Investigation of potentially catalytic residues of Uba5 through mutagenesis, purification, and structural characterization

Author

Grant Bradley, James Madison UniversityFollow

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.

Date of Graduation

5-8-2020

Publish

yes

Document Type

Thesis

Degree Name

Bachelor of Science (BS)

Department

Department of Biology

Advisor(s)

Christopher E. Berndsen

Ray Enke

Jon Monroe

Abstract

Ubiquitin-fold modifier 1 (Ufm1) is a member of the Ubiquitin (Ub) family of proteins whose primary function is degradation of proteins through a sequential mechanism of chemical reactions. Though Ufm1’s specific roles are largely unknown, this family of proteins has shown to play a part in a wide variety of processes, including regulation of the cell cycle¹, secretory functions of cells^2,3, and blood clotting⁴. Ufm1’s mechanism of action proceeds with the aid of three enzymes: an E1, E2, and E3. Uba5 is the E1 activating enzyme that is specific to Ufm1, and its mechanism of action and active site chemistry is not well understood^5,6. We do know that the enzyme contains a catalytic cysteine residue that is thought to be activated by another structure within the protein^5–7. The goal of this study was to investigate potentially catalytic residues within Uba5 through modeling with YASARA⁸, generate mutant proteins with point mutations within these residues, and purify these mutants for structural modelling and activity assays, to compare to the wild type. Such residues were identified and selected for purification as part of this study. These proteins were expressed; however, problems with the expression and purification protocol prevented us from successfully purifying the mutations, and SAXS data showed that what we did purify contained significant aggregation. As future directions, we suggest various adjustments to the expression and purification method that we feel will lead to better yields, and therefore continuation of these aims.

Recommended Citation

Bradley, Grant, "Investigation of potentially catalytic residues of Uba5 through mutagenesis, purification, and structural characterization" (2020). Senior Honors Projects, 2020-current. 69.
https://commons.lib.jmu.edu/honors202029/69