Interfaculty Institute of Biochemistry, University of Tübingen, 72076 Tübingen, Germany.
Toxins (Basel). 2021 Sep 18;13(9):669. doi: 10.3390/toxins13090669.
Equinatoxin II (EqtII) and Fragaceatoxin C (FraC) are pore-forming toxins (PFTs) from the actinoporin family that have enhanced membrane affinity in the presence of sphingomyelin (SM) and phase coexistence in the membrane. However, little is known about the effect of these proteins on the nanoscopic properties of membrane domains. Here, we used combined confocal microscopy and force mapping by atomic force microscopy to study the effect of EqtII and FraC on the organization of phase-separated phosphatidylcholine/SM/cholesterol membranes. To this aim, we developed a fast, high-throughput processing tool to correlate structural and nano-mechanical information from force mapping. We found that both proteins changed the lipid domain shape. Strikingly, they induced a reduction in the domain area and circularity, suggesting a decrease in the line tension due to a lipid phase height mismatch, which correlated with proteins binding to the domain interfaces. Moreover, force mapping suggested that the proteins affected the mechanical properties at the edge, but not in the bulk, of the domains. This effect could not be revealed by ensemble force spectroscopy measurements supporting the suitability of force mapping to study local membrane topographical and mechanical alterations by membranotropic proteins.
海葵毒素 II（EqtII）和 Fragaceatoxin C（FraC）是来自肌动蛋白孔毒素家族的孔形成毒素（PFT），它们在存在神经鞘磷脂（SM）时增强了膜亲和力，并在膜中具有共存相。然而，人们对这些蛋白质对膜域纳米尺度性质的影响知之甚少。在这里，我们使用共聚焦显微镜和原子力显微镜的力映射结合来研究 EqtII 和 FraC 对相分离的磷脂/SM/胆固醇膜的组织的影响。为此，我们开发了一种快速、高通量的处理工具，以关联力映射的结构和纳米力学信息。我们发现这两种蛋白质都改变了脂质域的形状。引人注目的是，它们导致域面积和圆度减小，这表明由于脂质相高度不匹配，线张力减小，这与蛋白质结合到域界面有关。此外，力映射表明，蛋白质在域的边缘而不是在域的体部影响机械性能。这一效应不能通过整体力谱测量来揭示，这支持了力映射在研究膜转导蛋白对局部膜拓扑和机械改变的适用性。
