Creation of Chemically Reactive Ubiquitin Probes by Genetic Incorporation of Unnatural Amino Acids
Author ORCID Identifier
Doctor of Philosophy
Thomas Ashton Cropp
Ubiquitin is a small, 76 amino acid post-translational modification (PTM) found in all eukaryotes. Ubiquitin is added to a protein substrate through a series of three enzymes, activating enzymes (E1s), conjugating enzymes (E2s), and ligases (E3s). These enzymes attach ubiquitin to a protein substrate by forming an amide bond between the C-terminal glycine of ubiquitin and the ε-amino group on the lysine of a target substrate. In addition, ubiquitin can be linked to itself in a variety of ways by way of its seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) or its N-terminal methionine. The attachment of each of these different chains leads to differential outcomes of the target protein. In addition, ubiquitin can be cleaved from proteins by deubiquitinases (DUBs). Within the human genome there are expected to be hundreds of different E2s, E3s, and DUBs, each with specificity to a particular set of proteins and/or ubiquitin chain conformations. The structure and function of many of these enzymes, however, is poorly understood or unknown. Furthermore, many of these enzymes have been linked with various diseases. It is therefore imperative to create tools for probing the interactions between ubiquitinating enzymes, their substrates, and ubiquitin.
This dissertation seeks to create ubiquitin probes containing unnatural amino acids that have been genetically incorporated. Here we were able to synthesize two ubiquitin trimers. One consisting of UbK11ThzK, UbK48R, and Ub1-75 and the other consisting of UbK48ThzK, UbHis, and Ub1-75. Each of these trimers will contain a reactive cysteine residue that can further be reduced to create a reactive dehydroalanine (Dha) residue. When combined with enzymes within the ubiquitination pathway, these probes should covalently react. In addition, this work attempts to determine the optimal conditions for UbK48Thzk-48UbHis purification from the monomeric UbHis. Here a monomeric ubiquitin containing propargyl lysine, UbK48Prk, is also successfully produced. In the presence of an azide, the alkyne of UbK8Prk should undergo a Click reaction to produce a triazole linkage.
This dissertation also seeks to improve the synthetic route of Nε -L-thiaprolyl-L-lysine methyl ester (ThzKOMe) that can be done on large-scale and that does not require expensive chemicals, special conditions, or lengthy purification steps. Because unnatural amino acids are incorporated into proteins at a low rate, attempts were made to recover these amino acids from media after fermentation and reuse them in new cultures and to use different strains of bacteria to increase the expression of proteins containing unnatural amino acids.
© Eve Nicholson
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Available for download on Wednesday, December 15, 2027
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