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）：本提案的科学目标是通过同时遵循蛋白质的结构动力学和催化剂的化学动力学来理解发生在酶中的自然控制良好的化学反应。我们的目标是利用x射线自由电子激光器（XFELs）的x射线晶体学和x射线光谱学技术从自然界中理解这些设计概念。虽然酶的结构和催化位点的化学已经得到了深入的研究，但要理解原子尺度的化学，需要一种超越传统的稳态x射线晶体学和低温x射线光谱学的新方法。了解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（直线相干光源）x射线自由电子激光器的强烈和超短飞秒（fs） x射线脉冲为克服常规同步加速器x射线源室温生物样品数据收集的局限性提供了一个机会。fs x射线脉冲使得在样品被破坏之前获得信号成为可能。本课题的目的是利用晶体学研究金属酶的蛋白质结构和动力学，并利用光谱学研究反应过程中催化配合物（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机理。我们将设计和应用一套完整的时间分辨x射线衍射和x射线吸收/发射