With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Michael Massiah from the George Washington University to investigate factors that contribute to the function of a key class of enzymes involved in cellular protein recycling. All living organisms employ a common mechanism to maintain protein concentration and function to ensure a normal cell life cycle. A protein marked for recycling is covalently modified with a protein called ubiquitin (Ub). Proteins with multiple Ubs are cleaved into small fragments. The class of proteins that facilitates protein Ub modification are called RING E3 enzymes, and they are overall similar in structure. However, each RING E3 marks a specific set of proteins for modification. Despite structural similarity, the basis for substrate specificity and varying levels of activities by E3s are unknown. With this project, graduate, undergraduate and high school students re-engineer a new class of E3 enzyme and determine how specific amino acid changes affect the enzyme function. Students gain important biotechnology skills and the use of numerous chemical tools to characterize protein structure and protein-protein interactions.
This research project focuses on the B-box1 domain from the MID1 protein. This new class of Ub E3 enzymes has the same overall structure as RING E3 but exhibits a much lower level of activity. Through the use of PCR-based mutagenesis, specific amino acids of the B-box1 domain are changed. Multi-dimensional nuclear magnetic resonance spectroscopy is utilized to determine the structures of the various mutants of the B-box1 domain, and used to characterize their interactions with an E2 enzyme that is part of the ubiquitination cascade. Isothermal titration calorimetry, fluorescence spectroscopy, and FTIR are among the tools employed to characterize binding interactions. Completion of this project leads to identification of amino acids and structural features important for E3 ligase activity and substrate binding. These findings can be applied to other B-box domains and RING E3s to predict activity and mechanism of action.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
