Assembly of a single protein pore

单个蛋白质孔的组装

• 批准号: BB/D010918/1
• 负责人: Mark Wallace
• 金额: $ 28.36万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD010918%2F1
• 关键词: Assembly; single; protein; pore

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.

单分子荧光是了解生物分子功能的强大技术。在单个分子水平上研究生物系统有很多优点，例如：我们可以揭示在传统批量测量中无法检测到的亚群，并且我们可以跟踪单个分子经历特定的反应或构象变化，从而能够观察原本隐藏的瞬态中间态。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量。了解控制膜蛋白行为的机制非常重要。膜蛋白负责控制细胞中的许多功能，包括信号传导和分子跨细胞膜的运输。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。我建议构建一种能够同时测量位于人工双层中的荧光标记膜蛋白的单分子荧光和离子电流的仪器。通过这种方式，膜蛋白内的构象变化可以与其传导的变化相关。该技术将通过应用于特定的生物问题进行测试：七聚成孔蛋白、葡萄球菌 α-溶血素 (aHL) 的组装和插入。 aHL由7个相同的亚基组成，当这7个亚基结合在一起时，它们形成一个通道。该通道形成β-桶结构。为了了解这些亚基如何组装，我们将它们与荧光分子连接起来。通过简单地计算 aHL 形式的亚基，我们将能够判断这些亚基是一次聚集在一起，还是成对聚集在一起，还是以更大的群体聚集在一起。通过在使用荧光标记观察孔形成的同时测量通过孔的电流，我们将能够了解 β-桶通道的形成与孔组装步骤之间的关系。