ESTABLISH CONCURRENT UV, VISIBLE, AND RAMAN SPECTROSCOPY AND X-RAY DIFFRACTION

建立并行紫外、可见光、拉曼光谱和 X 射线衍射

• 批准号: 7957318
• 负责人: ALLEN M ORVILLE
• 金额: $ 34.95万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957318
• 关键词: 2-Nitropropane dioxygenase; Active Sites; Address; Aldehydes; Algorithms; Area; Arthrobacter; Back; Betaine; Biochemistry; Caliber; Cells; Chemistry; Choline; Collaborations; Collection; Complement; Complement 3d; Complement component C4a; Complex; Computer Analysis; Computer Retrieval of Information on Scientific Projects Database; Computer software; Crystallography; Data; Data Collection; Data Set; Databases; Dose; Electronics; Electrons; Enzymes; Exhibits; Face; Flavins; Fluorescence; Fluorescence Spectroscopy; France; Funding; Grant; Half-Life; Hydrogen Bonding; Hydrogen Peroxide; Institution; International; Kinetics; Leadership; Ligand Binding; Light; Link; Linux; Logic; Maps; Measurement; Microscope; Microspectrophotometry; Morphologic artifacts; Oceans; Operating System; Optics; Oxidases; Oxygen; Photons; Positioning Attribute; Process; Publications; Publishing; Quartz; Raman Spectrum Analysis; Reaction; Reactive Oxygen Species; Reporting; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Rotation; Running; Science; Simulate; Solutions; Source; Spectrum Analysis; Spottings; Structure; Synchrotrons; System; Technology; Time; Travel; United States; United States National Institutes of Health; Visible Radiation; X ray diffraction analysis; X-Ray Diffraction; absorption; adduct; base; beamline; design; detector; digital imaging; electron density; electronic structure; emission spectroscopy; improved; indexing; inhibitor/antagonist; insight; instrument; lens; optical fiber; oxidation; reactive oxygen intermediate; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objectives The objectives stated in the recent renewal proposal are these: We will improve our current single crystal UV/Vis microspectrophotometry facility to provide optical spectra from single crystals in an essentially seamless and routine fashion, at the X-ray beamline, will develop single crystal fluorescence spectroscopy, and will develop single crystal Raman spectroscopy. Results  Our current capability at beamline X26-C consists of a Crystal Logic diffractometer with an ADSC Q4R area detector, a 4DX-ray Systems AB optical system, a Newport 75W Xe research arc lamp, and an Ocean Optics USB 4000 CCD-based spectrophotometer running SpectraSuite software on Windows XP or LINUX operating systems. We designed it to support routine collection of X-ray diffraction and optical absorption spectroscopic data. We have integrated both modes into the beamline control software in order to coordinate the data collection, and to link the results to our database tracking system, the PXDB. Visible light (350 - 850 nm) from a Xe arc lamp travels to the crystal, and then to the spectrophotometer, through quartz optical fibers. The 15x microscope objectives are based upon the Schwarzschild parabolic mirror design, which uses an all-reflecting principle and are, therefore, free from chromatic aberration. The light is focused to a spot