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

项目总结/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， 生物大分子组装体的结构动力学研究，包括可溶性蛋白质、膜 蛋白质和蛋白质-核酸复合物。EPR光谱应用范围广泛，从 从核酸和生物膜到蛋白质研究。这项技术提供了关于 生物学过程的关键识别人类疾病的治疗目标。这种方法用于 表征蛋白质-蛋白质、蛋白质-脂质和蛋白质-配体相互作用， 生物金属离子中心或通过掺入定点自旋标记。在过去的十年里，脉冲EPR 方法已经成为一种非常敏感和通用的定量构象变化， 大的蛋白质复合物在它们的自然环境中。从脉冲- EPR是对从X射线晶体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NMR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着的增长 在灵敏度，从而显着扩大了可测量的距离范围，并提高了 这种方法的多样性。Bruker ELEXTONE 580 Q波段光谱仪能够进行CW和各种 脉冲EPR方法，包括双电子-电子共振（DEER）和电子-核双共振（E-NMR）。 共振（ENDOR）光谱。DEER对15 ~80 μ m的自旋-自旋距离敏感， 用于研究大型蛋白质和核酸复合物的构象变化。的高级功能 拟议的仪器，这是关键的成功拟议的实验，包括a）显着 相对于X波段的灵敏度提高，这将允许在较低的样品浓度下进行测量; B） 提高样品通量，以满足结构生物学家日益增长的需求; c）提高分辨率 将可测量范围扩展到更长的距离，这对于确定全球范围内的 构象变化NIH资助的研究项目将直接受益于这种仪器 包括一些临床相关的可溶性和膜蛋白的结构和动力学研究 （配体和电压门控通道，EGF和胰岛素受体，朊病毒，发动蛋白相关蛋白，HIV蛋白， 和免疫受体）。所要求的设备将是东北部第一台脉冲EPR光谱仪 俄亥俄州地区。这项技术将为NIH资助的几个国家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，扩大生物医学研究的范围， 加强我们科学界内部的多学科合作互动， 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.