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: 8673988
• 负责人: Junko Yano
• 金额: $ 38.38万
• 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/8673988
• 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; DNA Sequence Rearrangement; Data; Data Collection; Data Set; Detection; Development; Electron Transport; Electrons; Elements; Environment; Enzymes; Evolution; Goals; 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; geometric structure; high throughput analysis; interest; metalloenzyme; novel; novel strategies; photosystem II; pressure; protein complex; protein structure; public health relevance; 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)：这项提议的科学目标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，了解自然界中酶发生的良好控制的化学作用。我们的目标是通过使用X射线自由电子激光(XFELs)的X射线结晶学和X射线光谱学技术从自然中理解这些设计概念。虽然酶的结构和催化部位的化学已经被深入研究，但理解原子尺度的化学需要一种新的方法，而不是传统的稳态X射线结晶学和低温X射线光谱学。跟踪金属酶在常温下几何结构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破坏，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高强度和超短飞秒(Fs)X射线脉冲为克服目前在常规同步辐射X射线源上采集生物样品的室温数据的局限性提供了机会。飞秒X射线脉冲使在样品被破坏之前获取信号成为可能。这项建议的目的是利用结晶学研究金属酶的蛋白质结构和动力学，以及在反应过程中利用光谱学研究催化络合物的化学结构和动力学(电荷、自旋和共价性)，以了解电子转移过程并阐明其机理。我们将设计和应用全套时间分辨X射线衍射和X射线吸收/发射 光谱学方法跟踪室温下的反应，这将提供前所未有的蛋白质和辅助因子之间的相关数据组合，所有这些都是完全了解结构和机制所必需的。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，而同时的室温时间分辨X射线结晶学将提供整个蛋白质复合体几何结构的变化。该提案还将侧重于同时跟踪生物系统中多个地点发生的化学作用。这将使我们能够遵循 金属蛋白中不同时间尺度和水平的多个位置之间的电子转移；一个位置内、一个分子中两个位置之间或两个不同分子之间的电子转移。这些系统将 可用于发展这些方法学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)、核苷酸还原酶(Mn，Fe)、固氮酶(Mo，Fe)和血红素酶(Fe)、环色素c过氧化物酶和一氧化氮合酶。