Ubiquitin (Ub) is a remarkably conserved protein found in nearly all tissues in eukaryotic cells. Human ubiquitin shares 96% sequence identity to yeast which reflects an extraordinary evolutionary pressure for conservation suggesting the importance of ubiquitin for maintaining cellular homeostasis. Ubiquitin Carboxy terminal Hydrolase-L3 (UC H-L3) is a human deubiquitinase enzyme whose precise biological function remains unknown yet it is proposed to hydrolyze small proteins, peptide remnants, and chemical vestiges from the C-terminus of ubiquitin following proteasomal processing. The recycling of ubiquitin by UCH-L3 preps ubiquitin for subsequent rounds of ligation and proteolysis. There appears to be two main biophysical properties that greatly affect UCH-L3 substrate selectivity. Previous research demonstrated a correlation between substrate size and catalytic rate with smaller proteins being cleaved from the C-terminus of ubiquitin more quickly than larger proteins. But there was one caveat to this correlation – UCH-L3 cannot cleave ubiquitin itself off of a diubiquitin substrate even though it is relatively small (76 amino acids). Might not this be due to ubiquitin’s relatively high melting temperature (88 ⁰C)? Previous work from our laboratory showed that when testing UCH-L3 hydrolysis with relatively small protein substrates (56 amino acids) attached to the C-terminus of ubiquitin, a strong correlation exists between the UCH-L3 catalytic rate and substrate thermal stability – less stable protein substrates are cleaved at a much higher rate relative to substrates of higher thermal stability. Could the strong evolutionary pressure to maintain the exact amino acid composition of ubiquitin be due, in part, to the need to maintain diubiquitin within the cytosol because of its important signaling functions? My thesis research explored how mutations to conserved hydrophobic residues affected the thermal destabilization of the C-terminal ubiquitin in the tandem diubiquitin signaling molecule. I also demonstrated that these mutations gave rise to significant increases to the hydrolysis of diubiquitin by UCH-L3. These findings give insights into the remarkable conservation of ubiquitin and its importance in its myriad of biological functions.