LASER RAMAN SCATTERING FROM BIOLOGICAL MACROMOLECULES

这项建议的目标是获得有用的结构 生物大分子及其络合物的信息 X-射线衍射法无法测定的条件下的配体 被执行。我们将研究它的构象和动态变化 蛋白质和核酸在溶液和晶体中的含量 各州。我们相信，这些信息将对 理解构象、动力学和构象之间的关系 这些大分子的生物学功能。这反过来又应该 使我们更好地理解生物学的分子基础 功能和辅助设计更加合理的医疗保健。我们的 主要的实验方法将是经典和紫外线 共振拉曼(UVRR)光谱。我们建议获得UVRR 酶促过程中形成的硫代中间体的光谱 含有硫醇的酶的催化作用。这样的中间体 在240 nm处吸收光线，并使用 239 nm的调频激光激发线。我们 建议从酶开始，甘油醛-3-磷酸 脱氢酶来回答一些基本的问题 催化过程中形成的硫代中间体的结构 以及它们与辅因子NAD+和NADH在 活动站点。我们建议将这项新技术应用于 在含有金属的酶催化过程中形成的中间体。 我们开发的第二种新的拉曼技术测量了 一种酶单晶在溶液中的拉曼光谱 含有底物的母液浴液。一种酶底物 中间体在晶体中形成稳定状态，如 底物扩散进来，产物扩散出去。我们已经证明了 这项技术在单晶体上的可行性 羧基肽酶。在每一个实验中，我们都希望找到证据 具有特殊结构的中间体的形成 更完整地解释这种酶的催化活性 举止。 我们还建议开发一种新的方法来定量 一种异常A型或Z型微量DNA的测定 在B型DNA占优势的情况下通过 光化学交联实验与UVRR相结合 光谱学。

The objective of this proposal is to obtain useful structural information on biological macromolecules and their complexes with ligands under conditions where X-ray diffraction methods cannot be carried out. We will study the conformation and dynamical changes of proteins and nucleic acids in both solution and crystalline states. We believe that such information would be of great help in understanding the relation between the conformation, dynamics and biological function of these macro-molecules. This in turn should lead to a better understanding of the molecular basis of biological function and aid in the design of more rational health care. Our primary experimental method will be classical and ultraviolet resonance RAMAN (UVRR) spectroscopy. We propose to obtain the UVRR spectra of thiointermediates that are formed during enzymatic catalysis involving thiol containing enzymes. Such intermediates absorb light at 240 nm and produce excellent UVRR spectra using the 239 nm exciting line from a frequency modified Nd-YAG laser. We propose to start with the enzyme, glyceraldehyde-3-phosphate dehydrogenase in order to answer some fundamental questions about the structure of the thio-intermediates formed during catalysis and their interaction with the cofactors NAD+ and NADH at the active site. We propose to apply this new technique to intermediates formed during catalysis by metal containing enzymes. A second new Raman technique that we have developed measures the Raman spectrum of an enzyme single crystal that is immersed in a bath of mother liquor containing the substrate. An enzyme-substrate intermediate builds up to a steady state in the crystal as substrate diffuses in and product diffuses out. We have demonstrate the feasibility of this technique with single crystals of carboxypeptidase. In each experiment we hope to find evidence for the formation of intermediates of unusual structures which will explain the catalytic activity of the enzyme in a more complete manner. We further propose to develop a new method for the quantitative determination of trace amounts of DNA in an unusual A or Z type conformation in the presence of a preponderance of B form DNA by combining photochemical crosslinking experiments with UVRR spectroscopy.