SUBMOLECULAR CA++ ATPASE STUDIES--STRUCTURE OF ION MOTIVE ATPASE/ION TRANSPORT

本项目的目标是确定活性的实际机制， （能量依赖性）离子跨生物膜的转运，至少 亚分子水平。 采用肌浆网Ca+2 ATP酶 离子泵的原型。 现代时间分辨同步辐射X射线 衍射研究确定了圆柱平均轮廓结构 对于钙泵，中等分辨率（约15埃） 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 负责活性钙的部分反应的循环系列 转运，即所谓的E1、（Ca+2）xE 1和（Ca+2）xE 1左右的P中间体。 这些研究扩展到确定镧系离子的位置 (La+3，Tb+3），取代酶的高亲和性金属上的钙离子 结合位点，在钙泵的轮廓结构内， 酶磷酸化对金属离子占据位置的影响 这些网站。 总的来说，这些研究提出了一种新的机制， 主动离子转运 由于这项工作，加上关键的 高计数率、快速时间帧X射线探测器的开发， 洗涤剂增溶的 Ca+2ATPase，我们现在准备研究的不仅是 所有酶中间体的结构），而且位置和金属 离子占有率的钙结合位点本身的轮廓 这些酶中间体的结构。 这个关键的时间分辨 结构信息，结合新兴的高分辨率3- 三维结构的酶，无疑将提供深刻的见解 研究Ca+2 ATP酶的活性钙转运机制， 可能与其他成员的运输机制高度相关， P型离子泵家族也包括质膜Na+、K+- ATP酶和Ca+2 ATP酶。

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 (about 15 equal angstroms) 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 about P intermediates. These studies were extended to determine the locations of lanthanide ions (La+3, Tb+3), replacing calcium ions on the enzyme's 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 a high count-rate, rapid time-framing x-ray detector and vectorially-oriented single monolayers of the detergent-solubilized Ca+2ATPase, we are now poised to investigate not only the profile structures of all enzyme 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 highly relevant to the transport mechanism for other members of the P-type family of ion pumps as well, including the plasma membrane Na+, K+- ATPases and Ca+2ATPases.