size that depends upon objective and the diameter of the optical fiber to which it is connected. For example, the incident photons are focused to 25 ¿m diameter spot through a 50 ¿m optical fiber, whereas photons are collected through a 75 ¿m diameter region focused by a 400 ¿m optical fiber. This arrangement yields full range electronic absorption spectra typically in less than one second. The standard experiments involve the following steps, which are integrated into our beamline control software (CBASS): 1) Mount and center a crystal with the goniometer on the diffractomer. 2) Record 72 digital images of the loop/crystal, one every 5¿ around a full 360¿ rotation. 3) CBASS calls the C3D algorithm, which determines the broadest, flat face of the loop/crystal and directs the goniometer to rotate the crystal so that this face is presented to the incident objective lens for spectroscopy. This establishes the "best" spectroscopy angle for the cryoloop and the crystal. It also avoids the cryoloop, which introduces artifacts if it were to intersect with the spectroscopy photon path. 4) Because macromolecular crystals often yield anisotropic optical spectra, several spectra are collected as a function of rotation angle; for example, best angle ¿ 30¿ in 5¿ or 10¿ increments, and another set centered at the best angle plus 180¿. From this point forward, two mutually exclusive types of data can be collected: time-resolved X-ray dose dependent (Step 5) or diffraction data-dependent (skip to Steps 6 - 7). 5) Optical spectra between 350 - 850 nm are collected from a stationary crystal, in the best orientation, in a time-dependent mode, during which the X-ray shutter is opened at a selected time and for a designated exposure time. This provides a data set from which reaction kinetics can be determined from the analysis of changes in optical spectra at one or more wavelengths. 6) Following best practices, the researcher screens the crystal for X-ray diffraction, indexes the unit cell, and determines the data collection strategy. After CBASS queries the user for input regarding the desired intervals to collect optical spectra, it starts the X-ray diffraction data collection. 7) At the preselected intervals CBASS rotates the crystal back to the best orientation and collects another optical spectrum. This occurs during the readout of the Q4 X-ray detector and while the X-ray shutter is closed. CBASS then continues executing the data collection. An example of a result from this facility is that obtained by Dr. Orville in collaboration with Drs. Gadda and Prabhakar. It was published in a report entitled, "Crystallographic, Spectroscopic, and Computational Analysis of a Flavin C4a-Oxygen Adduct in Choline Oxidase," that appeared recently in Biochemistry. Choline oxidase (CHO) from Arthrobacter globiformis is a FAD-dependent enzyme that catalyzes the two-step, four-electron oxidation of choline to glycine betaine, with betaine aldehyde as a two-electron oxidized intermediate. In the two oxidative half-reactions, two molecules of O2 are converted into two H2O2 molecules. We performed several spectroscopic measurements on a number