TIME RESOLVED X RAY INTERFEROMETRY AND SARCOPLASMIC RETICULUM CALCIUM ATPASE

该项目的总体目标是确定活性(依赖于能量的)离子通过生物膜传输到至少亚分子水平的实际机制。肌浆网Ca+2-ATPase被用作原型离子泵。用X射线结晶学测定了洗涤剂增溶的肌浆网Ca+2-ATPase的高分辨三维结构。我们最近开发了面向矢量的洗涤剂增溶的Ca+2-ATPase单层，以及利用非共振和共振X射线干涉和中子干涉测量研究这些单层中酶的所谓轮廓结构的方法，空间分辨率约为7埃。所使用的洗涤剂已被证明是维持酶功能的最佳选择。作为这项工作的结果，加上双聚焦波动同步辐射X射线光源和高计数速率、快速时间帧X射线探测器的关键开发，我们现在准备不仅研究寿命大于或等于1ms的动态活性钙转运过程中的所有酶中间体的轮廓结构(而不是仅选择几个瞬时捕获的中间体)到大约7埃的空间分辨率，而且还将研究钙结合位点本身在这些寿命大于或等于100ms的每个酶中间体的轮廓结构中的位置和金属离子占有率。钙的运输过程将通过合适的笼状底物的闪光光解在酶单层之间同步启动。关键的时间分辨结构信息只能从液晶膜中的Ca+2 ATPase的全功能研究(或在液晶膜(或非晶膜类)环境中的Ca+2 ATPase的全功能的非晶态研究中获得)。这一关键的时间解析的结构信息将通过详细的分子建模来解释，该模型利用通过X射线结晶学获得的酶的高分辨率三维结构作为关键的起点。此外，被证明对钙转运功能至关重要的残基的定点突变的影响将被用来促进这种解释。-这些研究是通过利用面向载体的酶的单分子层来实现的。因此，这些研究无疑将对Ca+2ATPase的主动钙转运机制提供更深入的了解，这很可能与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+1ATPase和Ca+2ATPase。这些关于跨膜离子梯度形成的实际机制的知识是在细胞水平上理解它们作为心脏生物能量学中心的调控的重要一步。

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.