Experimental test of the thermodynamic model of protein cooperativity using temperature-induced unfolding of a Ubq-UIM fusion protein.

This study describes the thermodynamic characterization of a Ubq-UIM fusion construct (Ubq-UIM), designed from the ubiquitin-UIM interaction system, to determine whether it exhibits cooperativity of folding. The Ubq-UIM fusion constructs exhibit higher stability than the core Ubq molecule, consistent with the finding that the UIM helix is docked to Ubq. Temperature-induced unfolding profiles of Ubq-UIM were monitored by DSC and far-UV and near-UV CD spectroscopies. Ubq-UIM appears to exhibit cooperative unfolding as indicated by results of global fits of a two-state model to far- and near-UV CD and DSC thermal unfolding data. The cooperativity of Ubq-UIM unfolding was further tested by the amino acid substitutions that selectively stabilize or destabilize Ubq, UIM, and/or the interface. The effects of these substitutions on the thermodynamic properties of Ubq-UIM are described well by a thermodynamic model for cooperativity in proteins. In particular, a substitution that lowered the stability of the Ubq-UIM interface indeed led to a decrease in cooperativity.