Elucidating molecular level details of eukaryotic nucleobase ascorbate transporter function.

所有细胞都被一层由脂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有效地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。这种机制有可能被用作将药物送入细胞的一种手段，这使得转运体成为制药公司非常感兴趣的主题。然而，我们对高等生物--真核生物的转运蛋白功能的了解目前还很有限。UapA是一种研究非常广泛的真核膜转运蛋白，负责在真菌nidulans中输入黄嘌呤和尿酸。这种真菌特别容易受到基因操纵，这使得在其自然环境中对UapA突变进行了广泛的分析。这类研究为了解UapA的功能、其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提供了见解。然而，我们仍然缺乏关于UAPA如何工作的详细图景。增加对UapA运作的理解的中心是一种称为X射线结晶学的技术，它使我们能够获得关于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如此详细的结构仍然是非常具有挑战性的，因为必须将这些蛋白从其膜环境中移除到含有洗涤剂的溶液中，以便能够分离和结晶它们。我们在获得UapA的详细结构方面取得了实质性进展，分离和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。我们还证明了分离的UapA与黄嘌呤结合，并在溶液中形成两个蛋白质分子的复合体，一个二聚体。这项研究的目的是通过X射线结晶学获得高分辨率的UapA结构。获得的UapA结构将用于指导进一步研究蛋白质关键区域在结构和功能中的作用。我们将把作为这项研究的一部分获得的结构和突变信息与目前关于UapA功能的现有数据相结合，以便建立一个关于真核膜转运蛋白如何工作的独特详细的图景。

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.