RAMAN STUDIES OF ENZYME COMPLEXES IN SOLUTION & CRYSTALS

溶液中酶复合物的拉曼研究

• 批准号: 6386316
• 负责人: PAUL R CAREY
• 金额: $ 25.7万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386316
• 关键词: Escherichia coli; Raman spectrometry; Streptococcus lactis; active sites; coenzyme A; conformation; crystallization; enzyme activity; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate analog; flavoproteins; halogenation; hydrolase; oxidoreductase; oxidoreductase inhibitor; quantum chemistry; solutions; thermodynamics

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.

描述：（逐字摘自申请人摘要）本项目的总体目标 研究的目的是为基础知识提供新的、详细的见解 通过拉曼光谱法测定酶的作用。技术创新 为该技术的发展和应用提供了前所未有的机遇 拉曼法。此处将利用这些来研究两类主要的 酶，一种具有潜在环境重要性的脱卤酶，因为它 降解氯化烃；和黄素蛋白，其中一些是 药物设计的有希望的目标。在这两个领域，拉曼差分光谱 将用于获得与酶结合的配体的拉曼光谱 稳定的 1:1 配合物，或反应中间体。拉曼的解释 数据在量子力学计算的辅助下，将提供详细的 有关构象、电子分布和分子的信息 配体的相互作用。初步研究表明，一些 酶-配体复合物以不止一种构象存在于溶液中 状态。这些构象的热力学性质将定义为 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来获取 使用显微镜光学器件获得样品的拉曼光谱。初步实验 使用拉曼显微镜表明可以得到高质量的拉曼光谱 从单个蛋白质晶体中获得。黄素蛋白的拉曼光谱 对羟基苯甲酸羟化酶是从悬滴晶体中获得的 他们的生长室。这些晶体的尺寸只有 30 微米。两个都 在晶体拉曼光谱中可以看到蛋白质和黄素峰。而且， 可以辨别埋藏或暴露于溶剂的黄素基团的标记带 在两种不同的酶-配体复合物中，每种复合物都已知有利于埋藏或 暴露的构象。因此，对于脱卤酶和黄素蛋白，我们 提议使用拉曼数据来比较配体化学的细节 溶液中的配合物与单晶。我们也会认真做 构象群体的比较，其中不止一种构象 状态被发现。