STRUCTURAL STUDIES OF ARTIFICIAL RNA LIGASE

人工RNA连接酶的结构研究

• 批准号: 8362401
• 负责人: Burckhard Seelig
• 金额: $ 0.03万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362401
• 关键词: Amino Acids; Apoenzymes; Binding; Enzymes; Event; Evolution; Funding; Grant; Hydroxyl Radical; In Vitro; Ions; Laboratories; Libraries; Ligase; Ligation; Metals; Monitor; NMR Spectroscopy; National Center for Research Resources; Nature; Nucleotides; Polymerase; Principal Investigator; Process; Proteins; RNA; RNA Ligase (ATP); Radiation; Randomized; Reaction; Research; Research Infrastructure; Resources; Sequence Analysis; Site; Source; Structure; Tertiary Protein Structure; United States National Institutes of Health; Work; Zinc; Zinc Fingers; base; cost; mutant; polymerization; research study; scaffold; structural biology; tripolyphosphate

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The objective of this collaborative project is to structurally characterize the zinc coordination of an artificial RNA ligase enzyme that has no counterpart in nature. This enzyme is the first example of a de novo enzyme generated by solely relying on the functional diversity of very large libraries of random protein mutants. The enzyme was isolated from a synthetic library of 4 trillion different randomized proteins by an in vitro selection and evolution process. The library was based on a small stable protein domain containing two zinc fingers with completely randomized loops of 9 and 12 amino acids. The enzyme performs a template-dependent ligation of a 5?-triphosphate-activated RNA to the 3?-hydroxyl end of a second RNA. There are no enzymes known in nature that catalyze this reaction. Yet, this reaction is related to chain elongation by one nucleotide during RNA polymerization. Polymerase enzymes typically require Mg2+ for a two-metal-ion reaction mechanism. In contrast, the artificial RNA ligase is inhibited by Mg2+ and instead requires Zn2+ for its activity. Sequence analysis of the artificial ligase suggests that the original scaffold was lost entirely during the selection and evolution process. NMR spectroscopy of the artificial enzyme reveals two highly structured regions, embedded in more dynamic sections. We observe two distinct zinc binding events during the addition of zinc to the zinc-free apoenzyme by monitoring structural changes in an HSQC experiment. We hypothesize that the two highly structured regions are each coordinated to a Zn2+, one site potentially being structural and the other catalytic. Elucidating the structure of this artificial RNA ligase will be crucial to investigate if a de novo laboratory-generated enzyme follows the same principles of structure and mechanism as proteins that were formed by natural evolution. This work will be the first XAS study of this artificial enzyme.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 这个协作项目的目的是在结构上表征人工RNA连接酶的锌协调，该酶在本质上没有对应物。该酶是仅依靠非常大的随机蛋白质突变体的功能多样性而产生的从头酶的第一个例子。通过体外选择和进化过程，从4万亿不同随机蛋白质的合成库中分离酶。该文库基于一个小的稳定蛋白质结构域，该结构域包含两个锌指，其完全随机的环为9和12氨基酸。该酶对第二个RNA的3？ - 羟基端进行5？三磷酸激活的RNA进行模板依赖性连接。本质上没有酶来催化这种反应。然而，该反应与RNA聚合过程中一个核苷酸的链伸长有关。两米离子反应机制通常需要Mg2+聚合酶。相比之下，人工RNA连接酶被MG2+抑制，而需要Zn2+的活性。人工连接酶的序列分析表明，原始支架在选择和进化过程中完全丢失。人工酶的NMR光谱揭示了两个高度结构化区域，这些区域嵌入了更动态的切片中。我们通过监测HSQC实验中的结构变化，观察到将锌添加到无锌载酶的过程中，观察到两个不同的锌结合事件。我们假设这两个高度结构化区域分别与Zn2+协调，一个位点可能是结构性的，另一种是催化性的。阐明这种人工RNA连接酶的结构对于研究从头实验室生成的酶是否遵循与自然进化形成的蛋白质相同的结构和机制原理。这项工作将是该人工酶的首次XAS研究。