Capturing eukaryotic transporters in action

所有细胞都被一层由脂肪类分子组成的膜所包围。这种膜起到了有效的屏障作用，将细胞内的内容物与外部环境隔开。除了有限数量的分子外，类脂膜本身对所有分子都是不可渗透的，然而细胞需要有一种吸收关键营养物质和清除废物的方法。跨膜的各种分子的进出口是通过一种被称为膜转运蛋白的特殊蛋白质系统来调节的，这些蛋白质嵌入到脂层中。这些转运蛋白结合在膜一侧的特定底物或货物上，经历重新配置，然后释放膜另一侧的底物。将关键营养物质运入和运出细胞的能力是细胞功能的基础。这些内置的转运机制也代表了一种将药物直接输送到细胞内的潜在方式。在理解单个膜转运蛋白的作用机制方面取得了实质性进展，特别是通过结构研究，然而这些方法未能捕捉到运输活动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，氢氚交换-质谱学(HDX-MS)，它提供了蛋白质的哪些区域可以被外部环境访问的信息。这些区域会随着蛋白质构象的变化而改变，这种信息可以被捕捉到，作为氚标记水平的变化。因此，不同形式的蛋白质的氚标记的差异可以让研究人员准确地建立起一幅蛋白质如何改变其形状以执行其功能的图景。HDX-MS在膜蛋白方面的应用代表了一种新兴技术，对于复杂生物体的膜蛋白尤其如此。我们将在这些研究中使用的蛋白质是一种真菌UapA的核苷酸转运蛋白，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优秀模型。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，可用于获取蛋白质采用的不同构象的信息。已知构成膜的脂分子对包括UapA在内的许多膜蛋白的结构和功能有重要影响。我们以前已经发现了一系列对UapA重要的脂类，然而这些脂类对蛋白质的构象状态有什么影响尚不清楚。我们将使用HDX-MS来询问UapA在人工脂环境中的构象机制。我们的研究将为UAPA如何执行其运输功能以及如何受到周围环境的影响提供独特的详细图景。这些信息也与其他密切相关的蛋白质相关，并可能有助于针对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.