The integrated efforts of biologists and chemists have resulted in a greater understanding of the function, structure, regulation and biomedical relevance of the ubiquitin-proteasome system (UPS). Ubiquitin, a key and central component of the UPS, is normally described primarily as a molecular tag that directs proteins to the proteasome for the purpose of degradation. Over the past 20 years several authors have speculated on the role ubiquitin possibly plays in triggering substrate unfolding to facilitate target degradation by the proteasome. The overall goal of my thesis project was to provide insights into the potential influence ubiquitin on the biophysical and biochemical properties of proteins to which it is covalently attached. To achieve this goal we study the more straightforward means by which ubiquitin is attached to target proteins, i.e., N-terminal mono-ubiquitination. By means of rational design we engineered a panel of small test proteins that exhibited variable thermal stabilities. These 'test' proteins were biophysically characterized as stand-alone, 'free' proteins in addition to being characterized as genetic fusions to the C-terminus of ubiquitin. Our results show that the thermal stabilities of the designed variants, both before and after monoubiquitination, are very similar and, more importantly, the addition of ubiquitin at the N-terminus of the test variants did not grossly alter the thermal stabilities of the test proteins. To investigate the biochemical relevance of mono-ubiquitinated proteins that exhibit variable thermal stabilities we characterized the engineered mono-ubiquitinated substrates in the context of the deubiquitinase Human Carboxy Hydrolase-L3 (UCH-L3). UCH-L3 removes small and unfolded peptide extensions from the C-terminus of ubiquitin. However no systematic analysis has been performed to evaluate the effect of the thermal stability of these extensions beyond their amino acid sequence and size. The hydrolysis assays performed on the engineered ubiquitin fusions demonstrate that differences in thermal stabilities of the proteins attached to ubiquitin greatly affect the capacity of UCH-L3 to process the substrates. Generally, unstable fusions are hydrolyzed at a significantly faster rate relative to stable fusions. Finally, we speculate on how these findings might provide additional insights into the roles that ubiquitin and the enzyme UCH-L3 play in natural systems.