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遵循蛋白质的结构动力学和催化剂的化学动力学来理解大自然控制的化学反应。我们的目标是使用X射线晶体学和X射线光谱技术从自然界了解这些设计概念，并使用X射线游离电子激光器（XFELS）了解这些概念。尽管已经对催化部位的酶的结构和化学的结构进行了深入的研究，但对原子尺度化学的理解还需要除传统的稳态X射线晶体学和低温温度下的X射线光谱以外的新方法。遵循在环境条件下金属酶的几何和电子结构的动态变化，同时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机制的关键。 LCLS（Linac Coolent Light Source）X射线的无X射线脉冲X射线X射线脉冲X射线X射线脉冲X射线X射线激光为克服常规同步X射线X射线的生物样品的当前限制。 FS X射线脉冲使在样品破坏之前可以获取信号。该提案的目的是使用晶体学以及在反应过程中使用光谱法研究催化络合物（电荷，自旋和共价）的化学结构和动力学的蛋白质结构和动力学，以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 光谱法在室温下遵循反应的方法，这将提供蛋白质，副因子之间相关数据的前所未有的组合，所有这些都需要完全理解结构和机制。光谱法将包括K-边缘发射和L边缘吸收光谱，以完全了解电子结构的时间进化，而同时室温时间分辨时间分辨X射线晶体学将提供整体蛋白质复合物的几何结构的变化。该提案还将集中于同时遵循生物系统中多个地点发生的化学反应。这将使我们遵循 金属蛋白在各个时间尺度和水平上的多个位点之间的电子转移；在一个位点中，分子或两个不同分子中的两个位点之间。将会 用于开发这些方法论是生物学中最重要的金属酶。细胞色素C氧化酶（Fe，Cu），核糖核苷酸还原酶（Mn，Fe），氮酶（MO，FE）和血红素酶（Fe），Cyctochrome C过氧化物酶和一氧化氮合成酶。