Proton Crystallography of Membrane Proteins Using High Frequency ENDOR

描述(申请人提供)：膜蛋白在许多细胞和生理过程中扮演着重要的角色，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媒介。虽然膜蛋白结构的获得是明显可行的，但由于膜蛋白结构测定的速度快于膜蛋白，导致我们对膜蛋白的认识水平与膜蛋白的可溶性有一定的差距。我们建议将一种称为高频ENDOR(电子核双共振)的间接检测磁共振波谱扩展到膜蛋白质结构的测定中。这种结构方法被称为恩多尔结晶学，主要用于分子晶体。高频(HF)Endor结晶术非常适合于许多膜蛋白的大尺寸、其晶体的小尺寸以及其有限的X射线衍射能力。它在很大程度上没有来自脂类和洗涤剂的非蛋白质背景信号，但可以产生原子分辨结构，包括在许多蛋白质最感兴趣的区域中实验确定的质子位置。我们建议通过以下三个具体目标开发用于膜蛋白晶体结构确定的高频Endor晶体：1)开发适合于膜蛋白晶体Endor晶体分析的高分辨率Endor方法，以便在高频/场下实施；2)构建并实现优化的高频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)开发自动化的峰分配方法，并分析从膜蛋白获得的实验测量的HFENDOR晶体数据。Endor结晶学数据将从几个典型的膜蛋白晶体(细胞色素c氧化酶、bc1复合体和b6f复合体)中收集，并将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许多先进的技术，以促进膜蛋白结构测定的最新水平。

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.