NMR and EPR Studies of Ribozyme Catalytic Mechanisms

核酶催化机制的 NMR 和 EPR 研究

• 批准号: 7680052
• 负责人: CHARLES G HOOGSTRATEN
• 金额: $ 24.76万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7680052
• 关键词: Address; Affect; Binding; Binding Sites; Biochemical Energetics; Biological Assay; Catalysis; Catalytic Domain; Catalytic RNA; Chemistry; Complement; Complex; Coupled; Coupling; DNA Sequence Rearrangement; Data; Development; Disease; Docking; Goals; Heteronuclear NMR; Ions; Kinetics; Label; Left; Ligation; Link; Magnetic Resonance; Maps; Messenger RNA; Metal Ion Binding; Metals; Methods; Molecular; Molecular Conformation; Molecular Structure; Mutation; Pathway interactions; Physiologic pulse; Play; Positioning Attribute; Process; Property; RNA; RNA Splicing; Reaction; Relaxation; Research Personnel; Role; Scheme; Site; Spliceosomes; Structure; Substrate Domain; System; Techniques; Testing; Therapeutic; Thermodynamics; U6 small nuclear RNA; Ursidae Family; Variant; Work; catalyst; cofactor; comparative; design; divalent metal; fascinate; hairpin ribozyme; interest; leadzyme; mRNA Precursor; new technology; novel; programs; research study; stem; tool

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）：本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。目前正在开发核酶衍生物，作为许多毁灭性疾病的治疗药物。对核酶机制的基本理解在衍生物的设计中是至关重要的，在药理学性质上有必要的改进，同时保持所需的催化活性。我们的观点是，核糖酶的静态结构不足以理解其作用机制，结构工作必须与分子动力学，其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相结合。我们将应用当代磁共振技术，包括异核磁共振自旋弛豫和先进的EPR实验，提供直接与这些问题有关的独特信息，从而将结构研究与生物化学能量学联系起来。将强调核磁共振自旋弛豫研究的核酶构象的动力学性质。在表征良好的发夹核酶中，我们将使用核磁共振自旋弛豫来研究分子在活性和非活性序列变体中的动态特性，以及作为三级结构形成（对接）的函数。我们将继续使用综合的EPR和NMR方法来描述多价金属离子对核酶的结构和动力学的影响，多价金属离子对催化至关重要，但不直接参与该系统的化学反应。我们也对U6 snRNA感兴趣，这是真核mRNA剪接装置的催化关键成分。在这个系统中，我们将研究生物化学鉴定的金属离子的结构，它似乎参与了反应化学，无论是在形成其直接结合位点的内部茎环中，还是在更大的复合物中，包括前mrna序列。这两种系统的研究将为RNA的构象动力学、金属离子辅因子连接和催化功能之间的相互关系提供互补和协同的视角。简而言之，我们提出了一个技术进步和新应用的综合生物物理计划，这将显著推进核酶领域对这些迷人催化剂的分子水平理解。