LASER RAMAN SCATTERING FROM BIOLOGICAL MACROMOLECULES

生物大分子的激光拉曼散射

• 批准号: 3268788
• 负责人: WARNER L PETICOLAS
• 金额: $ 9.69万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3268788
• 关键词: DNA; Raman spectrometry; carboxypeptidase; chemical kinetics; conformation; enzyme mechanism; enzyme structure; enzyme substrate; enzyme substrate complex; glyceraldehyde 3 phosphate dehydrogenase; laser spectrometry; nicotinamide adenine dinucleotide; nucleic acid structure; oligonucleotides; protein folding; thiols; ultraviolet spectrometry; zinc

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.

该建议的目的是获得有用的结构 生物大分子及其与 在X射线衍射方法无法使用的条件下进行配体 贯彻我们将研究其构象和动力学变化 蛋白质和核酸在溶液和晶体中 states.我们认为，这些信息将有助于 理解构象、动力学和 这些大分子的生物学功能。这反过来又应 从而更好地理解生物学的分子基础 功能和帮助设计更合理的医疗保健。我们 主要的实验方法将是经典和紫外 共振拉曼（UVRR）光谱。我们建议获得UVRR 在酶促反应期间形成的硫代中间体的光谱 涉及含硫醇的酶的催化。此类中间体 吸收240 nm的光并使用 239 nm激发线从频率修改的Nd-YAG激光器。我们 我建议从甘油醛-3-磷酸酶开始 脱氢酶，以回答一些基本问题， 在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的相互作用， 活性部位我们建议将这项新技术应用于 在含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 浸没在一个或多个激光器中的酶单晶的拉曼光谱。 含有底物的母液浴。酶-底物 中间体在晶体中积累到稳定状态， 底物扩散进入而产物扩散出来。我们展示了 这种技术的可行性与单晶， 羧肽酶在每一个实验中，我们希望找到证据， 形成不寻常结构的中间体，这将 更完整地解释酶的催化活性 方式 我们进一步提出了一种新的方法， 不寻常的A或Z型中微量DNA的测定 在B型DNA占优势的情况下， 结合光化学交联实验和UVRR 谱