SITE-SPECIFIC INVESTIGATIONS OF UBIQUITIN ACTIVATION, CARBAMYLATION, AND INTERACTIONS WITH UBIQUITIN-BINDING DOMAINS

Abstract

The post-translational modification of proteins with ubiquitin (Ub) can induce a multitude of cellular signaling processes. Ubiquitination involves the attachment of the C-terminus of Ub to lysines on the substrate protein through an isopeptide linkage. This process is facilitated by a multitude of enzymes which work in concert to write and erase these linkages. The power of Ub signaling is that Ub itself can be modified by additional Ub units to generate polyubiquitin chains through any of the seven lysines or N-terminal amine, and each of these attachment points produces polyubiquitin (polyUb) chains with unique orientations of the internal Ub. This allows for K48 polyUb chains to mark a substrate for proteasomal degradation or K63 polyUb chains to trigger DNA repair and maintenance processes. The Ub signaling system is an amalgamation of post-translational modifications, enzymatic activity, and carefully curated protein-protein binding interactions for this small 76 amino acid protein. My work presented in this disseration involves harnessing the power of nuclear magnetic resonance (NMR) experimentation to observe interactions of multiple components of the Ub system with site-specific resolution and selective kinetics. To this end, I have implemented some standard and atypical NMR experiments to observe the potential for carbon dioxide carbamates to modulate the Ub signaling system. I have determined the kinetics of the enzymatic Ub-activating process, and this was extrapolated to understand how ubiquitiun-like proteins, which share a similar fold to Ub, are discriminated from erroneously taking the place of Ub. I have solved the solution structure of an unusual ubiquitin-like domain and explored how it interacts with Ub. Lastly, I will report on the implementation of an unnatural amino acid that is a photosensitive cross-linker and demonstrate that this technology can be used to detect novel ubiquitin-binding proteins.