3D ELECTRON MICROSCOPY OF THE ATPase HRS-2

ATPase HRS-2 的 3D 电子显微镜

• 批准号: 6622949
• 负责人: PAWEL A. PENCZEK
• 金额: $ 28.25万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6622949
• 关键词: adenosine triphosphate; adenosinetriphosphatase; bioimaging /biomedical imaging; chemical association; conformation; cryoelectron microscopy; electron microscopy; enzyme activity; enzyme inhibitors; monomer; mutant; phosphoproteins; point mutation; protein binding; protein protein interaction; protein structure function; structural biology; syntaxin

DESCRIPTION (provided by applicant): Hrs-2 is an ATPase that has been implicated in a variety of cellular processes by virtue of its functional protein interactions. We have determined that the form of hrs-2 with the highest specific ATPase activity is an oligomer of six subunits. However, the structure of this complex is unknown. Consequently, the extensive functional studies lack a structural basis for their interpretation. Our goal is to use three-dimensional electron microscopy to determine the structure of hrs-2 and its functional complexes. We show in the preliminary results that hrs-2 can be isolated in sufficient quantities to visualize it in the electron microscope and that the data collected is of sufficient quality to perform a three-dimensional reconstruction of this protein. Association of hrs-2 with interacting proteins as well as its intrinsic ATPase activity likely plays a role in its function. Within the framework of this proposal, we will investigate the role of the ATPase activity and interacting proteins in the structure of the hrs-2 oligomer. To achieve this goal, we will employ three-dimensional electron microscopy and further refine the results using computational structure prediction methods. This structural information will provide insight into the role of interacting proteins and enzyme activity on the conformation of the hrs-2 oligomer and ultimately on its function. The Specific Aims are: (1) Three-dimensional electron microscopy of the native structure of hrs-2. (2) High-resolution structure of the hrs-2 by cryo-electron microscopy and structure prediction methods. (3) Determination of the role of hrs-2-interacting proteins on the structure of hrs-2. (4) Determination of the effect of ATP binding on the structure of hrs-2. (5) Determination of the structural basis for the mechanism by which hrs-2 inhibits SNARE complex formation.

描述（由申请人提供）：Hrs-2 是一种 ATP 酶，已被 凭借其功能参与多种细胞过程 蛋白质相互作用。我们已经确定 hrs-2 的形式为 最高特异性 ATP 酶活性是六个亚基的寡聚物。然而， 该复合物的结构未知。因此，广泛的功能 研究缺乏解释的结构基础。我们的目标是使用 三维电子显微镜测定hrs-2和 其功能复合物。我们在初步结果中表明 hrs-2 可以 分离出足够的量以在电子显微镜下观察 并且收集的数据质量足以执行 该蛋白质的三维重建。 hrs-2 与相互作用蛋白及其内在 ATP 酶的关联 活动可能在其功能中发挥作用。在此框架内 提案中，我们将研究 ATPase 活性和相互作用的作用 hrs-2 寡聚体结构中的蛋白质。为了实现这一目标，我们将 采用三维电子显微镜并进一步完善结果 使用计算结构预测方法。这个结构信息 将深入了解相互作用的蛋白质和酶活性的作用 hrs-2 寡聚物的构象及其功能。这 具体目标是： (1) 原生体的三维电子显微镜 hrs-2 的结构。 (2) 低温电子高分辨率HRS-2结构 显微镜和结构预测方法。 (3)角色的确定 hrs-2 结构上的 hrs-2 相互作用蛋白。 (4) 的确定 ATP结合对hrs-2结构的影响。 (5) 的确定 hrs-2 抑制 SNARE 复合物机制的结构基础 形成。