Proton Crystallography of Membrane Proteins Using High Frequency ENDOR

使用高频 ENDOR 进行膜蛋白质子晶体学分析

• 批准号: 7011282
• 负责人: GARY J. GERFEN
• 金额: $ 18.4万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7011282
• 关键词: NIH Roadmap Initiative tag; automated data processing; electron crystallography; electron spin resonance spectroscopy; hydrogen ions; membrane proteins; method development; molecular probes; protein structure

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.

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，它是细胞及其环境之间，细胞内部区域之间以及包含器官系统的隔室之间的材料和信息传递的介体。尽管获得膜蛋白结构显然是可行的，但是可溶性蛋白结构的测定速率超过了膜蛋白的速率，因此我们对膜蛋白及其可溶性对应物的了解水平之间存在差距。我们建议扩展一种间接检测磁共振光谱法被称为高频endor（电子核双共振），以应用于膜蛋白结构的测定。这种结构方法称为endor晶体学，主要用于分子晶体。高频（HF）内多的晶体学非常适合许多膜蛋白的大尺寸，其晶体的小尺寸以及衍射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，包括许多蛋白质最有趣的区域中实验确定的质子位置。我们建议通过以下三个特定目的开发高频内多的晶体学来用于膜蛋白晶体的结构测定：1）开发适用于膜蛋白晶体的endor晶体学以在高频/场上实现的高分辨率endor方法； 2）构建并实施了优化的高频内多探针，以方便晶体负载和旋转，高灵敏度和自动数据收集； 3）开发了从膜蛋白的实验测得的Hfendor晶体学数据的自动峰分配和分析的方法。将从几种代表性的膜蛋白晶体（细胞色素C氧化酶，BC1复合物和B6F复合物）中收集Endor晶体学数据，将从该数据中提取蛋白质结构坐标。这些特定的目的汇集了许多先进的技术，以推动膜蛋白结构测定中的最新技术。