Cynthia Wolberger studies the molecular mechanisms underlying transcription regulation and ubiquitin signaling.
Structural Role of the Catalytic Asparagine in Ubiquitin E2 Conjugating Enzymes
Ubiquitin is covalently attached to protein substrates by a cascade of reactions catalyzed by E1, E2, and E3 enzymes. The E1 ubiquitin-activating enzyme transfers ubiquitin to the active site cysteine of the E2 ubiquitin-conjugating enzyme, and then the E3 ubiquitin ligase promotes transfer of the ubiquitin to a substrate lysine, with which it forms an isopeptide bond. All E2 enzymes contain an asparagine residue near the active site cysteine which, when mutated, decreases the catalytic efficiency of the enzyme. Although this residue was postulated to play a direct role in catalysis, structural studies of wild-type enzymes have failed to establish a definitive role for the residue in the wild-type enzyme. The goal of this project will be to explore whether mutations of this residue have a structural effect on the enzyme, which then indirectly affects catalytic activity. The student will learn how to generate point mutations in plasmids and how to purify proteins and set up crystallization trials. The student will also learn how to collect and process x-ray diffraction data and determine structures using the method of molecular replacement. In addition, the student will learn how to do ubiquitin conjugation assays.