Proton Crystallography of Membrane Proteins Using High Frequency ENDOR

描述（由申请人提供）：膜蛋白在许多细胞和生理过程中发挥关键作用，作为细胞与其环境之间、细胞内隔室之间以及组成器官系统的隔室之间的物质和信息传递的介质。虽然获得膜蛋白结构显然是可行的，但可溶性蛋白结构测定的速度超过了膜蛋白的速度，因此我们对膜蛋白及其可溶性对应物的理解水平之间存在差距。我们建议扩展一种称为高频ENDOR（电子核双共振）的间接检测磁共振光谱法用于膜蛋白结构测定。这种结构方法被称为ENDOR晶体学，主要用于分子晶体。高频（HF）ENDOR晶体学非常适合于许多膜蛋白的大尺寸，其晶体的小尺寸以及其对X射线的有限吸收能力。它在很大程度上不含来自脂质和去污剂的非蛋白质背景信号，但可以产生原子分辨率结构，包括许多蛋白质最感兴趣区域中实验确定的质子位置。我们提出通过以下三个具体目标来开发用于膜蛋白晶体结构测定的高频ENDOR晶体学：1）开发适合于在高频/场下实施的膜蛋白晶体的ENDOR晶体学的高分辨率ENDOR方法; 2）构建和实施优化的高频ENDOR探针，以便于晶体加载和旋转、高灵敏度和自动化数据收集; 3）开发用于自动峰分配和分析来自膜蛋白的实验测量的HFENDOR晶体学数据的方法。将从几种代表性膜蛋白晶体（细胞色素c氧化酶、bc 1复合物和b6 f复合物）中收集ENDOR晶体学数据，并从该数据中提取蛋白质结构坐标。这些特定的目标汇集了许多先进的技术，以推进膜蛋白结构测定的最新技术水平。

DESCRIPTION (provided by applicant): Membrane proteins play a crucial role in many cellular and physiological processes as mediators of material and information transfer between cells and their environment, between compartments within cells, and between compartments comprising organ systems. Although it is clearly feasible to obtain membrane protein structures, the rate of soluble protein structure determination is outpacing that of membrane proteins so that there is a gap between our level of understanding of membrane proteins and their soluble counterparts. We propose to extend a form of indirect-detection magnetic resonance spectrometry known as High Frequency ENDOR (electron nuclear double resonance) for application to membrane protein structure determination. This structural method has been called ENDOR Crystallography and has been used primarily in molecular crystals. High Frequency (HF) ENDOR Crystallography is well-suited to the large size of many membrane proteins, the small size of their crystals, and their limited ability to diffract X-rays. It is largely free of non-protein background signals from lipids and detergents yet can yield atomic resolution structures including experimentally determined proton locations in the most interesting regions of many proteins. We propose to develop High Frequency ENDOR Crystallography for structural determination in membrane protein crystals through the following three specific aims: 1) Develop high-resolution ENDOR methods suitable for ENDOR Crystallography of membrane protein crystals for implementation at high frequency/field; 2) Construct and implement an optimized High Frequency ENDOR probe for convenient crystal loading and rotation, high sensitivity, and automated data collection; 3) Develop methods for automated peak assignment and analysis of experimentally measured HFENDOR crystallographic data from membrane proteins. ENDOR Crystallography data will be collected from several representative membrane protein crystals (Cytochrome c Oxidase, the bc1 complex and the b6f complex) and protein structural coordinates will be extracted from that data. These specific aims bring together a number of advanced techniques in order to advance the state of the art in membrane protein structure determination.