Assembly of a single protein pore

单分子荧光是了解生物分子功能的一种强有力的技术。例如，在单个分子水平上研究生物系统有许多优势；我们可以揭示在传统整体测量中无法检测到的亚群，并且我们可以在单个分子经历特定反应或构象变化时跟踪它，使我们能够观察到原本被隐藏的瞬时中间状态。为了将生物分子结构的变化与其功能的变化联系起来，需要能够同时监测结构和功能的技术。对于膜蛋白通道，有一个明显的蛋白质功能指标，即离子通过通道的流动。了解控制膜蛋白行为的机制是非常重要的。膜蛋白负责控制细胞中的许多功能，包括信号和跨细胞膜的分子运输。然而，由于它们的复杂性，人们对它们的结构、相互作用或行为知之甚少。我建议建造一种仪器，能够同时测量位于人工双层中的带有荧光标记的膜蛋白的单分子荧光和离子电流。通过这种方式，膜蛋白内的构象变化可以与其传导的变化联系起来。这项技术将通过应用于一个特定的生物学问题来测试：组装和插入七聚体形成孔的蛋白，葡萄球菌α-溶血素(AHL)。AHL由7个相同的亚基组成，当这7个亚基结合在一起时，它们形成了一个通道。该通道形成了一种贝塔桶结构。为了了解这些亚基是如何组装的，我们将把它们与荧光分子联系起来。通过简单地计算AHL形成的亚基，我们将能够区分这些亚基是一次一个地聚集在一起，还是成对地聚集在一起，还是以更大的群体形式聚集在一起。通过测量通过孔的电流，同时我们使用荧光标签观察它的形成，我们将能够理解β-桶通道的形成是如何与孔组装的步骤相关的。

Single-molecule fluorescence is a powerful technique for understanding the function of biomolecules. Studying biological systems at the level of individual molecules has many advantages, for example; we can reveal sub-populations that would otherwise be undetectable in conventional bulk measurements, and we can follow a single molecule as it undergoes a particular reaction or conformational change, enabling the observation of transient intermediate states that would otherwise be hidden. In order to relate changes in the structure of a biomolecule to changes in its function, techniques capable of monitoring both structure and function are required. For membrane protein channels, there is an obvious indicator of protein function, the flow of ions through the channel. Understanding the mechanisms that govern the behaviour of membrane proteins is very important. Membrane proteins are responsible for controlling many functions in the cell, including signalling and the transport of molecules across the cell membrane. However, due to their complexity, relatively little is known about their structure, interactions or behaviour. I propose to construct an instrument capable of making simultaneous measurements of both the single-molecule fluorescence and ion current from a fluorescently labelled membrane protein situated in an artificial bilayer. In this way, conformational changes within the membrane protein can be related to changes in its conduction. This technique will be tested by application to a specific biological problem: The assembly and insertion of the heptameric pore-forming protein, staphylococcal alpha-hemolysin (aHL). aHL is composed of 7 identical subunits, and when these 7 subunits combine they form a channel. This channel forms a beta-barrel structure. To understand how these subunits assemble, we will link them to fluorescent molecules. By simply counting the subunits as aHL forms we will be able to tell if the subunits come together one at a time, or in pairs, or in larger groups. By measuring the electrical current through a pore at the same time as we watch it form using fluorescent labels, we will be able to understand how the formation of a beta-barrel channel is related to the steps of pore assembly.