Instituto de Ciencia Molecular, Universidad de Valencia, Paterna, Spain.
Biophys J. 2010 Nov 3;99(9):2917-25. doi: 10.1016/j.bpj.2010.08.068.
Pores made by amphipathic cationic peptides (e.g., antimicrobials and fragments of pore-forming proteins) are typically studied by examining the kinetics of vesicle leakage after peptide addition or obtaining structural measurements in reconstituted peptide-lipid systems. In the first case, the pores have been considered transient phenomena that allow the relaxation of the peptide-membrane system. In the second, they correspond to equilibrium structures at minimum free energy. Here we reconcile both approaches by investigating the pore activity of the α5 fragment from the proapoptotic protein Bax (Baxα5) before and after equilibrium of peptide/vesicle complexes. Quenching assays on suspensions of large unilamellar vesicles suggest that in the presence of Baxα5, the vesicles maintain a leaky state for hours under equilibrium conditions. We proved and analyzed stable pores on single giant unilamellar vesicles (GUVs) in detail by monitoring the entrance of dyes added at different times after incubation with the peptide. When the GUVs came in contact with Baxα5, leakage started stochastically, was delayed for various periods of time, and in the majority of cases proceeded rapidly to completion. After hours in the presence of the peptide, the same individual GUVs that refilled completely at first instance maintained a porated state, which could be observed in subsequent leak-in events for serially added dyes. However, these long-term pores were smaller in size than the initial equilibration pores. Stable pores were also detected in GUVs made in the presence of Baxα5. The latter pores can be considered equilibrium states and may correspond to structures measured previously in bilayer stacks. Although pore formation may occur as a kinetic process, equilibrium pores may also be functionally relevant structures, especially in highly regulated systems such as the apoptotic mitochondrial pores induced by Bax.
两亲性阳离子肽（例如，抗菌肽和孔形成蛋白的片段）形成的孔通常通过在添加肽后检查囊泡泄漏的动力学或在重建的肽-脂质系统中获得结构测量来研究。在第一种情况下，这些孔被认为是允许肽-膜系统松弛的瞬态现象。在第二种情况下，它们对应于最小自由能的平衡结构。在这里，我们通过研究促凋亡蛋白 Bax 的α5 片段（Baxα5）在肽/囊泡复合物达到平衡前后的孔活性来调和这两种方法。在大单层囊泡悬浮液上的猝灭测定表明，在 Baxα5 的存在下，囊泡在平衡条件下保持泄漏状态数小时。我们通过监测在与肽孵育不同时间后添加的染料的进入，详细证明并分析了单个巨大单层囊泡（GUV）上的稳定孔。当 GUV 与 Baxα5 接触时，泄漏会随机开始，延迟不同的时间段，并且在大多数情况下迅速完成。在肽存在数小时后，最初完全重新填充的相同单个 GUV 保持穿孔状态，可以在随后的连续添加染料的泄漏事件中观察到。然而，这些长期孔的尺寸比初始平衡孔小。在存在 Baxα5 的情况下也检测到 GUV 中的稳定孔。这些后一种孔可以被认为是平衡状态，并且可能对应于以前在双层堆栈中测量的结构。尽管孔形成可能作为动力学过程发生，但平衡孔也可能是功能相关的结构，特别是在像 Bax 诱导的凋亡线粒体孔这样的高度调节系统中。
