Department of Micro- and Nanotechnology, DTU Nanotech, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.
Langmuir. 2015 Mar 3;31(8):2472-83. doi: 10.1021/la504752u. Epub 2015 Feb 20.
The capability of membrane-active peptides to disrupt phospholipid membranes is often studied by investigating peptide-induced leakage of quenched fluorescent molecules from large unilamellar lipid vesicles. In this article, we explore two fluorescence microscopy-based single-vesicle detection methods as alternatives to the quenching-based assays for studying peptide-induced leakage from large unilamellar lipid vesicles. Specifically, we use fluorescence correlation spectroscopy (FCS) to study the leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles dispersed in aqueous solution, and we use confocal imaging of surface-immobilized large unilamellar lipid vesicles to investigate whether there are heterogeneities in leakage between individual vesicles. Of importance, we design an experimental protocol that allows us to quantitatively correlate the results of the two methods; accordingly, it can be assumed that the two methods provide complementary information about the same leakage process. We use the two methods to investigate the membrane-permeabilizing activities of three well-studied cationic membrane-active peptides: mastoparan X, melittin, and magainin 2. The FCS results show that leakage induced by magainin 2 is less dependent on the size of the encapsulated fluorescent molecules than leakage induced by mastoparan X and melittin. The confocal imaging results show that all three peptides induce leakage by a heterogeneous process in which one portion of the vesicles are completely emptied of their contents but another portion of the vesicles are only partially emptied. These pieces of information regarding leakage induced by mastoparan X, melittin, and magainin 2 could not readily have been obtained by the established assays for studying peptide-induced leakage from lipid vesicles.
膜活性肽破坏磷脂膜的能力通常通过研究肽诱导大单层脂质囊泡中猝灭荧光分子的泄漏来研究。在本文中，我们探索了两种基于荧光显微镜的单囊泡检测方法，作为研究大单层脂质囊泡中肽诱导泄漏的基于猝灭测定的替代方法。具体来说，我们使用荧光相关光谱（FCS）研究不同大小的荧光分子从分散在水溶液中的大单层脂质囊泡中的泄漏，并且我们使用表面固定的大单层脂质囊泡的共焦成像来研究单个囊泡之间的泄漏是否存在异质性。重要的是，我们设计了一个实验方案，使我们能够定量关联两种方法的结果；因此，可以假设两种方法提供了关于同一泄漏过程的互补信息。我们使用这两种方法研究了三种研究充分的阳离子膜活性肽：蜂毒素 X、蜂毒素和抗菌肽 2 的膜透化活性。FCS 结果表明，抗菌肽 2 诱导的泄漏对包裹荧光分子的大小的依赖性小于蜂毒素 X 和蜂毒素诱导的泄漏。共焦成像结果表明，所有三种肽都通过一种不均匀的过程诱导泄漏，其中一部分囊泡完全排空其内容物，而另一部分囊泡仅部分排空。关于蜂毒素 X、蜂毒素和抗菌肽 2 诱导的泄漏的这些信息，如果没有脂质囊泡中肽诱导泄漏的既定测定法，就很难获得。
