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

本项目的目标是确定主动控制的实际机制 (能量依赖)离子通过生物膜传输到至少 亚分子水平。肌浆网Ca+2ATPase 作为典型的离子泵。最新时间分辨同步加速器x射线 衍射研究确定了柱面平均轮廓结构 使钙泵达到中等分辨率(约15埃) 在功能齐全的分离肌浆网膜内 重要的、瞬间捕获的酶中间体出现在 与活性钙有关的一系列循环部分反应 转运，即所谓的E1、(Ca+2)XE1和(Ca+2)XE1左右的P中间体。 这些研究扩展到确定稀土离子的位置。 (La+3，Tb+3)，取代酶的高亲和力金属上的钙离子 在钙泵的轮廓结构内的结合位置 酶磷酸化对金属离子位置和占有率的影响 这些网站。总而言之，这些研究提出了一种新的机制 主动离子传输。作为这项工作的结果，连同关键的 高计数率、快速定帧X射线探测器的研制 洗涤剂增溶的矢量取向单层膜 CA+2ATPase，我们现在准备调查的不仅仅是配置文件 所有酶中间体的结构)，但也包括位置和金属 钙结合部位本身在剖面内的离子占有率 每一种酶中间体的结构。此密钥已时间解析 结构信息，结合新兴的高分辨率3- 对于酶的空间结构，无疑会提供深刻的洞察力 探讨Ca+2ATPase的主动钙转运机制 可能与联合国其他成员的运输机制高度相关 P型离子泵家族，包括质膜Na+，K+- ATPase和Ca+2ATPase。

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.