Protein Stability Regulated by Tag- and Site-Specific Ubiquitination and SUMOylation

Post-translational modification by ubiquitin and small ubiquitin-like modifiers (SUMOs) is central to the regulation of cellular protein function, but the mechanisms by which these tags modulate protein stability remain incompletely understood. Here, we show that ubiquitination alters the stability of the B1 domain of protein L in a site- and structure-specific manner by conjugating ubiquitin to seven individual lysines. Conjugation at K28, located within the α-helix, markedly increased thermal stability and enhanced resistance to mechanical unfolding, whereas modification at other sites had negligible effects. NMR and fluorescence analyses revealed local rearrangements near the α-helix and β3 sheet upon K28 ubiquitination, while conjugation of SUMO1 at the same site produced minimal effects, suggesting ubiquitin-specific substrate interactions. These results demonstrate that ubiquitin can act as an allosteric modulator of protein structure and mechanics, illustrating how modification site and tag identity together encode diverse functional outcomes in the proteome.