Elucidating molecular level details of eukaryotic nucleobase ascorbate transporter function.

所有的细胞都被一层由脂质分子组成的膜包围着。这层膜作为一个有效的屏障，将细胞内的内容物与外界环境隔离开来。除了有限数量的分子外，脂质膜本身对所有分子都是不渗透的，然而细胞需要一种有效地吸收关键营养物质并去除废物的方法。通过一种被称为膜转运蛋白的特殊蛋白质系统，各种分子通过膜的输入和输出被嵌入到脂质层中。这些转运蛋白在膜的一侧与特定的底物或货物结合，经过重新配置，然后将底物释放到膜的另一侧。这种机制有可能被利用作为一种将药物进入细胞的手段，这使得转运体成为制药公司非常感兴趣的主题。然而，我们对高阶生物（真核生物）的转运蛋白功能的了解目前是有限的。UapA是一种被广泛研究的真核生物膜转运蛋白，在真菌芽曲霉中负责黄嘌呤和尿酸的进口。这种真菌特别适合基因操作，这使得在其原生环境中对UapA突变体进行广泛分析成为可能。这些研究提供了对UapA功能的深入了解，它在细胞内的位置，以及蛋白质的哪些部分负责运输到正确的位置。然而，我们仍然缺乏UapA如何工作的详细图片。增加对UapA操作的理解的核心是一种叫做x射线晶体学的技术，它使我们能够获得蛋白质结构中原子排列的非常详细的信息。获得转运蛋白等膜蛋白的详细结构仍然非常具有挑战性，因为必须将蛋白质从膜环境中去除到含有洗涤剂的溶液中，这样它们才能被分离和结晶。我们在获得UapA的详细结构方面取得了实质性进展，分离并结晶了两种不同形式的蛋白质，并获得了一些高分辨率的结构数据。我们还表明，分离的UapA与黄嘌呤结合，并在溶液中形成两种蛋白质分子的复合物，即二聚体。本文提出的研究目的是通过x射线晶体学获得高分辨率的UapA结构。获得的UapA结构将用于指导进一步研究蛋白质结构和功能关键区域的作用。我们将把作为这项研究的一部分获得的结构和诱变信息与目前可用的UapA功能数据结合起来，以建立真核生物膜转运蛋白如何工作的独特详细图像。

All cells are surrounded by a membrane made up of 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 efficiently 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 specialized 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. Such a mechanism has the potential to be exploited as a means of getting drugs into cells and this has made transporters the subject of significant interest by pharmaceutical companies. However, our understanding of transporter function for higher-order organisms, the eukaryotes, is currently limited. UapA is an unusually well studied eukaryotic membrane transporter responsible for import of xanthine and uric acid in the fungus Aspergillus nidulans. This fungus is particularly amenable to genetic manipulation and this has allowed extensive analysis of mutants of UapA in its native environment. Such studies have provided insights into UapA function, its location within the cell and which parts of the protein are responsible for trafficking to the correct location. However we still lack a detailed picture of how UapA works. Central to increasing understanding of the operation of UapA is a technique called X-ray crystallography which allows us to obtain very detailed information on the arrangement of the atoms within a protein structure. Obtaining such detailed structures of membrane proteins such as transporters remains very challenging as it is necessary to remove the proteins from their membrane environment into a detergent containing solution so they can be isolated and crystallised. We have made substantial progress towards obtaining a detailed structure of UapA, having isolated and crystallised two different forms of the protein and obtained some high resolution structural data. We have also shown that isolated UapA binds xanthine and forms a complex of two protein molecules, a dimer, in solution. The aim of the research outlined in this proposal is to obtain a high resolution structure of UapA by X-ray crystallography. The structure of UapA obtained will be used to guide further investigations into the roles of key regions of the protein in structure and function. We will combine the structural and mutagenic information obtained as part of this study with currently available data on UapA function in order to build up a uniquely detailed picture of how a eukaryotic membrane transporter works.