Submission Date

4-24-2023

Document Type

Paper- Restricted to Campus Access

Department

Biochemistry & Molecular Biology

Adviser

Amanda Reig

Committee Member

Samantha Wilner

Committee Member

Meghan Tierney

Committee Member

Eric Williamsen

Department Chair

Eric Williamsen

Department Chair

Anthony Lobo

Project Description

The de novo Due Ferri Single Chain protein model has been shown to be a viable protein scaffold to study functional characteristics through its thermodynamically favored and structurally sound four-helix bundle motif. Previous studies have shown that DFsc proteins exhibit DNA cleavage activity in time-controlled experiments. Additionally, phosphate bond cleavage activity has been demonstrated through small molecule studies using bis-4-nitrophenylphosphate (BNPP) cleavage assays. In this study, the phosphatase activity of several DFsc proteins was further quantified using 6,8-difluoro-4-methylumbelliferyl phosphate (difMUP) fluorescence absorption spectroscopy assays. From these small molecule studies, it was concluded that 1 μM 3-His G4DFsc exhibits higher phosphatase activity than 4-His and a bovine serum albumin (BSA) control at the same concentration. However, metal coordination to the DF active site did not appear to confer higher phosphatase activity. In addition to these phosphatase activity studies, cobalt binding assays for the DFY and G4Y proteins were conducted. It was determined from UV-vis absorption spectroscopy that DFY binds two equivalents of metal tightly to its active site. In contrast, cobalt did not exhibit high affinity for G4Y, therefore, a binding ratio was unable to be determined through UV-vis spectroscopy. As an extension of these studies and to substantiate results by Paredes et al. and Huynh 2022, DNA cutting assays were conducted for the DFsc, G4DFsc, and the tyrosine substituted G4Y and DFY proteins. From these DNA cutting assays, the results suggested that G4Y exhibits increased cleavage activity when complexed to binuclear metal as compared to the unsubstituted G4DFsc protein. In addition, increasing concentration of binuclear metal conferred higher cleavage activity. However, increased cleavage activities in DFY against DFsc were unable to be substantiated due to a lack of sufficient agarose electrophoresis data.

Comments

This research was funded by a grant from the National Institutes of Health (Grant# R15-GM114906).

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