Biological Spectroscopy and Crystallography Using an X-ray Free Electron Laser

使用 X 射线自由电子激光进行生物光谱学和晶体学

• 批准号: 10587180
• 负责人: Junko Yano
• 金额: $ 54.13万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10587180
• 关键词: Active Sites; Binding; Biochemical Reaction; Biological; Biology; Catalytic Domain; Charge; Chemical Dynamics; Chemicals; Chemistry; Collaborations; Complex; Crystallography; Cytochrome P450; Data; Data Analyses; Data Collection; Data Correlations; Development; Diffusion; Electron Transport; Electronics; Electrons; Elements; Engineering; Environment; Enzymes; Event; Evolution; Fluorescence; Generations; Geometry; Goals; Heme; Homo; Hydrogen Bonding; Hydrogenase; Hydroxyl Radical; In Situ; Kinetics; Measurement; Membrane; Metalloproteins; Metals; Methane; Methane Metabolism Pathway; Methane hydroxylase; Methodology; Methods; Monitor; Mononuclear; Nature; Nuclear; Oxidases; Oxidation-Reduction; Oxidoreductase; Oxygen; Oxygenases; Physiologic pulse; Physiological; Property; Protein Dynamics; Proteins; Protocols documentation; Protons; Radiation induced damage; Reaction; Resolution; Ribonucleotide Reductase; Roentgen Rays; Sampling; Signal Transduction; Site; Source; Specificity; Spectrum Analysis; Speed; Structure; Synchrotrons; System; Techniques; Temperature; Time; Transition Elements; Visualization; Water; Width; X ray diffraction analysis; X ray spectroscopy; X-Ray Crystallography; Xray Emission Spectroscopy; absorption; analog; biological systems; catalyst; cofactor; copper oxidase; cryogenics; design; electronic structure; emission spectroscopy; enzyme structure; experimental study; flexibility; geometric structure; insight; interest; isopenicillin N; metal complex; metallicity; metalloenzyme; novel; novel strategies; oxidation; protein structure; spectroscopic data; tool; x-ray free-electron laser

Project Summary/Abstract The goal of this proposal is to understand nature’s well-controlled chemistry that occurs in metalloenzymes, by developing the necessary tools and applying them to follow the structural dynamics of the protein and chemical dynamics of the metal-catalyst. For this purpose, we will use X-ray crystallography and X-ray spectroscopy techniques at X-ray Free Electron Lasers (XFELs). Although the structure of enzymes and the chemistry at the catalytic sites have been studied intensively, an understanding of the atomic-scale chemistry requires a new approach beyond the conventional steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure of metalloenzymes at ambient temperature, while overcoming the severe X-ray damage to the redox active catalytic center, is key for deriving the reaction mechanism. The intense and ultra-short femtosecond (fs) X-ray pulses from XFELs provide an opportunity to overcome the current limitations of room temperature data collection for biological samples at synchrotron X- ray sources. The fs X-ray pulses with shot-by-shot sample replacement make it possible to acquire the signal before damage occurs and the sample is destroyed. We will design and apply a suite of time-resolved X-ray diffraction and X-ray absorption/emission spectroscopy methods, that make use of the unique properties of the XFEL beam. This will provide an unprecedented combination of correlated data between the protein and the co-factors, all of which are necessary to understand the interplay between the geometric structure of the protein and electronic structure of the metal complex, and the functional consequences. Spectroscopy, both emission and absorption spectroscopy, will provide the time-evolution of the electronic structure, while simultaneous room temperature X-ray crystallography will help us visualize the changes in the geometric structure of the overall protein. We will use these methodologies to study some of the most important metalloenzymes in biology to gain insights into the catalytic mechanisms, including mono, and dinuclear systems both with homo- and hetero-metallic centers. A representative example of these systems are Fe enzymes for oxygen and C-H bond activation where the involvement of high-valent Fe are proposed, such as the heme containing Cyt P450 systems, non-heme enzymes ribonucleotide reductase (Mn/Fe and Fe/Fe), and methane mono oxygenase (Fe/Fe). We will also focus on Ni and Ni/Fe enzymes relevant to H2 generation and methane metabolism, and functional analogs of the important class of heme-copper oxidase systems engineered into simpler proteins.

项目总结/摘要 这个提议的目的是了解自然界中发生的控制良好的化学反应， 金属酶，通过开发必要的工具，并应用它们来遵循的结构动力学的 蛋白质和金属催化剂的化学动力学。为此，我们将使用X射线晶体学， X射线自由电子激光器（XFEL）的X射线光谱技术。 尽管人们已经研究了酶的结构和催化位点的化学性质， 从本质上讲，对原子尺度化学的理解需要一种超越传统方法的新方法。 稳态X射线晶体学和低温下的X射线光谱学。跟随动态 在环境温度下金属酶的几何和电子结构的变化，而 克服X射线对氧化还原活性催化中心的严重损伤，是推导该反应的关键 机制来自XFEL的强烈和超短飞秒（fs）X射线脉冲提供了一个机会， 克服了目前在同步加速器X射线加速器上收集生物样品室温数据的局限性， 射线源。飞秒X射线脉冲与逐炮样品替换使得获取信号成为可能 在损坏发生和样本被破坏之前。 我们将设计和应用一套时间分辨X射线衍射和X射线吸收/发射 光谱学方法，利用XFEL光束的独特性质。这将提供一个 蛋白质和辅助因子之间的相关数据的前所未有的组合，所有这些都是 了解蛋白质的几何结构和电子结构之间的相互作用是必要的 金属络合物的性质和功能性后果。光谱学，包括发射和吸收 光谱，将提供电子结构的时间演变，而同时室温 X射线晶体学将帮助我们可视化整个蛋白质几何结构的变化。 我们将使用这些方法来研究生物学中一些最重要的金属酶， 深入了解催化机制，包括单核和双核系统， 异金属中心这些系统的代表性实例是用于氧和C-H键的Fe酶 提出涉及高价Fe的活化，例如含有Cyt P450的血红素 系统，非血红素酶核糖核苷酸还原酶（Mn/Fe和Fe/Fe）和甲烷单加氧酶 (Fe/Fe）。我们还将关注与H2生成和甲烷代谢相关的Ni和Ni/Fe酶， 血红素-铜氧化酶系统的重要类别的功能类似物被工程化为更简单的蛋白质。