肽对细胞质膜的破坏和扰动在阐明生物学现象方面引起了极大的兴趣。通常,使用脂质体作为模型膜研究这些复杂的过程-通过封装荧光染料或通过其他光谱方法,比如核磁共振。尽管含有生理相关的脂质,没有合成模型真正概括了质膜的全部复杂性和分子多样性。这里,生物代表性膜模型,巨大质膜囊泡(GPMV),通过用化学应激源诱导出芽事件从真核细胞制备。然后隔离GPMV,和双层用荧光亲脂性示踪剂标记,并在具有膜活性肽的微孔板中孵育。随着膜受损和/或聚集,所产生的两种示踪剂之间的荧光共振能量转移(FRET)增加并且在微板中周期性地测量。当膜复杂性是要考虑的重要变量时,这种方法提供了一种特别有用的方法来检测扰动。此外,它提供了一种动态检测质膜损伤的方法,这可能与肽自组装或结构重排的动力学相关。允许测试各种肽-膜相互作用条件(肽:磷脂比率,离子强度,缓冲区,等。)马上。•使用可以从各种细胞系制备的完整质膜囊泡。•可以提供与传统合成脂质模型相当的吞吐量(例如,染料包封的脂质体)。
Disruptions and perturbations of the cellular plasma membrane by peptides have garnered significant interest in the elucidation of biological phenomena. Typically, these complex processes are studied using liposomes as model membranes-either by encapsulating a fluorescent dye or by other spectroscopic approaches, such as nuclear magnetic resonance. Despite incorporating physiologically relevant lipids, no synthetic model truly recapitulates the full complexity and molecular diversity of the plasma membrane. Here, biologically representative membrane models, giant plasma membrane vesicles (GPMVs), are prepared from eukaryotic cells by inducing a budding event with a chemical stressor. The GPMVs are then isolated, and bilayers are labelled with fluorescent lipophilic tracers and incubated in a microplate with a membrane-active peptide. As the membranes become damaged and/or aggregate, the resulting fluorescence resonance energy transfer (FRET) between the two tracers increases and is measured periodically in a microplate. This approach offers a particularly useful way to detect perturbations when the membrane complexity is an important variable to consider. Additionally, it provides a way to kinetically detect damage to the plasma membrane, which can be correlated with the kinetics of peptide self-assembly or structural rearrangements. Key features • Allows testing of various peptide-membrane interaction conditions (peptide:phospholipid ratio, ionic strength, buffer, etc.) at once. • Uses intact plasma membrane vesicles that can be prepared from a variety of cell lines. • Can offer comparable throughput as with traditional synthetic lipid models (e.g., dye-encapsulated liposomes).