PROTON TRANSLOCATION THROUGH F1F0 ATP SYNTHASE

描述：（来自申请人的摘要）：这项工作的长期目标是了解F1F0 ATP合酶的偶联机制。大多数生物体通过F1F0 ATP合酶合成ATP，因此这些酶在很大程度上负责能量代谢的核心功能。在人类中，能量代谢的失败与衰老和各种遗传疾病有关。例如，某些利氏综合征患者的疾病直接归因于影响F1F0 ATP合酶的突变。原核生物和真核生物的F1F0 ATP合成酶具有共同的结构和机制。因此，我们选择大肠杆菌F1F0 ATP合酶作为模型。大肠杆菌系统提供了一种特性良好的酶，并使用了最广泛的分子遗传技术。近年来，人们对连接该酶的F1和F0部分的两个茎结构关注较多。中心茎被认为在催化过程中在酶复合体内旋转，而外围茎则充当定子，将大部分F1固定在适当的位置。这导致了广泛持有的观点，即外周茎是一种刚性静态结构特征，但这种解释与所有实验观察结果并不一致。假设外周茎在耦合中起动态作用。提出以下具体目标来研究这一概念：1)确定b亚基缺失的酶的外周茎的长度；2)研究a亚基与b亚基相互作用的位点；3)通过寻找外周茎短暂解离的证据，研究b亚基与F1相互作用的动力学；4)研究F1F0 ATP合酶组装的优选中间体。

DESCRIPTION: (from the applicant's abstract): The long-term goal of this work is to understand the mechanism of coupling in F1F0 ATP synthase. Most organisms synthesize ATP via F1F0 ATP synthases so these enzymes are largely responsible for the central function of energy metabolism. In humans, failures in energy metabolism are associated with aging and a variety of inherited disorders. For example, the disease in certain individuals with Leigh's Syndrome is directly attributed to mutations affecting F1F0 ATP synthase. Prokaryotic and eucaryotic F1F0 ATP synthases share common structures and mechanisms. Therefore, the Escherichia coli F1F0 ATP synthase has been chosen as a model. The E. coli system offers a well-characterized enzyme and the use of the broadest range of molecular genetic techniques. Recently, much attention has been focussed on the two stalk structures linking the F1 and F0 sectors of the enzyme. The central stalk is thought to rotate within the enzyme complex during catalysis, while the peripheral stalk serves as a stator holding the bulk of F1 in place. This has led to the widely held view that the peripheral stalk is a rigid static structural feature, but this interpretation is not consistent with all experimental observations. It is hypothesized that the peripheral stalk plays a dynamic role in coupling. The following Specific Aims are proposed to investigate this concept: 1) determining the length of the peripheral stalk in enzymes with deletions in the b subunits; 2) studying the site of interaction between the a and b subunits; 3) investigating the dynamics of the b subunit interaction with F1 by looking for evidence of transient dissociation of the peripheral stalk; 4) studying the preferred intermediates in the assembly of F1F0 ATP synthase.