MECHANISM OF ACTIVE ION TRANSPORT--STRUCTURAL STUDIES

描述：本项目的目标是确定实际机制 的活性（能量依赖）离子运输通过生物膜， 至少是亚分子水平。 肌浆网Ca+2 ATP酶是 用作原型离子泵。 现代时间分辨同步加速器 X-射线衍射研究确定了圆柱平均轮廓 钙泵的结构，以中等分辨率（约。 15%） 在三个完全功能的分离的肌浆网膜内， 重要的，短暂捕获的酶中间体发生在 负责活性钙转运的部分反应的循环系列， 即所谓的E1、（Ca+2）xE 1和（Ca+2）xE 1-P中间体。 这些研究 扩展到测定镧系离子（La+3，Tb+3）的位置， 取代酶高亲和力金属结合位点上的钙离子， 内钙泵的轮廓结构和酶的作用 这些位点上的磷酸化和金属离子占据。 总的来说，这些研究提出了一种新的机制， 运输 由于这项工作，加上关键的 高计数率、快速时帧X射线探测器的发展 洗涤剂增溶的 Ca+2 ATP酶，Blasie博士现在准备研究，不仅是轮廓 动态活性钙内所有酶中间体的结构 传输过程（而不是只有少数选定的瞬时捕获 中间体），而且位置和金属离子占据的 钙结合位点本身的轮廓结构内的每一个 这些酶的中间体。 这个关键的时间分辨结构信息， 结合新兴的高分辨率三维结构， 酶，无疑将提供深入了解的机制， Ca+2 ATP酶的钙转运，最可能与 用于P型离子泵家族的其他成员的输送机构， 包括质膜Na+1/K+1ATP酶和Ca+2ATP酶。 等 关于跨膜蛋白形成的实际机制的知识 离子梯度是了解其调节的重要一步 作为细胞水平心脏生物能量学的核心。

DESCRIPTION: The goal of this project is to determine the actual mechanism of active (energy dependent) ion transport across biological membranes to at least the submolecular level. The sarcoplasmic reticulum Ca+2ATPase is employed as the prototypical ion pump. Recent time-resolved synchrotron x-ray diffraction studies determined the cylindrically-averaged profile structure for the calcium pump to moderate resolution (ca. 15 percent) within fully-functional isolated sarcoplasmic reticulum membranes for three important, transiently-trapped enzyme intermediates occurring within the cyclic series of partial reactions responsible for active calcium transport, the so-called E1, (Ca+2)xE1 and (Ca+2)xE1-P intermediates. These studies were extended to determine the locations of lanthanide ions (La+3, Tb+3), replacing calcium ions on the enzyme high-affinity metal binding sites, within the profile structure of the calcium pump AND the effect of enzyme phosphorylation on the positions and metal ion occupancies of these sites. Collectively, these studies suggest a novel mechanism for active ion transport. As a result of this work, together with the critical developments of high count-rate, rapid time-framing x-ray detector and vectorially-oriented single monolayers of the detergent-solubilized Ca+2ATPase, Dr. Blasie is now poised to investigate, not only the profile structures of all enzyme intermediates within the dynamic active calcium transport process (as opposed to only a few selected transiently trapped intermediates), but also the positions and metal ion occupancies of the calcium binding sites themselves within the profile structures of each of these enzyme intermediates. This key time-resolved structural information, combined with the emerging high-resolution 3-dimensional structure for the enzyme, will undoubtedly provide deep insight into the mechanism of active calcium transport by the Ca+2ATPase, most likely high relevant to the transport mechanism for other members of the P-type family of ion pumps as well, including the plasma membrane Na+1/K+1ATPases and Ca+2ATPases. Such knowledge concerning the actual mechanism for the formation of transmembrane ionic gradients is an important step toward understanding their regulation as central to cardiac bioenergetics at the cellular level.