Submission Date

4-26-2024

Document Type

Paper- Restricted to Campus Access

Department

Chemistry

Adviser

Amanda J. Reig

Committee Member

Samantha Wilner

Committee Member

Marcus J. Wagner

Department Chair

Amanda J. Reig

External Reviewer

Rachel N. Austin

Distinguished Honors

This paper has met the requirements for Distinguished Honors.

Project Description

Metalloproteins use metal cations or metal containing cofactors to confer specific structural and reactive properties to a protein. Their natural prevalence makes them of interest to scientists, and one way to study their roles in biological systems is through de novo protein design. This method uses novel, lab designed proteins to model natural behaviors, and the Due Ferri single chain (DFsc) protein is one such example. The DFsc protein is a small, four-helix bundle protein that can bind a wide range of transition metals to its active site and is tolerant to site-directed mutagenesis. It was recently shown that the DFsc protein family is capable of hydrolyzing phosphate bonds in plasmid DNA and small-molecule substrates. Our goal was to better understand this hydrolytic activity through structural modifications of the DFsc protein and the development of new quantitative activity assays. To better mimic the trans-His active site structure of naturally occurring hydrolases, four new variants of the DFsc protein were created (E11H/H107E-G4, E11H/H107D-G4, E44H/H77E-G4, and E44H/H77D-G4). To complement previous small molecule activity assays using plasmid DNA, bis(4-nitrophenyl) phosphate (BNPP) and 6,8-difluoro-4-methylumelliferyl phosphate (DiFMUP), an assay was developed using fluorophore-tagged oligomeric DNA strands. These DNase assays were then used to determine the hydrolytic activity of both cis-His and trans-His DFsc protein variants. Initial studies indicate that the trans-His E11H/H107E-G4 variant has higher phosphatase activity than the cis-His G4DFsc protein, but additional quantitative studies are needed to confirm these results and determine if the enhanced activity is due to the His configuration or other factors.

Comments

This work was funded by the NIH Grant R15-GM144906.

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