3D ELECTRON MICROSCOPY OF THE ATPase HRS-2

描述(由申请人提供)：HRS-2是一种已被 通过其功能与多种细胞过程有关 蛋白质的相互作用。我们已经确定HRS-2的形式与 ATPase比活力最高的是六个亚基的寡聚体。然而， 这个复合体的结构尚不清楚。因此，广泛的功能 研究缺乏对它们的解释的结构基础。我们的目标是利用 用三维电子显微镜确定hRS-2和HRS-2的结构 它的功能复合体。我们在初步结果中表明，hars-2可以 以足够的数量被分离出来，以便在电子显微镜中观察到它 并且所收集的数据具有足够的质量来执行 这种蛋白质的三维重建。 HRS-2与相互作用蛋白及其固有的ATPase的关系 活动可能在其功能中发挥作用。在这一框架内 建议，我们将研究ATPase的活性和相互作用的作用 HrS-2低聚体结构中的蛋白质。为了实现这一目标，我们将 使用三维电子显微镜并进一步改进结果 使用计算结构预测方法。这一结构信息 将深入了解相互作用的蛋白质和酶活性的作用 对hrS-2低聚体的构象和最终功能的影响。这个 具体目标是：(1)原生生物的三维电子显微镜 Hrs-2的结构。(2)HRS-2的低温电子高分辨结构 显微镜和结构预测方法。(三)角色定位 HrS-2结构上的相互作用蛋白。(4)确定 三磷酸腺苷结合对hars-2结构的影响。(5)确定 HRS-2抑制SNARE复合体机制的结构基础 队形。

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.