STRUCTURE--FUNCTION ANALYSIS OF RIBONUCLEASE A

核糖核酸酶A的结构--功能分析

• 批准号: 2392131
• 负责人: Ronald T Raines
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392131
• 关键词: X ray crystallography; active sites; chemical cleavage; enzyme activity; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate complex; gene mutation; hybrid enzyme; ionic bond; oligonucleotides; pancreatic ribonuclease; protein engineering; protein structure function; stable isotope; synthetic enzyme; thermodynamics

The overall objective of this research project is to reveal the chemical basis for the recognition and cleavage of nucleic acids by proteins, and to create novel proteins with properties useful to biology and medicine. The object of this proposal is ribonuclease A (RNase A), a small protein that catalyzes the cleavage of the P-O5 bond of RNA specifically after pyrimidine residues. Classic work on RNase A has contributed much information on enzymatic catalysis; on the chemistry, folding, and stability of proteins; and on molecular evolution. This wealth of information, combined with the ability to produce mutant enzymes and synthesize substrate analogs, make this system ideal for revealing atomic determinants of both enzymatic catalysis and protein - nucleic acid interactions. The specific aims of this research project are to elucidate (1) the structure of the transition state in RNA cleavage, (2) the mechanism and energetics of enzymatic processivity, (3) the importance of electrostatics in protein - nucleic acid interactions, and (4) how active-site residues cooperate during catalysis. In addition, hybrid proteins will be used to determine whether the divergence of surface loops has allowed RNase.A to acquire special biological activities. Also, semisynthetic ribonucleases will be created that can suffer specific site. Finally, a most important aim is to determine three-dimensional structures of meaningful protein - nucleic acid complexes. The proposed experiments use techniques from organic chemistry, biochemistry, biophysics, and molecular biology to test hypotheses on the relationship between protein structure and protein function (or dysfunction). The results of these experiments will provide new insights into this relationship as well as novel proteins for biomedical analyses and therapies.

该研究项目的总体目标是揭示化学 蛋白质识别和切割核酸的基础，以及 创造具有对生物学和医学有用的特性的新型蛋白质。 该提案的对象是核糖核酸酶 A (RNase A)，一种小蛋白质 催化 RNA P-O5 键的裂解 嘧啶残基。 RNase A 的经典工作贡献良多 有关酶催化的信息；关于化学、折叠和 蛋白质的稳定性；以及分子进化。 这份财富 信息，结合产生突变酶的能力和 合成底物类似物，使该系统成为揭示原子的理想选择 酶催化和蛋白质-核酸的决定因素 互动。 本研究项目的具体目标是阐明（1） RNA切割中过渡态的结构，(2)机制和 酶促过程的能量学，（3）静电学的重要性 蛋白质-核酸相互作用，以及（4）活性位点残基如何 催化过程中相互配合。 此外，混合蛋白将用于 确定表面环的分歧是否允许 RNase.A 获得特殊的生物活性。 此外，半合成核糖核酸酶 将创建可以遭受特定站点影响的站点。 最后还有一个最重要的 目的是确定有意义的蛋白质的三维结构 - 核酸复合物。 拟议的实验使用有机化学技术， 生物化学、生物物理学和分子生物学来检验关于 蛋白质结构和蛋白质功能之间的关系（或 功能障碍）。 这些实验的结果将提供新的见解 研究这种关系以及用于生物医学分析的新型蛋白质 和疗法。