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有效地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特殊蛋白质系统介导的，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。这种机制有可能被开发为一种使药物进入细胞的手段，这使得转运蛋白成为制药公司感兴趣的主题。然而，我们的理解，转运功能的高阶生物，真核生物，目前是有限的。UapA是一种被广泛研究的真核细胞膜转运蛋白，在构巢曲霉中负责黄嘌呤和尿酸的转运。这种真菌是特别适合遗传操作，这使得广泛的分析突变体的UapA在其原生环境。这些研究提供了对UapA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位置的见解。然而，我们仍然缺乏关于UAPA如何工作的详细描述。增加对UapA运作的理解的核心是一种称为X射线晶体学的技术，该技术使我们能够获得关于蛋白质结构内原子排列的非常详细的信息。获得膜蛋白如转运蛋白的这种详细结构仍然非常具有挑战性，因为必须将蛋白质从其膜环境中除去到含有去污剂的溶液中，以便它们可以被分离和结晶。我们已经取得了实质性的进展，以获得详细的结构UapA，分离和结晶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结合黄嘌呤，并在溶液中形成两个蛋白质分子的复合物，即二聚体。本提案中概述的研究目的是通过X射线晶体学获得UapA的高分辨率结构。获得的UapA的结构将用于指导进一步研究蛋白质的关键区域在结构和功能中的作用。我们将结合联合收割机的结构和诱变信息，作为本研究的一部分，目前可用的数据UapA功能，以建立一个独特的详细的图片如何真核细胞膜转运蛋白的工作。

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.