Dynamics and pathways of assembly in membrane pore formation

孔形成蛋白是生物体与威胁其健康和生存的病原体之间持续战斗的关键武器。这些蛋白质作用于细胞，细胞是所有生命形式的微米尺度的基本单位。细胞被一层薄膜隔开并与环境隔离。病原体如细菌可以释放成孔蛋白（“毒素”），其在宿主生物体中的健康细胞的膜中钻孔，以释放细菌的营养物质，侵入这些细胞和/或杀死它们。例如，感染细菌性肺炎的患者遭受这种毒素肺炎球菌溶血素对肺组织的破坏性影响。然而，免疫系统使用类似的机制来杀死细菌和感染或癌细胞，从而防止它们对生物体造成进一步的损害。它分泌相关的，但有些不同的孔形成蛋白来覆盖这些不想要的入侵者的膜。为了执行这些任务，孔形成蛋白已经发展了复杂的钻孔机制。这些蛋白质可以从水溶性形式转化为一种非常不同的形式，其中20-50个蛋白质分子组装成一个环形孔结合到膜上。我们可以用X射线或电子来观察这些形式，以推断它们的三维结构。多亏了这样的实验，我们现在对膜中的可溶性蛋白质及其孔结构有了相当清晰的了解。对于某些形成气孔的蛋白质，科学家们甚至已经确定了使这种转变成为可能的蛋白质内部的变化。然而，如果我们希望设计出阻止这种气孔形成的药物，就像上面提到的细菌性肺炎的例子一样，了解更多关于它们形成的步骤将是有用的。正是这种孔隙组合在很大程度上仍然是个谜。在这个项目中，我们将试图回答一些关于膜孔形成的具体问题。我们想知道蛋白质是如何在膜上组装的。它们是一个接一个地组装，还是先形成更大的单元，然后再组装成一个孔？蛋白质是先在膜上组装，还是先在膜上停靠，然后在孔中组装？在这个过程中，被组装好的蛋白质包围的膜会在什么时候被挤出，形成一个洞？为了研究这一过程的动力学，我们依赖于一种称为原子力显微镜的技术。原子力显微镜相当于小规模的阅读盲文：用一个微小的人造手指，我们感觉到孔形成蛋白质，而他们组装在膜上。虽然X射线晶体学和电子显微镜仅限于静态样品，但原子力显微镜可以探测工作中的活性蛋白质。因此，我们将应用原子力显微镜的膜，是暴露在攻击的孔形成蛋白质。与此同时，我们将受益于电子显微镜提供的更详细的视图，以识别由化学键或降低温度捕获的成孔蛋白质的中间组件。因此，电子显微镜将提供不同组装状态下的成孔蛋白质的高度详细的图像，原子力显微镜将使我们能够看到蛋白质如何在这些不同状态之间转换。

Pore-forming proteins are crucial armaments in the continuous battle between living organisms and the pathogens that threaten their fitness and survival. These proteins act on cells, which are the micrometre-scaled, basic units of all forms of life. Cells are separated and protected from their environment by a thin membrane. Pathogens such as bacteria can release pore-forming proteins ("toxins") that drill holes in the membranes of healthy cells in the host organism, to release nutrients for the bacteria, to invade these cells and/or kill them. Patients affected by bacterial pneumonia, for example, suffer from the devastating effects of such a toxin, pneumolysin, on lung tissue. The immune system, however, uses a similar mechanism to kill germs and infected or cancerous cells, thus preventing them from doing further damage to the organism. It secretes related, but somewhat different pore-forming proteins to perforate the membranes of such unwanted invaders.To perform these tasks, pore-forming proteins have developed sophisticated drilling mechanism. These proteins can convert from a soluble form in the aqueous, cellular environment into a very different form, in which 20-50 protein molecules assemble into a ring-shaped pore bound to the membrane. We can look at these forms with X-rays or electrons to deduce their three-dimensional structures. Thanks to such experiments, we now have a reasonably clear picture of the soluble proteins and their pore structure in the membrane. For some pore-forming proteins, scientists have even identified the changes inside the proteins which make this transition possible.However, if we wish to design drugs that prevent such pores from being formed, as in the example of bacterial pneumonia indicated above, it would be useful to know more about the steps in their formation. It is exactly this pore assembly that is still largely enigmatic. In this project, we will try to answer some specific questions about membrane pore formation. We would like to know how the proteins assemble on the membrane. Do they assemble one by one, or do they first form larger units that subsequently assemble in a pore? Do the proteins first need to assemble on the membrane, or can they dock in the membrane and assemble in pores afterwards? And at what point in this process will the membrane that is surrounded by the assembled protein be extruded to create a hole?To investigate the dynamics of this process, we rely on a technique called atomic force microscopy. Atomic force microscopy is the small-scale equivalent of reading Braille: With a tiny artificial finger, we feel the pore-forming proteins while they assemble on the membrane. Whereas X-ray crystallography and electron microscopy are limited to static samples, atomic force microscopy can probe active proteins while they are at work. We will thus apply atomic force microscopy to the membranes that are being exposed to attack by pore-forming proteins. Meanwhile, we will benefit from the more detailed views provided by electron microscopy to identify intermediate assemblies of pore-forming proteins, that are trapped by chemical bonds or by lowering the temperature. Electron microscopy will thus provide highly detailed pictures of pore forming proteins in different states of assembly and atomic force microscopy will enable us to see how the proteins transit between these different states.