STRUCTURE OF THE SODIUM PUMP NUCLEOTIDE BINDING DOMAIN

描述：（逐字来自申请人的摘要）钠泵已经被 充血性心力衰竭的治疗目标 强心苷已经有超过世纪的历史了。不幸的是， 已知强心苷抑制的机制。实验研究 在过去的三十年里， 钠泵催化的生化反应和转运反应 它会调解。然而，ATP水解的分子机制 与离子的“上坡”运动相结合仍然是一个谜。这可能 这是因为人们对它知之甚少。 这种蛋白质的结构-功能关系。我们的长期目标是 对钠泵转运机制的完整理解， 需要酶在其各种形态中的高分辨率X射线衍射图 构象和特定氨基酸残基的鉴定 与配体结合有关。然而，获得质量所需的技术 膜蛋白的晶体尚未完善。具体 该提案中概述的实验将利用细菌遗传学， 过表达Na，K-ATP酶的大胞质环（Aim I）。所有的 到目前为止，已经发现了与ATP结合和水解有关的残基， 这部分蛋白质。这同样适用于本组织的所有成员。 重要的蛋白质家族（即，P型ATP酶）。ATP结合 野生型和突变型ATP结合结构域的特征将被 使用CD和X射线晶体学测定和结构表征 技术（目标三）。此外，我们还将确定 Na，K-ATP酶与ATP结合结构域（Aim II）物理相互作用。的 这项工作的结果将提供一个ATP所涉及的氨基酸图谱 协调，从而有助于阐明ATP之间的耦合机制 水解和阳离子转运。钠泵在各种 体液和电解质平衡的器官。这些过程是动态的 这种平衡可能会在各种疾病中被破坏 states.在充分描述这些病理情况之前， 因此，需要对钠泵的功能有更全面的了解。

DESCRIPTION: (Verbatim from the Applicant's Abstract) The sodium pump has been the target for the therapeutic treatment of congestive heart failure with cardiac glycosides for more than a century. Unfortunately there is still little known about the mechanism of cardiac glycoside inhibition. Experimental studies over the past three decades have established some relationships between the biochemical reactions catalyzed by the sodium pump and the transport reactions it mediates. However, the molecular mechanisms by which the hydrolysis of ATP is coupled to the "uphill" movement of ions remains a mystery. This probably stems from the fact that there remains little known about the structure-function relationship of this protein. Our long term goal is to gain a complete understanding of the sodium pump transport mechanism which will require high-resolution x-ray diffraction patterns of the enzyme in its various conformations and the identification of specific amino acids residues associated with ligand binding. However, the techniques required to get quality crystals of membrane proteins have not yet been perfected. The specific experiments outlined in this proposal will exploit bacterial genetics to overexpress the large cytoplasmic loop of the Na,K-ATPase (Aim I). All the residues thus far implicated in ATP binding and hydrolysis have been found in this section of the protein. The same holds true for all members of this important protein family (i.e., P-type ATPases). The ATP binding characteristics of the wild type and mutant ATP binding domains will be determined and structurally characterized using CD and x-ray crystallographic techniques (Aim III). In addition, we will determine which other sections of the Na,K-ATPase physically interact with the ATP binding domain (Aim II). The results from this work will provide a map of the amino acids involved in ATP coordination and thus help elucidate the coupling mechanism between ATP hydrolysis and cation transport. The sodium pump is vital in a variety of organs for fluid and electrolyte balance. These processes are in a dynamic equilibrium and this equilibrium can become disrupted in a variety of disease states. Before an adequate description of these pathological situations can be made, a more complete understanding of sodium pump function is required.