NMR and EPR Studies of Ribozyme Catalytic Mechanisms

核酶催化机制的 NMR 和 EPR 研究

• 批准号: 6970416
• 负责人: CHARLES G HOOGSTRATEN
• 金额: $ 25.3万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6970416
• 关键词: cofactor; conformation; electron spin resonance spectroscopy; enzyme mechanism; manganese; molecular dynamics; molecular site; nuclear magnetic resonance spectroscopy; nucleic acid structure; ribozymes; structural biology

DESCRIPTION (provided by applicant): The overarching goal of this project is to develop a detailed biophysical understanding of the mechanism of action of catalytic RNA molecules, or ribozymes. Ribozyme derivatives are currently under development as therapeutics for a number of devastating diseases. A fundamental understanding of ribozyme mechanism is critical in the design of derivatives, with necessary improvements in pharmacological properties that simultaneously maintain the desired catalytic activity. Our thesis, is that, the static structure of a ribozyme is insufficient to understand its mechanism of action, and that structural work must be coupled with studies of the molecule's dynamics, its interactions with multivalent metal ions, and the effects of those metal ions on molecular structure and dynamics. We will apply contemporary magnetic resonance techniques, including heteronuclear NMR spin relaxation and advanced EPR experiments, to provide unique information that bears directly on these issues and, thus, to link structural studies with biochemical energetics. The dynamic properties of ribozyme conformations, as studied by NMR spin relaxation, will be emphasized. In the well characterized hairpin ribozyme, we will investigate the dynamic properties of the molecule using NMR spin relaxation in active vs. inactive sequence variants, and as a function of tertiary structure formation (docking). We will proceed to use an integrated EPR and NMR approach, to delineate the effects of multivalent metal ions, which are crucial for catalysis, but do not participate directly in chemistry in this system, on the ribozyme's structure and dynamics. We are also interested in the U6 snRNA, a catalytically critical component of the eukaryotic mRNA splicing apparatus. In this system, we will investigate the structure of a biochemically-identified metal ion, which appears to participate in reaction chemistry, both in the internal stemloop that forms its immediate binding site, and in larger complexes, including pre-mRNA sequences. Studies of these two systems will provide complementary and synergistic perspectives on the interrelationships among conformational dynamics, metal ion cofactor ligation, and catalytic function in RNA. In short, we propose an integrated biophysical program of technological progress and novel applications, that will significantly advance the ribozyme field toward a molecular-level understanding of these fascinating catalysts.

描述（由申请人提供）：该项目的总体目标是对催化RNA分子或核酶的作用机制进行详细的生物物理理解。核酶衍生物目前正在开发中，作为许多毁灭性疾病的治疗剂。对核酶机制的基本理解在衍生物的设计中是至关重要的，同时保持所需的催化活性的药理学性质的必要改进。我们的论点是，静态结构的核酶是不足以理解其作用机制，结构工作必须结合分子的动力学，它与多价金属离子的相互作用，以及这些金属离子对分子结构和动力学的影响的研究。我们将应用当代的磁共振技术，包括异相NMR自旋弛豫和先进的EPR实验，提供直接涉及这些问题的独特信息，从而将结构研究与生物化学能量学联系起来。核酶构象的动力学性质，如核磁共振自旋弛豫研究，将被强调。在充分表征的发夹核酶中，我们将使用NMR自旋弛豫在活性与非活性序列变体中研究分子的动态特性，并作为三级结构形成（对接）的函数。我们将继续使用一个集成的EPR和NMR方法，描绘多价金属离子的影响，这是催化的关键，但不直接参与化学在这个系统中，对核酶的结构和动力学。我们也对U6 snRNA感兴趣，它是真核生物mRNA剪接装置的催化关键组分。在这个系统中，我们将调查的结构的生化鉴定的金属离子，这似乎参与化学反应，无论是在内部茎环，形成其直接结合位点，并在较大的复合物，包括前mRNA序列。这两个系统的研究将提供互补和协同作用的观点之间的相互关系的构象动力学，金属离子辅因子连接，和催化功能的RNA。简而言之，我们提出了一个综合的生物物理程序的技术进步和新的应用，这将显着推进核酶领域对这些迷人的催化剂的分子水平的理解。