Capturing eukaryotic transporters in action

捕获行动中的真核转运蛋白

• 批准号: BB/V006487/1
• 负责人: Argyris Politis
• 金额: $ 52.04万
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV006487%2F1
• 关键词: Capturing; eukaryotic; transporters; action; Capturing; eukaryotic; transporters; action

All cells are surrounded by a membrane made up of fatty lipid molecules. This membrane acts as an effective barrier separating the contents of the cell from the external environment. The lipid membrane itself is impermeable to all but a limited number of molecules, however cells need to have a means of taking up key nutrients and removing waste products. The import and export of a wide range of molecules across the membrane is mediated via a system of specialised proteins called membrane transporters, which are embedded into the lipid layer. These transporter proteins bind a specific substrate or cargo on one side of the membrane, undergo a reconfiguration and then release the substrate on the other side of the membrane. The ability to transport key nutrients into and out of cells is fundamental to cellular function. These inbuilt transport mechanisms also represent a potential means of direct drug delivery into cells. Substantial progress is being made in understanding the mechanism of action of individual membrane transporters in particular through structural studies, however these approaches fail to capture the full dynamic range of different protein conformations required for transport activity. Here we will use a novel technique, Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) which provides information on which regions of a protein are accessible to the external environment. These regions change as the protein changes conformations and this information can be captured as a change in the level of deuterium labelling. Thus, differences in the deuterium labelling of different forms of the protein can allow researchers to build up a picture of precisely how a protein changes its shape to performs its function. The application of HDX-MS to membrane proteins represents an emerging technology and this is particularly true with regard to membrane proteins from complex organisms. The protein we will use for these studies is a nucleobase transporter from a fungus, UapA, which is an important transporter in its own right but is also an excellent model for a large number of human and other mammalian transporters. Our groups have studied UapA for a number of years and have many tools including a range of different substrates, inhibitors and mutant forms that can be used to obtain information on the different conformations adopted by the protein. The lipid molecules that make up the membrane are known to have important effects on structure and function of many membrane proteins including UapA. We have previously identified a series of lipids important for UapA, however it is not known what effect these lipids have on the conformational state of the protein. We will use HDX-MS to interrogate the conformational mechanism of UapA in artificial lipid environments. Our research will provide a uniquely detailed picture of how UapA carries out its transport function and how this is affected by its surrounding environment. This information is also relevant to other closely related proteins and may facilitate drug discovery efforts targeting UapA and other membrane transporters.

所有细胞都被膜脂质分子组成的膜包围。该膜充当有效的屏障，将细胞的内容与外部环境区分开。除了有限的分子以外，脂质膜本身对所有人都无法渗透，但是细胞需要一种吸收关键营养和去除废物产物的方法。跨膜的各种分子的导入和导出是通过称为膜转运蛋白的专用蛋白系统介导的，这些系统嵌入脂质层中。这些转运蛋白在膜的一侧结合特定的底物或货物，进行重新配置，然后在膜的另一侧释放底物。将关键营养物质输送到细胞的能力是细胞功能的基础。这些内置的运输机制也代表了直接药物递送到细胞的潜在手段。在理解单个膜转运蛋白的作用机理方面，特别是通过结构研究取得了重大进展，但是这些方法无法捕获运输活性所需的不同蛋白质构象的全部动态范围。在这里，我们将使用一种新型的技术，即氢氘交换质谱法（HDX-MS），该技术提供了有关外部环境可访问哪些蛋白质区域的信息。这些区域随着蛋白质的变化构象的变化而发生变化，并且可以将这些信息捕获为氘标记水平的变化。因此，不同形式的蛋白质标记的差异可以使研究人员能够确切地构建蛋白质如何改变其形状以执行其功能的图片。 HDX-MS在膜蛋白上的应用代表了一种新兴技术，而对于复杂生物体的膜蛋白尤其如此。我们将用于这些研究将使用的蛋白质是来自真菌UAPA的核碱酶转运蛋白，它本身就是重要的转运蛋白，但也是大量人类和其他哺乳动物转运蛋白的绝佳模型。我们的小组已经研究了UAPA多年，并且拥有许多工具，包括一系列不同的底物，抑制剂和突变体形式，可用于获取有关蛋白质采用的不同构型的信息。众所周知，构成膜的脂质分子对包括UAPA在内的许多膜蛋白的结构和功能具有重要影响。我们以前已经确定了一系列对UAPA重要的脂质，但是尚不清楚这些脂质对蛋白质构象状态具有什么影响。我们将使用HDX-MS在人工脂质环境中询问UAPA的构象机制。我们的研究将为UAPA如何执行其运输功能以及如何影响周围环境的影响提供独特的详细图片。该信息也与其他密切相关的蛋白质有关，并可能促进针对UAPA和其他膜转运蛋白的药物发现工作。