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

使用 X 射线自由电子激光进行生物光谱学和晶体学 - 补充设备

• 批准号: 9027669
• 负责人: Junko Yano
• 金额: $ 12.23万
• 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 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9027669
• 关键词: Active Sites; Area; Biochemical Reaction; Biocompatible Materials; Biological; Biology; Catalytic Domain; Charge; Chemical Dynamics; Chemical Structure; Chemicals; Chemistry; Complex; Crystallography; Cytochrome c Peroxidase; Data; Data Collection; Data Set; Detection; Development; Electron Transport; Electrons; Elements; Environment; Enzymes; Equipment; Evolution; Goals; Health; Heme; Lasers; Light; Measurement; Metalloproteins; Metals; Methodology; Methods; Modeling; Nature; Nitric Oxide Synthase; Nitrogenase; Oxidation-Reduction; Peroxidases; Phase; Physiologic pulse; Physiological; Planning Techniques; Process; Protein Dynamics; Proteins; Radiation induced damage; Reaction; Relaxation; Ribonucleotide Reductase; Roentgen Rays; Sampling; Signal Transduction; Site; Solutions; Source; Spectrum Analysis; Speed; Structure; Synchrotrons; System; Techniques; Temperature; Testing; Time; Transition Elements; X ray diffraction analysis; X ray spectroscopy; X-Ray Crystallography; X-Ray Diffraction; Xray Emission Spectroscopy; absorption; aqueous; biological systems; catalyst; chemical reaction; cofactor; cryogenics; cytochrome c oxidase; design; electronic structure; emission spectroscopy; enzyme structure; flexibility; free-electron laser; geometric structure; high throughput analysis; interest; metalloenzyme; novel; novel strategies; photosystem II; pressure; protein complex; protein structure; synchrotron radiation

DESCRIPTION (provided by applicant): The scientific aim 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 these 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 of the LCLS (Linac Coherent Light Source) X-ray free electron laser provide an opportunity to overcome the current limitations of room temperature data collection for biological samples at regular 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 to follow the reaction at room temperature, that will provide an unprecedented combination of correlated data between the protein, the co-factors, all of which are necessary for a complete understanding of structure and mechanism. Spectroscopy will include both K-edge-emission and L-edge 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 complex. The proposal will also focus on simultaneously following the chemistry that occurs at multiple sites in biological systems. This will allow us to follow the electron transfer between the multiple sites in metalloproteins at various time-scales and levels; within a site, between two sites in a molecule or in two different molecules. The systems that will be used for developing these methodologies are some of the most important metallo-enzymes in biology; cytochrome c oxidase (Fe, Cu), ribonucleotide reductase (Mn, Fe), nitrogenase (Mo, Fe) and heme enzymes (Fe), cyctochrome c peroxidase and nitric oxide synthase.

描述（由申请人提供）：该提案的科学目的是通过同时跟踪蛋白质的结构动力学和催化剂的化学动力学，了解酶中发生的自然良好控制的化学反应。我们的目标是利用 X 射线自由电子激光器 (XFEL) 通过 X 射线晶体学和 X 射线光谱技术从自然界中了解这些设计概念。尽管酶的结构和催化位点的化学已经被深入研究，但对原子尺度化学的理解需要一种超越传统稳态 X 射线晶体学和低温下 X 射线光谱学的新方法。跟踪金属酶在环境条件下几何和电子结构的动态变化，同时克服X射线对氧化还原活性催化中心的严重损伤，是推导反应机理的关键。 LCLS（直线加速器相干光源）X 射线自由电子激光器的强超短飞秒 (fs) X 射线脉冲为克服当前在常规同步加速器 X- 下采集生物样品室温数据的局限性提供了机会。射线源。飞秒 X 射线脉冲可以在样品被破坏之前获取信号。该提案的目的是使用晶体学研究金属酶的蛋白质结构和动力学，以及在反应过程中使用光谱学研究催化复合物（电荷、自旋和共价）的化学结构和动力学，以了解电子-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 光谱方法在室温下跟踪反应，这将提供蛋白质、辅助因子之间的相关数据的前所未有的组合，所有这些对于完整理解结构和机制都是必要的。光谱学将包括 K 边发射光谱和 L 边吸收光谱，以全面了解电子结构的时间演化，而同步室温时间分辨 X 射线晶体学将提供电子结构几何结构的变化。整个蛋白质复合物。该提案还将重点关注同时跟踪生物系统中多个位点发生的化学反应。这将使我们能够遵循 不同时间尺度和水平的金属蛋白多个位点之间的电子转移；在一个位点内、在一个分子的两个位点之间或在两个不同的分子中。这些系统将 用于开发这些方法的是生物学中一些最重要的金属酶；细胞色素c氧化酶（Fe、Cu）、核糖核苷酸还原酶（Mn、Fe）、固氮酶（Mo、Fe）和血红素酶（Fe）、细胞色素c过氧化物酶和一氧化氮合酶。