STRUCTURAL STUDIES OF ARTIFICIAL RNA LIGASE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 这个合作项目的目标是从结构上表征人工RNA连接酶的锌配位，这种酶在自然界中没有对应的配位。这种酶是第一个仅依靠非常大的随机蛋白质突变体文库的功能多样性而产生的从头酶的例子。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的蛋白质结构域，包含两个锌指，具有9个和12个氨基酸的完全随机环。该酶依赖于模板将5？三磷酸激活的RNA连接到第二个RNA的3？羟基端。自然界中没有已知的酶能催化这一反应。然而，这个反应与RNA聚合过程中一个核苷酸的链延长有关。聚合酶通常需要镁离子来实现双金属离子反应机制。相反，人工RNA连接酶被镁离子抑制，而需要锌离子才能发挥活性。人工连接酶的序列分析表明，在选择和进化过程中，原始支架完全丢失。人造酶的核磁共振波谱显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，观察到在向无锌酶添加锌的过程中发生了两个不同的锌结合事件。我们假设这两个高结构区域每个都配位到一个锌离子上，一个位置可能是结构的，另一个是催化的。阐明这种人工RNA连接酶的结构对于研究实验室从头产生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造酶的第一次XAS研究。

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.