Capturing eukaryotic transporters in action

所有的细胞都被一层由脂肪脂质分子组成的膜所包围。这种膜作为一个有效的屏障，将细胞内容物与外部环境隔开。脂质膜本身是不可渗透的，除了有限数量的分子，但是细胞需要有一种吸收关键营养物质和去除废物的方法。各种分子跨膜的输入和输出是通过一种称为膜转运蛋白的专门蛋白质系统介导的，该系统嵌入脂质层中。这些转运蛋白在膜的一侧结合特定的底物或货物，经历重新配置，然后在膜的另一侧释放底物。运输关键营养物质进出细胞的能力是细胞功能的基础。这些内在的转运机制也代表了将药物直接递送到细胞中的潜在手段。在理解单个膜转运蛋白的作用机制方面正在取得实质性进展，特别是通过结构研究，然而这些方法未能捕获转运活性所需的不同蛋白质构象的全部动态范围。在这里，我们将使用一种新的技术，氢氘交换质谱（HDX-MS），它提供了蛋白质的哪些区域可以进入外部环境的信息。这些区域随着蛋白质改变构象而改变，并且该信息可以被捕获为氘标记水平的变化。因此，不同形式蛋白质的氘标记差异可以使研究人员准确地了解蛋白质如何改变其形状以执行其功能。HDX-MS对膜蛋白的应用代表了一种新兴技术，对于来自复杂生物体的膜蛋白尤其如此。我们将用于这些研究的蛋白质是来自真菌的核碱基转运蛋白UapA，它本身就是一种重要的转运蛋白，但也是大量人类和其他哺乳动物转运蛋白的极好模型。我们的团队已经研究UapA多年，并拥有许多工具，包括一系列不同的底物，抑制剂和突变形式，可用于获得蛋白质所采用的不同构象的信息。已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的脂质UapA的重要性，但它是不知道这些脂质对蛋白质的构象状态有什么影响。我们将使用HDX-MS来询问UapA在人工脂质环境中的构象机制。我们的研究将提供UapA如何执行其运输功能以及如何受其周围环境影响的独特详细图片。这一信息也与其他密切相关的蛋白质有关，并可能有助于靶向UapA和其他膜转运蛋白的药物发现工作。

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.