Pulsed-Electron Paramagnetic Resonance Spectrometer for Distance Determination in Biological Macromolecules

项目摘要/摘要 我们建议在凯斯西储大学购买一台最先进的脉冲EPR光谱仪 包括可溶性蛋白质、膜在内的生物大分子组装体的外部结构动力学研究 蛋白质和蛋白质-核酸复合体。EPR光谱应用范围广泛，从 核酸和生物膜，到蛋白质研究。这项技术提供了以下有价值的信息 对确定人类疾病的治疗靶点至关重要的生物学过程。此方法用于 表征蛋白质-蛋白质、蛋白质-脂肪和蛋白质-配体的相互作用 生物金属离子中心或结合定点定向自旋标记。在过去的十年里，脉冲EPR 方法已经成为量化构象变化的一种特别敏感和通用的方法 在它们的自然环境中形成的大型蛋白质复合体。脉冲法获得的大分子动力学数据。 EPR是对从X射线结晶学获得的高分辨率但静态的信息的补充 低温电子显微镜。此外，这种方法非常适合于对于核磁共振来说太大的系统 测量。硬件和脉冲协议的最新发展导致了显著的增长 从而大大扩展了可测量距离的范围，并改进了 这种方法的多功能性。布鲁克ELEXSYSE580 Q波段光谱仪能够进行连续波和各种 脉冲EPR方法，包括双电子-电子共振(DER)和电子-核双重 共振(Endor)光谱仪。鹿对自旋距离的敏感度在15~~80？之间，这是适宜的 用于研究大型蛋白质和核酸复合体的构象变化。的高级功能 拟议的文书对拟议的实验的成功至关重要，包括a)显著 相对于X波段提高了灵敏度，这将允许在较低的样品浓度下进行测量；b) 增加样品处理量，以满足结构生物学家日益增长的需求；c)提高分辨率 用于将可测量范围扩展到更长的距离，这对于确定全局 构象变化。美国国立卫生研究院资助的研究项目将直接受益于这一仪器 包括一些临床上相关的可溶性蛋白和膜蛋白的结构和动态研究 (配体和电压门控通道、EGF和胰岛素受体、Pron、动力素相关蛋白、HIV蛋白、 和免疫受体)。所要求的设备将是东北地区的第一台脉冲EPR光谱仪 俄亥俄州地区。这项技术将为NIH资助的几个项目提供最先进的结构生物学工具 研究人员致力于基本的生物学问题，扩大生物医学研究和 加强我们大中华区科学界内的多学科协作互动 克利夫兰地区。

Project Summary/Abstract We propose to acquire a state-of-the-art pulsed-EPR spectrometer at Case Western Reserve University to carry out structural dynamic studies of bio-macromolecular assemblies that include soluble proteins, membrane proteins, and protein-nucleic acid complexes. EPR spectroscopic applications span a wide range of areas, from nucleic acids and biomembranes, to protein research. This technology provides valuable information about biological processes critical for identification of therapeutic targets in human diseases. This approach is used to characterize protein-protein, protein-lipid, and protein-ligand interactions either by using naturally occurring biological metal ion centers or by incorporating site-directed spin-labeling. In the last decade, pulsed-EPR methods have emerged as an exceptionally sensitive and versatile for quantifying conformational changes in large protein complexes in their native environment. The macromolecule dynamics data obtained from pulsed- EPR are complementary to the high-resolution, yet static, information gained from X-ray crystallography and cryo-electron microscopy. Additionally, this approach is ideally suited for systems too large for NMR measurements. The latest developments in the hardware and pulse-protocols have led to remarkable increases in sensitivity and thereby have significantly extended the range of measurable distances and improved the versatility of this method. The Bruker ELEXSYS E580 Q-band spectrometer is capable of CW- and a variety of pulsed- EPR methods, including double electron-electron resonance (DEER) and electron-nuclear double resonance (ENDOR) spectroscopies. DEER is sensitive to spin-spin distance from 15 to ~80 Å, which is suitable for studying conformational changes in large proteins and nucleic acid complexes. The advanced features of the proposed instrument, which are critical to the success of the proposed experiments, include a) significantly improved sensitivity relative to X-band, which would allow measurement at lower sample concentrations; b) enhanced sample throughput to cater to the growing needs of the structural biologists; c) increased resolution for extending measurable range to much longer distances, which is particularly important for determining global conformational changes. The NIH-funded research projects that will directly benefit from this instrumentation include the structural and dynamic studies of a number of clinically relevant soluble and membrane proteins (ligand- and voltage-gated channels, EGF and insulin receptors, prions, dynamin related proteins, HIV proteins, and immune receptors). The requested equipment will be the first pulsed-EPR spectrometer in the Northeast Ohio area. This technology will provide a state-of-the-art structural biology tool to several NIH-funded investigators to work on fundamental biological problems, expanding the scope of biomedical research and strengthening the multidisciplinary collaborative interactions within our scientific community here in the Greater Cleveland area.