Equilibrium and kinetic studies of protein cooperativity using urea-induced folding/unfolding of a Ubq-UIM fusion protein

► Urea-induced folding/unfolding monitored by far-UV circular dichroism and fluorescence spectroscopies. ► Equilibrium data for Ubq-UIM is inconsistent with a two-state model. ► Analysis of the kinetics of folding suggesting that formation of Ubq-domain is independent of UIM. ► A thermodynamic linkage model that involves four-states is applicable to the urea-induced unfolding of Ubq-UIM. ► The applicability of the model is further supported by variants of Ubq-UIM that have altered stabilities of domains.