Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
Department of Mechanical Engineering, Nagaoka University of Technology, Nagaoka, Niigata 840-2188, Japan.
Langmuir. 2020 Sep 1;36(34):10012-10021. doi: 10.1021/acs.langmuir.0c00833. Epub 2020 Aug 21.
Analysis of the pore formation mechanisms of biological nanopores can provide insight into pore-forming peptide-induced diseases and into the characterization of nanopores employed in sensing methods. Evaluation of pore formation mechanisms is typically performed using microscopy including atomic force microscopy, transmission electron microscopy, as well as electrically via channel current measurements using a patch-clamp amplifier. However, due to the relatively low temporal resolution of the above-mentioned microscopy techniques and the low analysis accuracy of the channel current measurements, new analytical methods are required. Here, we describe a new analytical strategy to measure and analyze both ionic currents associated with biological nanopore insertion and deinsertion into and out of lipid bilayers to determine pore formation mechanisms for several representative proteins. The current changes associated with protein deinsertion are monitored as the lipid membrane leaflets are pulled apart-a unique phenomenon enabled by our gold nanoneedle measurement probe. This deinsertion current analysis (DiCA) is performed using a gold nanoneedle-supported lipid bilayer at which a bilayer membrane is formed by bringing together two lipid monolayers on the surface of the nanoneedle and at the interface of an aqueous solution and a lipid/oil mixture. The lipid bilayer can be pulled apart by removing the nanoneedle from this interface. In this study, we demonstrate the determination of pore formation mechanisms for four different pore-forming proteins and peptides-α-hemolysin, streptolysin O, alamethicin, and amyloid β 1-42 using DiCA. As a result, we successfully discern the pore formation mechanism, either addition or expansion, for each protein/peptide by analyzing the ratio and magnitude of insertion and deinsertion current events.
生物纳米孔的孔形成机制分析可以深入了解孔形成肽诱导的疾病，并深入了解用于传感方法的纳米孔的特性。孔形成机制的评估通常使用显微镜技术进行，包括原子力显微镜、透射电子显微镜，以及通过使用膜片钳放大器的通道电流测量进行电测量。然而，由于上述显微镜技术的时间分辨率相对较低，以及通道电流测量的分析精度较低，因此需要新的分析方法。在这里，我们描述了一种新的分析策略，用于测量和分析与生物纳米孔插入和从脂质双层中插入和退出相关的离子电流，以确定几种代表性蛋白质的孔形成机制。当脂质膜小叶被拉开时，监测与蛋白质退出相关的电流变化——这是我们的金纳米针测量探针实现的独特现象。这种退出电流分析（DiCA）是在金纳米针支撑的脂质双层上进行的，其中双层膜是通过将两个脂质单层在纳米针表面上和在水溶液与脂质/油混合物的界面上组合形成的。通过从该界面移除纳米针，可以将脂质双层拉开。在这项研究中，我们使用 DiCA 演示了四种不同的孔形成蛋白和肽——α-溶血素、链球菌溶血素 O、短杆菌肽和淀粉样β 1-42 的孔形成机制的确定。结果，我们通过分析插入和退出电流事件的比率和幅度，成功区分了每种蛋白质/肽的孔形成机制，无论是附加还是扩展。
