成孔蛋白(PFP)包括由许多病原细菌产生的毒力因子。然而,PFP还包含非毒力因子，如凋亡的Bcl2样蛋白，也发生在非致病性细菌中，甚至发生在所有生命王国中。成孔蛋白是一类古老的蛋白质,在破坏细胞膜方面非常强大。总的来说,与脂质膜结合后，它们从可溶性单体形式转化为寡聚态，然后经历巨大的构象变化形成跨膜孔。因此，PFP使其靶细胞的质膜对溶质具有渗透性，可能导致细胞死亡，或者对细胞功能进行更微妙的操作。最近的冷冻EM和X射线晶体学研究揭示了几种PFP在其孔前和孔状态下的高分辨率结构，然而，关于诱导孔隙形成的线索的许多方面，孔前到孔的构象转变，膜渗透的机制和相关的动力学仍然知之甚少，和这些转变的动态的直接可视化缺失。使用高速原子力显微镜（HS-AFM），各种PFP的低聚动力学和孔前到孔的转变动力学，如李斯特溶素O(LLO)，lysenin,和穿孔素-2（PFN2），可以研究。这些研究表明，LLO不会形成规则形状或大小的孔，而是形成膜插入弧，这些弧作为线性体传播和破坏脂质膜。相比之下,lysenin形成稳定的前孔和孔非酰胺环，HS-AFM可以研究它们在膜上的扩散和pH依赖性插入。同样，PFN2在酸化时经历了孔前到孔的转变。HS-AFM系统的开放性允许对过渡进行实时成像，并揭示了所有观察到的分子在3s内过渡到孔状态。在这一章中,我们详细说明了制备脂质的方案，形成支持的脂质双层，并提供实时指南，实际空间HS-AFM观测PFP的作用。
Pore-forming proteins (PFPs) include virulence factors that are produced by many pathogenic bacteria. However, PFPs also comprise non-virulence factors, such as apoptotic Bcl2-like proteins, and also occur in non-pathogenic bacteria and indeed in all kingdoms of life. Pore-forming proteins are an ancient class of proteins, which are tremendously powerful in damaging cell membranes. In general, upon binding to lipid membranes, they convert from the soluble monomeric form into an oligomeric state, and then undergo a dramatic conformational change to form transmembrane pores. Thus, PFPs render the plasma membrane of their target cells permeable to solutes, potentially leading to cell death, or to more subtle manipulations of cellular functions. Recent cryo-EM and X-ray crystallography studies revealed high-resolution structures of several PFPs in their pre-pore and pore states, however many aspects regarding the cues that induce pore formation, the pre-pore to pore conformational transition, the mechanism of membrane permeation and associated dynamics are still less well understood, and direct visualization of the dynamics of these transitions are missing. Using high-speed atomic force microscopy (HS-AFM), the kinetics of oligomerization and the pre-pore to pore transition dynamics of various PFPs, such as Listeriolysin O (
LLO), lysenin, and Perforin-2 (PFN2), could be studied. These studies revealed that
LLO does not form pores of regular shape or size, but rather forms membrane inserted arcs that propagate and damage lipid membranes as lineactants. In contrast, lysenin forms stable pre-pore and pore nonameric rings and HS-AFM allowed to
study their diffusion on and the pH-dependent insertion into the membrane. Similarly, PFN2 underwent pre-pore to pore transition upon acidification. The openness of the HS-AFM system allowed the transition to be imaged in real time and revealed that all observed molecules transitioned into the pore state within 3s. In this chapter, we detail protocols to prepare lipids, form supported lipid bilayers, and provide guidelines for real-time, real-space HS-AFM observations of PFPs in action.
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