Submission Date

7-21-2022

Document Type

Paper- Restricted to Campus Access

Department

Chemistry

Faculty Mentor

Amanda Reig

Comments

Presented during the 24th Annual Summer Fellows Symposium, July 22, 2022 at Ursinus College.

This research was made possible through the National Institutes of Health Grant No. R15-GM144906. Any findings expressed in this paper are not necessarily reflective of the views of the National Institutes of Health.

Project Description

The Due Ferri single chain (DFsc) family of proteins was originally created to better understand the characteristics of carboxylate-bridged diiron proteins. It was observed that when zinc coordinates to the active site of this protein rather than iron, the protein cleaves phosphate bonds via hydrolysis. To better understand, and potentially enhance this reactivity, mutations have been made in or near the metal binding active site. In this project, we have developed an assay using phosphorus nuclear magnetic resonance spectroscopy (31P-NMR) to monitor the catalytic activity of DFsc by observing the conversion of bis(4-nitrophenyl) phosphate (BNPP) to para-nitrophenolate (PNP) and para-nitro-phenylphosphate (pNPP). Using integral calculations, rates of reaction for DFsc and its mutants were determined. Additionally, the developed 31P-NMR protocol was replicated on a UV-Vis plate reader. The NMR protocol indicated that DFsc is the most efficient protein for phosphatase activity, however, the UV-Vis data indicated that 3-HisG4DFsc is the most efficient protein for phosphatase activity. Further experimentation is necessary to further optimize these protocols and acquire more consistent data that can be compared across different approaches.

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