TIME RESOLVED X RAY INTERFEROMETRY AND SARCOPLASMIC RETICULUM CALCIUM ATPASE

本项目的总体目标是确定至少在亚分子水平上的活性（能量依赖性）离子跨生物膜传输的实际机制。肌浆网Ca+2 ATP酶被用作原型离子泵。洗涤剂溶解的肌浆网Ca+2 ATP酶的高分辨率三维结构刚刚被X射线晶体学确定。我们最近开发了洗涤剂增溶的Ca+2 ATP酶的矢量取向的单单层膜和用于研究这些单层膜中的酶的所谓的轮廓结构的方法，其空间分辨率为约7埃，采用非共振和共振X射线干涉测量法和中子干涉测量法。所用的洗涤剂已被证明是最佳的保持酶的功能。作为这项工作的结果，加上双聚焦波荡器同步加速器x射线源和高计数率、快速时间帧x射线探测器的关键发展，我们现在不仅要研究在动态活性钙转运过程中，寿命大于或等于1毫秒的所有酶中间体的轮廓结构，（与仅几个选择的瞬时捕获的中间体相反）到大约7埃的空间分辨率，而且还包括钙结合位点本身在这些酶中间体中的每一种的分布结构内的位置和金属离子占有率，钙转运过程将通过合适的笼状底物的闪光光解在酶单层系综之间同步启动。关键的时间分辨结构信息只能从液晶膜内Ca+2 ATP酶的功能形式的研究中获得（或在液晶膜（或非结晶膜样）环境中以其完全功能形式的Ca+2 ATP酶的非结晶研究。这一关键的时间分辨结构信息将通过详细的分子建模来解释，利用通过X射线晶体学获得的酶的高分辨率三维结构作为关键的起点。此外，将采用对钙转运功能至关重要的残基的定点突变的影响来促进这种解释。这些研究通过利用载体定向酶的单层而成为可能。因此，这些研究无疑将为深入了解Ca+2 ATP酶的活性钙转运机制提供帮助，该机制很可能与P型离子泵家族的其他成员的转运机制高度相关，包括质膜Na+1/K+1 ATP酶和Ca+2 ATP酶。关于跨膜离子梯度形成的实际机制的这种知识是理解它们在细胞水平上作为心脏生物能量学中心的调节的重要一步。

The overall 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+2 ATPase is employed as the prototypical ion pump. The high-resolution 3-dimensional structure of the detergent-solubilized sarcoplasmic reticulum Ca+2 ATPase has just been determined by x-ray crystallography. We have recently developed vectorially-oriented single monolayers of the detergent-solubilized Ca+2 ATPase and the methods for the study of the so-called profile structure of the enzyme in these monolayers to a spatial resolution of approximately 7 angstroms employing both non-resonance & resonance x-ray interferometry and neutron interferometry. The detergent utilized has been shown to be optimal for maintaining enzyme functionality. As a result of this work, together with the critical developments of a doubly-focused undulator synchrotron x-ray source and a high count-rate, rapid time-framing x-ray detector, we are now poised to investigated not only the profile structures of all enzyme intermediates within the dynamic active calcium transport processes with lifetimes greater than or equal to 1 ms (as opposed to only a few selected transiently trapped intermediates) to a spatial resolution of approximately 7 angstroms, but also the positions and metal ion occupancies of the calcium binding sites themselves within the profile structures of each of these enzyme intermediates with lifetimes greater than or equal to 100 ms. The calcium transport processes will be initiated synchronously among the enzyme monolayer ensemble via the flash-photolysis of suitable caged-substrates The key time-resolved structural information can only be obtained from such studies of the Ca+2 ATPase in its fully functional form within a liquid- crystalline membrane (or non-crystalline studies of the Ca+2 ATPase in its fully functional form within a liquid crystalline membrane (or non- crystalline membrane-like) environment. This key time-solved structural information will be interpreted via detailed molecular modeling utilizing the high-resolution 3-dimensional structure for the enzyme obtained via X-ray crystallography as a critical starting point.. In addition, the effects of site-directed mutations of residues shown to be critical to the calcium transport function will be employed to facilitate this interpretation.-these studies are made possible by the utilization of single monolayers of the vectorially-oriented enzyme. As a result, these studies 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+1/K+1ATPases and Ca+2ATPases. Such knowledge concerning the actual mechanisms for the formation of transmembrane ionic gradients is an important step toward understanding their regulation as central to cardiac bioenergetics at the cellular level.