Ubiquitin (Ub) is a small, 76 amino acid, and post-translational modification (PTM) protein in eukaryotes. Modification of a substrate protein via the covalent attachment of the C-terminal glycine of Ub to the ε-amino group of lysine residues in a substrate is termed ubiquitination. Unlike, other PTM proteins, Ub can form polyUb chains at one or more of its seven lysine residues. (K6, K11, K27, K29, K33, K48, and K68). The consequence of these different polymerization sites is altered biological response with different polyUb linkages conferring different fates to target proteins. Unfortunately, the study of these chains have been limited by the inability to generate homogeneous polyUbs chains linked at known lysine residues. Furthermore, a three step enzymatic cascade consisting of activating-enzymes (E1s), conjugating enzymes (E2s), and ligase enzymes (E3s) tightly controls this modification. In response, our laboratory has developed a system that creates polyUb chains through bacterial expression and "synthetic" building blocks. Now, the main questions are what do these chains interact with in the cell and how do these interactions mediate biological responses?
In an attempt to answer these questions, this dissertation looks at different molecular techniques created to capture the transient interactions of monoUb and diUb probes with Ub substrates, such as, ubiquitin binding domains (UBDs) and conjugating E2 enzymes. One molecular technique focuses on the use of incorporating a genetically encoded, photo-crosslinker, p-Benzoyl-L-phenylalanine (pBpa) into diUb probes to capture their interaction with UBDs. This sets the foundation for understanding Ub’s cellular signaling recognition of UBDs. Another technique is creating diUb probes that contain lysine derivatives, Nε-L-Thiaprolyl-L-lysine (ThzK) or Nε-L-Cysteinyl-L-lysine (CysK), and can form a disulfide bonds with E2 enzymes to capture their complex, opening an opportunity to understand mechanistically the role E2 enzymes have with polyUb chain formation. Herein, these techniques are established to help unravel the complexity of Ub signaling.
|01 January 2018
|Braxton, Courtney N
|VCU Scholars Compass
|Virginia Commonwealth University
|Theses and Dissertations
|© Courtney N. Braxton
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