STRUCTURAL STUDIES OF ARTIFICIAL RNA LIGASE

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 这个合作项目的目的是从结构上表征一种在自然界中没有对应物的人工RNA连接酶的锌配位。这种酶是通过完全依赖于非常大的随机蛋白质突变体库的功能多样性而产生的从头酶的第一个例子。该酶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。该文库基于含有两个锌指的小的稳定蛋白质结构域，所述锌指具有9和12个氨基酸的完全随机的环。该酶进行模板依赖性连接的5？三磷酸激活的RNA的3？第二RNA的羟基末端。自然界中没有已知的酶催化该反应。然而，该反应与RNA聚合过程中一个核苷酸的链延长有关。聚合酶通常需要Mg 2+进行双金属离子反应机制。相比之下，人工RNA连接酶被Mg 2+抑制，而是需要Zn 2+才能发挥其活性。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。人造酶的核磁共振光谱揭示了两个高度结构化的区域，嵌入在更动态的部分。我们观察到两个不同的锌结合过程中加入锌的无锌脱辅基酶的HSQC实验通过监测结构变化的事件。我们假设，这两个高度结构化的区域是每个协调的Zn 2+，一个网站可能是结构和其他催化。阐明这种人工RNA连接酶的结构对于研究从头实验室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则至关重要。这项工作将是这种人工酶的第一个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.