Structure and mechanism of the mitochondrial ATP synthase and Batten Disease gene product, Cln3p

研究策略 摘要 据估计，真核细胞中所有开放阅读框的20%-30%编码一个膜蛋白和一个 据估计，所有药物中有60%与膜蛋白结合。不足为奇的是，对当前 药物表明，疏水性是药物靶标常见的8种特性之一。然而， 膜蛋白的高分辨结构数量相对较少，结构数量较少 结合到膜蛋白上的疏水分子的比例甚至更小。这项提案有两个截然不同的 项目的唯一共同点是正在研究的蛋白质是膜蛋白和 参与细胞中的关键功能。第一个项目需要酵母线粒体ATP合成酶 我们已经解决了整个单体酶的第一个近原子模型，我们做到了这一点， 并且没有抑制剂结合到膜上嵌入Fo结构域。我们建议扩大这些范围 研究提供被抑制或捕获在各种反应中间体中的ATP合成酶的结构。 我们的方法是独一无二的，因为我们连接了ATP合成酶的两个亚单位，这使得我们能够 捕获处于扭曲状态的转子的中间体。因此，我们的方法允许捕获 反应中间体。抑制剂的使用将有助于捕获中间体并确定它们的模式。 有约束力的。第二个项目是对酵母Yhc3p的研究，Yhc3p是人类Cln3p的同源物，在人类中存在， 在青少年神经退行性疾病巴顿身上有缺陷。而Cln3在1995年被报道为 对于幼年型巴顿病的基因缺陷，这种蛋白的功能尚不清楚。基座 根据同源性和其他数据，我们假设Cln3p是一个小分子转运体，其功能是 与氧自由基的形成有关。我们有证据表明酵母基因YHC3受到严格的调控 并相信，通过识别调控，我们将了解其在细胞中的重要性。最后，我们有 建立了Yhc3p的高效表达系统和纯化方案，并对其进行了初步研究。 用于x射线衍射研究的功能和启动结晶试验。虽然不同，但这些项目进展顺利。 在我们的专业知识范围内，并将增加了解膜蛋白所需的基础 健康、疾病和作为药物发现的目标。

Research Strategy Abstract An estimated 20-30% of all open reading frames in the eukaryotic cell encode a membrane protein and an estimated 60% of all drugs bind to a membrane protein. Not surprisingly, statistical analysis of the current drugs indicate that hydrophobicity is one of 8 properties that is common in a drug target. However, the number of high-resolution structures of membrane proteins is relatively small and the number of structures of hydrophobic molecules bound to membrane proteins is even smaller. This proposal has 2 disparate projects whose only commonality is that the proteins under investigation are membrane proteins and involved in critical functions in the cell. The first project entails the yeast mitochondrial ATP synthase of which we have solved the first near atomic model of the entire monomeric enzyme and we did this, with and without inhibitors bound to the membrane embedded, Fo domain. We propose to expand these studies to provide structures of the ATP synthase inhibited or trapped in various reaction intermediates. Our approach is unique in that we have linked 2 subunits of the ATP synthase and this allows us to capture intermediates with the rotor in a twisted state. As such, our approach allows for the capture of reaction intermediates. The use of inhibitors will serve to trap intermediates and identify their mode of binding. The second project is studies on yeast Yhc3p, a homologue of human Cln3p, which is in humans, is defective in the juvenile neurodegenerative disease, Batten. While, Cln3 was reported in 1995 as the gene defective for the juvenile form of Batten disease, the function of this protein is still unknown. Based on homology and other data, we hypothesize that Cln3p is a small molecule transporter whose function is tied to the formation of oxygen radicals. We have evidence that the yeast gene, YHC3, is tightly regulated and believe that by identifying the regulation, we will understand its importance in the cell. Lastly, we have developed an over-expression system and purification scheme for Yhc3p, which we will use to study the function and start crystallization trials for x-ray diffraction studies. While disparate, these projects fall well within our expertise and will add to the base necessary for the understanding of membrane proteins in health, disease, and as targets for drug discovery.