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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 该合作项目的目标是从结构上表征人工 RNA 连接酶的锌配位，该酶在自然界中没有对应物。这种酶是仅依靠非常大的随机蛋白质突变体文库的功能多样性产生的从头酶的第一个例子。该酶是通过体外选择和进化过程从 4 万亿种不同随机蛋白质的合成文库中分离出来的。该文库基于一个小的稳定蛋白结构域，包含两个锌指，具有完全随机的 9 和 12 个氨基酸环。该酶将 5′-三磷酸激活的 RNA 与第二个 RNA 的 3′-羟基末端进行模板依赖性连接。自然界中没有已知的酶可以催化该反应。然而，这一反应与 RNA 聚合过程中一个核苷酸的链延长有关。聚合酶通常需要 Mg2+ 来实现双金属离子反应机制。相比之下，人工 RNA 连接酶受 Mg2+ 抑制，而需要 Zn2+ 才能发挥其活性。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。人工酶的核磁共振波谱揭示了两个高度结构化的区域，嵌入在更动态的部分中。我们通过监测 HSQC 实验中的结构变化，观察到在将锌添加到无锌脱辅酶过程中两个不同的锌结合事件。我们假设这两个高度结构化的区域各自与 Zn2+ 配位，一个位点可能是结构性的，另一个位点是催化性的。阐明这种人工 RNA 连接酶的结构对于研究实验室从头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至关重要。这项工作将是这种人工酶的首次 XAS 研究。