of choline oxidase crystals including before and after X-ray exposure. Importantly, the difference spectra (after  before) clearly shows a spectrum with ¿max at 400 nm that is nearly identical to spectra obtained from flavin C4a-OOH or C4a-OH enzyme reaction intermediates. Typically these reactive oxygen intermediates exhibit half-lives of only several ms in solution, but remarkably, it is stable in the crystal at 100K. The time-dependent results show that this species is generated very quickly upon X-ray exposure (Figure 2). The difference feature at 400 nm increases in an exponential process, and with a t1/2 of approximately 40 seconds. This rate is approximately commensurate with the decrease of the 460 and 485 nm features attributed to oxidized FAD with an apparent t1/2 of approximately 100 seconds. The resulting electron density maps (unbiased simulated annealing at 1.8 ¿ resolution) and the interpretation of the atomic structure is consistent with two possible reactive oxygen species, namely a covalent flavin C4a-OOH and/or C4a-OH adducts. Either structure correlates well with the spectroscopic observations. Moreover, each type of data reinforces the conclusions; whereas either type of data alone would yield somewhat ambiguous results. Plans  Non-resonance and Resonance Raman Spectroscopy - Structure and function are undeniably linked. Since the Raman effect involves interactions between atomic positions, electron distribution, and intermolecular forces, it is ideally suited to provide insights into function that a crystal structure alone cannot. Indeed, the information available from Raman spectroscopy can be very detailed, exceeding the level of resolution found in all but the highest resolution X-ray crystal structures. It can also reveal changes in the distribution of electrons in a bound ligand as well as details about hydrogen bonding strengths between active site residues and substrate or inhibitor molecules. We have begun procurement of a micro-beam Raman instrument, which we hope to commission within the next half year. Off-line single-crystal spectroscopy  We plan to construct a station for spectroscopy that duplicates the stations inside the x-ray hutch at beamline X26-C. Single Crystal, Fluorescence-Emission Spectroscopy  Fluorescence spectroscopy complements the electronic absorption and Raman spectroscopy capabilities. Dr. Orville has just submitted a Challenge Grant to obtain apparatus to accomplish this at beamline X26-C, hoping to obtain apparatus for fluorescence spectroscopy at both the on-line and off-line single-crystal spectroscopy stations. Significance  The technology we are developing will be unique in the United States, and will be matched only by the equivalent system at the ESRF in France. The scientific problems that we and our users will address are central to the progress of macromolecular sciences in the United States. The national resource we envision will support unprecedented, highly correlated studies. The results will provide much needed data on the complex relationships among macromolecular atomic structure, electronic structure and chemistry. These data will be used by the large number of national and international researchers in the field. Our plans will place the United States in a leadership position in this area. Publications  H¿roux, A., Bozinovski, D.M., Valley, M.P., Fitzpatrick, P.F. and Orville, A.M. Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction, Biochemistry 48, 3407-3416 (2009). Orville, A.M., Lountos, G.T., Finnegan, S., Gadda, G.,Prabhakar R. Crystallographic, Spectroscopic, and Computational Analysis of a Flavin-C4a-Oxygen Adduct in Choline Oxidase, Biochemistry 48, 720-728 (2009).

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 目的是在最近的续签建议中所述的物体是：我们将改善当前的单晶紫外线/Vis微光谱测量学设施，以在本质上无缝且常规的方式中提供来自单晶的光谱，在X射线束线上，将开发单晶荧光光谱，并会发展单晶晶体频谱。 结果我们在Beamine X26-C上的当前功能由具有ADSC Q4R区域检测器，4DX射线系统AB光学系统，Newport 75W XE Research Arc Lamp和Ocean Optics USB 4000 CCD的基于CCD的基于CCD的Emplepophotometoper Spectrasuite软件在Windows XP或Linux操作系统上的Spectrasuite软件组成的晶体逻辑衍射仪。我们设计它是为了支持X射线衍射仪和光学滥用光谱数据的常规收集。我们已经将两种模式集成到光束线控制软件中，以协调数据收集，并将结果链接到我们的数据库跟踪系统PXDB。 可见光（350-850 nm）从Xe Arc灯向晶体传播，然后通过石英光纤到分光光度计。 15倍显微镜对象基于Schwarzschild抛物面镜设计，该镜像使用了全反射原理，因此没有色差。光聚焦到斑点大小，该点大小取决于物镜和连接的光纤的直径。例如，事件照片通过50`m光纤聚焦到直径25•直径斑点，而照片是通过75»M直径的区域收集的，该区域由400座光纤聚焦。这种布置通常在不到一秒钟的时间内产生全范围电子滥用光谱。 标准实验涉及以下步骤，这些步骤已集成到我们的光束线控制软件（CBASS）中： 1）将晶体安装在衍射剂上的晶体中。 2）记录72个循环/晶体的数字图像，每5到360“旋转，每5？。 3）CBAS称为C3D算法，该算法确定了环/晶体的最宽，平坦的表面，并指示Goniiomenter旋转晶体，以便将此面呈现给入射物镜以进行光谱镜头。这为冻结和晶体建立了“最佳”光谱角。它还避免了冻结，如果它与光谱光子路径相交，它会引入伪影。 4）由于大分子晶体通常产生各向异性光谱，因此收集了几个光谱作为旋转角的函数。例如，最佳角度€30€以5或10的增量，另一组以最佳角度加180。 从这一点开始，可以收集两种相互排斥的数据类型：时间分辨X射线剂量依赖性（步骤5）或衍射数据依赖性（跳过步骤6-7）。 5）在最佳方向以时间依赖的模式以固定晶体收集350-850 nm之间的光谱，在此期间，在选定的时间和指定的暴露时间开放X射线快门。这提供了一个数据集，可以从一个或多个波长的光谱变化的分析中确定反应动力学。 6）遵循最佳实践，研究人员筛选了X射线衍射的晶体，为单位单元格索引并确定数据收集策略。 CBAS查询后，用户以获取有关收集光谱的所需间隔的输入，它启动了X射线衍射数据收集。 7）在提出的间隔中，CBAS将晶体旋转回到最佳方向，并收集另一个光谱。这发生在Q4 X射线检测器的读数期间，并且在关闭X射线快门时。然后，CBAS继续执行数据集合。 该设施的结果的一个例子是Orville博士与Drs合作获得了。 Gadda和Prabhakar。它发表在一份题为“胆碱氧化酶中黄素C4a-氧气添加的晶体学，光谱和计算分析”的报告中，最近出现在生物化学中。来自节肢动物球菌素的胆碱氧化物（CHO）是一种依赖FAD的酶，可催化胆碱的两步，四电子氧化物到甘氨酸甲虫，其甲醛作为两种电力的氧化物中间体。在两个氧化半反应中，将两个O2分子转化为两个H2O2分子。我们对包括X射线前后的许多胆碱氧化酶晶体进行了多次光谱测量。重要的是，差异光谱（以后）清楚地显示了在400 nm处具有最大值的光谱，该光谱与从黄素C4A-OOH或C4A-A-OH酶反应中间体获得的光谱几乎相同。 通常，这些活性氧中间体在溶液中仅显示几个MS的半衰期，但值得注意的是，它在100K的晶体中稳定。时间依赖性结果表明，X射线暴露会很快生成该物种（图2）。 400 nm处的差异特征在指数过程中增加，而T1/2的差异约为40秒。该速率与氧化物FAD的460和485 nm特征的降低大致相称，其明显的T1/2约为100秒。所得的电子密度图（以1.8的分辨率取消模拟退火）和原子结构的解释与两个可能的活性氧物种一致，即共价黄素C4A-OOH和/或C4A-A-OH加合物。任何一种结构都与光谱观测良好相关。此外，每种类型的数据都加强了结论。而单独的两种数据都会产生一些模棱两可的结果。 计划非谐振和共振拉曼光谱 - 结构和功能无可否认地联系在一起。由于拉曼效应涉及原子位置，电子分布和分子间力之间的相互作用，因此理想情况下，它可以提供对仅晶体结构不能仅能的功能的见解。实际上，拉曼光谱可用的信息可以非常详细，超过了除最高分辨率X射线晶体结构以外的所有分辨率水平。它还可以揭示结合配体中电子分布的变化，以及有关活性位点保留和底物或抑制剂分子之间氢键强度的细节。我们已经开始采购微型梁拉曼仪器，我们希望在未来半年内进行委托。 离线单晶光谱法我们计划构建一个用于光谱的站点，该电视检查复制了Beamine X26-C处的X射线厨房内部的站点。 单晶，荧光发射光谱荧光光谱完成电子滥用和拉曼光谱能力。 Orville博士刚刚提出了一项挑战资助，以获取在Beam Line X26-C上完成此设备，希望在线和离线单晶光谱站获得荧光光谱的设备。 意义在美国，我们正在开发的技术将是独一无二的，并且只能与法国ESRF的同等系统匹配。我们和我们的用户将解决的科学问题是美国大分子科学进展的核心。我们设想的国家资源将支持前所未有的高度相关研究。结果将提供有关大分子原子结构，电子结构和化学之间复杂关系的急需数据。这些数据将由该领域的大量国家和国际研究人员使用。我们的计划将使美国在这一领域处于领导地位。 出版物，A.，Bozinovski，D.M。和A.M. Orville硝基烷氧化酶反应中中间体的晶体结构，生物化学48，3407-3416（2009）。 Orville，A.M.，Lountos，G.T.，Finnegan，S.，Gadda，G.，Prabhakar R.晶体学，光谱和计算分析，以及在胆碱氧化酶中添加黄素-C4A-氧气的计算分析，生物化学48，720-728（2009）。