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.
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