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起着至关重要的作用，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，但可溶性蛋白结构的确定速度超过了膜蛋白的确定速度，因此我们对膜蛋白的理解水平与可溶蛋白之间存在差距。我们建议扩展一种间接检测的磁共振光谱法，称为高频ENDOR（电子核双共振），用于膜蛋白结构的测定。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大尺寸的膜蛋白，小尺寸的晶体，以及它们衍射x射线的有限能力。它在很大程度上没有来自脂质和洗涤剂的非蛋白质背景信号，但可以产生原子分辨率结构，包括在许多蛋白质最感兴趣的区域实验确定的质子位置。我们提出开发用于膜蛋白晶体结构测定的高频ENDOR晶体技术，主要有以下三个具体目标：1)开发高分辨率ENDOR方法，适用于膜蛋白晶体的高频/场ENDOR晶体分析；2)构建并实现优化的高频ENDOR探头，方便晶体加载和旋转，灵敏度高，数据采集自动化；3)开发自动峰分配和分析膜蛋白实验测量的hendor晶体数据的方法。ENDOR晶体学数据将从几个具有代表性的膜蛋白晶体（细胞色素c氧化酶、b1复合物和b6f复合物）中收集，并从这些数据中提取蛋白质结构坐标。这些特定的目标汇集了一些先进的技术，以推进膜蛋白结构测定的艺术状态。

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.