在各种生物技术和临床应用中发挥作用。为了实现这种控制，我们使用合成分子进化来鉴定 pHD 肽，这是一个在低肽浓度下组装成膜中大分子大小的孔的肽家族，但仅在 pH < ∼6 时。进一步的进步还需要对特定膜有更好的选择性。在这里，我们通过测量结合、二级结构和大分子穿孔来确定阴离子头基和双层厚​​度对 pHD 肽作用机制的影响。肽 pHD15 在两性离子和阴离子膜中的分配和折叠效果一样好，但在含磷脂酰丝氨酸的膜中形成孔的效力较低。该肽也在不同厚度的膜中以类似方式结合和折叠，但其释放大分子的能力发生了显着变化。它在由具有 14 个碳酰基链的 PC 制成的囊泡中引起有效的大分子穿孔，但随着双层厚度的增加，大分子穿孔急剧减少，并且在由具有 20 个碳酰基链的 PC 脂质制成的流体双层中不会发生任何肽浓度。头基和双层厚​​度对大分子穿孔的影响不能由结合的肽量来解释，而是反映了肽插入跨膜孔状态的固有选择性。分子动力学模拟表明，厚度的影响是由于跨膜肽和双层烃之间的疏水匹配/不匹配。这种基于头基特别是双层厚度的显着选择性程度是不寻常的，它表明可以设计或进化对特定膜具有精细选择性的成孔肽。