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.

项目摘要/摘要 该提案的目的是了解大自然在酶中发生的良好控制的化学反应， 通过同时遵循催化剂的蛋白质和化学动力学的结构动力学。这是 我们的目标是通过X射线晶体学和X射线光谱了解自然的设计概念 使用X射线游离电子激光器（XFELS）的技术。 尽管已经研究了酶的结构和催化位点的化学 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 在环境条件下，金属酶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。来自X射线免费电子激光器的强烈而超短的飞秒（FS）X射线脉冲提供 克服生物样品室温数据收集的当前局限性的机会 在Synchrotron X射线源中。 FS X射线脉冲使在样品为之前获取信号成为可能 被摧毁。该建议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复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法了解电子转移过程 并阐明机制。 我们将设计并应用一套时间分辨的X射线衍射和X射线吸收/发射 利用Xfel束的独特特性的光谱法进行了遵循反应 室温。这将提供蛋白质和 共同因素，所有这些都是对结构和机制的完整理解所必需的。 光谱法将包括发射和吸收光谱，以完全了解 电子结构的时间进化，同时室温时间分辨X射线 晶体学将提供整体蛋白质的几何结构的变化。 将用于开发这些方法的系统是一些最重要的系统 生物学中的金属酶使用高价值Fe和其他元素进行氧和C-H键激活。 我们将专注于非血红素酶，例如核糖核苷酸还原酶（Mn/Fe和Fe/Fe）和甲烷单声道 氧合酶（Fe/Fe）和含有Cyt P450系统的血红素和血红素 - 铜氧化酶的功能类似物 系统设计为更简单的蛋白质。