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.

项目摘要/摘要 该提议的目的是了解发生大自然的良好控制的化学反应 金属酶，通过开发必要的工具并应用它们遵循结构动态 金属催化剂的蛋白质和化学动力学。为此，我们将使用X射线晶体学和 X射线光谱技术在X射线游离电子激光器（XFELS）处。 尽管酶的结构和催化位点的化学已经研究了 强烈地，对原子级化学的理解需要一种新的方法 稳态X射线晶体学和X射线光谱在低温温度下。遵循动态 金属酶在环境温度下的几何和电子结构的变化，而 克服对氧化还原活性催化中心的严重X射线损伤是推导反应的关键 机制。 Xfels的强烈而超短的飞秒（FS）X射线脉冲提供了机会 克服同步子x-上生物样品的室温数据收集的当前局限性 射线来源。通过射击样品更换的FS X射线脉冲使获得信号成为可能 在发生损坏之前，样本被破坏。 我们将设计并应用一套时间分辨的X射线衍射和X射线遗憾/排放 光谱法，利用Xfel束的独特性能。这将提供一个 蛋白质与副因素之间相关数据的空前组合，所有这些都是 了解蛋白质和电子结构的几何结构之间的相互作用所需 金属复合物以及功能后果。光谱，发射和滥用 光谱法将提供电子结构的时间进化，而同时进行室温 X射线晶体学将帮助我们可视化整体蛋白质的几何结构的变化。 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 对催化机制（包括单声道和双核系统）都有洞察力的见解 异质金属中心。这些系统的代表性示例是氧和C-H键的Fe酶 提出高价值Fe的激活，例如包含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），以及甲烷单加氧酶 （fe/fe）。我们还将专注于与H2代和甲烷代谢相关的Ni和Ni/Fe酶，以及 重点氧化酶系统的重要类别的功能类似物设计为简单的蛋白质。