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: 10246411
• 负责人: Junko Yano
• 金额: $ 37.91万
• 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 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10246411
• 关键词: Active Sites; Biochemical Reaction; Biological; Biology; Catalytic Domain; Charge; Chemical Dynamics; Chemical Structure; Chemistry; Collaborations; Communities; Complex; Crystallography; Cytochrome P450; Data; Data Analyses; Data Collection; Development; Diffusion; Electron Transport; Electrons; Elements; Engineering; Enzymes; Event; Evolution; Feedback; Goals; Heme; Hydrogen Bonding; Hydroxyl Radical; In Situ; Measurement; Metals; Methane; Methodology; Methods; Nature; Oxidation-Reduction; Oxygen; Oxygenases; Photons; Physiologic pulse; Physiological; Process; Property; Protein Dynamics; Proteins; Protocols documentation; Protons; Radiation induced damage; Reaction; Resort; Ribonucleotide Reductase; Roentgen Rays; Sampling; Signal Transduction; Site; Source; Spectrum Analysis; Speed; Spottings; Structure; Synchrotrons; System; Techniques; Temperature; Time; Water; Width; X ray diffraction analysis; X ray spectroscopy; X-Ray Crystallography; Xray Emission Spectroscopy; absorption; analog; biological systems; catalyst; chemical reaction; cofactor; copper oxidase; cryogenics; design; electronic structure; emission spectroscopy; enzyme structure; experimental study; flexibility; geometric structure; interest; metalloenzyme; novel; novel strategies; 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 enzymes, by following the structural dynamics of the protein and chemical dynamics of the catalyst simultaneously. It is our goal to understand the design concepts from nature with X-ray crystallography and X-ray spectroscopy techniques using 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 metallo-enzymes at ambient conditions, 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 X-ray free electron lasers 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 make it possible to acquire the signal before the sample is destroyed. The objective of this proposal is to study the protein structure and dynamics of metallo-enzymes using crystallography, as well as the chemical structure and dynamics of the catalytic complexes (charge, spin, and covalency) using spectroscopy during the reaction to understand the electron-transfer processes and elucidate the mechanism. We will design and apply a full 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, to follow the reaction at room temperature. This will provide an unprecedented combination of correlated data between the protein and the co-factors, all of which are necessary for a complete understanding of structure and mechanism. Spectroscopy will include both emission and absorption spectroscopy to get a complete understanding of the time-evolution of the electronic structure, while simultaneous room temperature time-resolved X-ray crystallography would provide the changes in the geometric structure of the overall protein. The systems that will be used for developing these methodologies are some of the most important metallo-enzymes in biology that use high-valent Fe and other elements for oxygen and C-H bond activation. We will focus on non-heme enzymes such as ribonucleotide reductase (Mn/Fe and Fe/Fe) and methane mono oxygenase (Fe/Fe), and heme containing Cyt P450 systems, and functional analogs of heme-copper oxidase systems engineered into simpler proteins.

项目概要/摘要 该提案的目标是了解自然界中酶中发生的良好控制的化学反应， 同时跟踪蛋白质的结构动力学和催化剂的化学动力学。这是 我们的目标是通过 X 射线晶体学和 X 射线光谱学了解自然界的设计概念 使用 X 射线自由电子激光器 (XFEL) 的技术。 尽管酶的结构和催化位点的化学性质已经被研究 深入地讲，对原子尺度化学的理解需要一种超越传统方法的新方法 低温下的稳态 X 射线晶体学和 X 射线光谱学。跟随动态 在环境条件下金属酶的几何和电子结构发生变化，同时 克服X射线对氧化还原活性催化中心的严重损伤，是衍生该反应的关键 机制。来自 X 射线自由电子激光器的强超短飞秒 (fs) X 射线脉冲提供 克服当前生物样品室温数据收集局限性的机会 在同步加速器 X 射线源处。飞秒 X 射线脉冲可以在样品被扫描之前获取信号 被毁了。该提案的目的是研究金属酶的蛋白质结构和动力学 使用晶体学以及催化配合物的化学结构和动力学（电荷， 自旋和共价）在反应过程中使用光谱学来了解电子转移过程 并阐明其机制。 我们将设计并应用全套时间分辨 X 射线衍射和 X 射线吸收/发射 利用 XFEL 光束的独特性质的光谱方法来跟踪反应 室温。这将提供蛋白质和蛋白质之间相关数据的前所未有的组合。 辅助因素，所有这些都是完整理解结构和机制所必需的。 光谱学将包括发射光谱和吸收光谱，以全面了解 电子结构的时间演化，同时室温时间分辨 X 射线 晶体学将提供整个蛋白质几何结构的变化。 将用于开发这些方法的系统是一些最重要的系统 生物学中的金属酶，利用高价 Fe 和其他元素来激活氧和 C-H 键。 我们将重点关注非血红素酶，例如核糖核苷酸还原酶（Mn/Fe 和 Fe/Fe）和甲烷单酶 加氧酶 (Fe/Fe) 和含有 Cyt P450 系统的血红素，以及血红素铜氧化酶的功能类似物 系统被设计成更简单的蛋白质。