RAMAN STUDIES OF ENZYME COMPLEXES IN SOLUTION & CRYSTALS

描述：(逐字摘自申请者的摘要)这项工作的总体目标 研究的目的是提供新的、详细的关于 用拉曼光谱方法研究酶的作用。技术创新 为发展和应用提供了前所未有的机遇 拉曼法。我们将在这里利用它们来研究两类主要的 酶，一种具有潜在环境重要性的脱卤酶，因为它 降解氯代烃；以及黄素蛋白，其中一些是 药物设计的前景看好的目标。在这两个领域，拉曼差异光谱 将被用来获得与酶结合的配体的拉曼光谱 稳定的1：1络合物，或反应中的中间体。对拉曼效应的解读 在量子力学计算的帮助下，数据将提供详细的 关于构象、电子分布和分子的信息 配基的相互作用。初步研究显示，有几个 酶-配体复合体以多种构象存在于溶液中 州政府。这些构象的热力学性质将由下式定义 研究种群随温度的变化。技术创新 还允许建造灵敏的拉曼显微镜，以获得 使用显微镜光学的样品的拉曼光谱。初步实验 拉曼显微镜已经表明，高质量的拉曼光谱可以 从单一蛋白质晶体中获得。黄素蛋白的拉曼光谱 从悬浮液中的晶体中获得对羟基苯甲酸羟基酶。 它们的生长室。这些晶体的尺寸只有30微米。两者都有 在晶体拉曼光谱中可以看到蛋白质峰和黄素峰。此外， 可以辨别埋藏的或暴露在溶剂中的黄素基的标记带 在两个不同的酶-配体复合体中，每个已知有利于掩埋或 暴露的构象。因此，对于脱卤酶和黄素蛋白，我们都是 建议使用拉曼数据来比较配体化学的细节 溶液中的络合物与单晶。我们也会认真做一次 构象群体的比较，其中不止一个构象 状态已找到。

DESCRIPTION: (Verbatim from the Applicant's Abstract)The broad goal of this research is to provide new and detailed insights into the fundamentals of enzyme action by the approach of Raman spectroscopy. Technical innovations offer unprecedented opportunities for the development and application of the Raman method. These will be exploited here to investigate two main classes of enzymes, a dehalogenase that is of potential environmental import, since it degrades a chlorinated hydrocarbon; and flavoproteins, some of which are promising targets for drug design. In both areas, Raman difference spectroscopy will be used to obtain the Raman spectrum of a ligand bound to the enzyme in stable 1:1 complexes, or in reaction intermediates. Interpretation of the Raman data, aided by quantum mechanical calculations, will provide detailed information on the conformation, electron distribution and molecular interactions for the ligand. Preliminary studies reveal that several enzyme-ligand complexes exist in solution in more than one conformational state. The thermodynamic properties of these conformations will be defined by studying the variation of populations with temperature. Technical innovations also permit the construction of sensitive Raman microscopes that acquire the Raman spectrum of a sample using microscope optics. Preliminary experiments with the Raman microscope have shown that high-quality Raman spectra can be obtained from single protein crystals. Raman spectra of the flavoprotein parahydroxybenzoate hydroxylase were obtained from crystals in hanging drops in their growth chambers. The crystals were only 30 microns in dimensions. Both protein and flavin peaks could be seen in the crystal Raman spectra. Moreover, marker bands for the buried or solvent-exposed flavin group could be discerned in two different enzyme-ligand complexes, each known to favor the buried or exposed conformation. Thus, for both dehalogenase and flavoproteins we are proposing to use Raman data to compare the details of ligand chemistry for complexes in solution versus single crystals. We will also make a careful comparison of conformational populations, where more than one conformational state is found.