RAMAN STUDIES OF ENZYME COMPLEXES IN SOLUTION & CRYSTALS

描述：（逐字来自申请人的摘要）本发明的广泛目标是： 研究是为了提供新的和详细的见解的基本面， 通过拉曼光谱法研究酶的作用。技术创新 为发展和应用提供了前所未有的机遇， 拉曼法。这些将在这里被利用来研究两个主要类别的 酶，脱卤酶是潜在的环境进口，因为它 降解氯代烃;和黄素蛋白，其中一些是 有希望的药物设计目标。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复合物或反应中间体。拉曼光谱解析 在量子力学计算的帮助下，数据将提供详细的 构象、电子分布和分子结构信息 配体的相互作用。初步研究显示， 酶-配体复合物以一种以上的构象存在于溶液中 状态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 还允许构造灵敏的拉曼显微镜， 使用显微镜光学器件的样品的拉曼光谱。初步实验 与拉曼显微镜已经表明，高质量的拉曼光谱可以 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中的晶体中获得的， 他们的生长室这些晶体的尺寸只有30微米。两 在晶体拉曼光谱中可以看到蛋白质和黄素峰。此外，委员会认为， 可以辨别出埋藏的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，每一种都有利于埋藏的或 暴露的构造。因此，对于脱卤酶和黄素蛋白， 建议使用拉曼数据来比较配体化学的细节， 溶液中的复合物与单晶体。我们也会仔细 构象群体的比较，其中一个以上的构象 国找到了。

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.