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

Understanding the origins of cooperativity in proteins remains an important topic in protein folding. This study describes experimental folding/unfolding equilibrium and kinetic studies of the engineered protein Ubq–UIM, consisting of ubiquitin (Ubq) fused to the sequence of the ubiquitin interacting motif (UIM) via a short linker. Urea-induced folding/unfolding profiles of Ubq–UIM were monitored by far-UV circular dichroism and fluorescence spectroscopies and compared to those of the isolated Ubq domain. It was found that the equilibrium data for Ubq–UIM is inconsistent with a two-state model. Analysis of the kinetics of folding shows similarity in the folding transition state ensemble between Ubq and Ubq–UIM, suggesting that formation of Ubq domain is independent of UIM. The major contribution to the stabilization of Ubq–UIM, relative to Ubq, was found to be in the rates of unfolding. Moreover, it was found that the kinetic m-values for Ubq–UIM unfolding, monitored by different probes (far-UV circular dichroism and fluorescence spectroscopies), are different; thereby, further supporting deviations from a two-state behavior. A thermodynamic linkage model that involves four states was found to be applicable to the urea-induced unfolding of Ubq–UIM, which is in agreement with the previous temperature-induced unfolding study. The applicability of the model was further supported by site-directed variants of Ubq–UIM that have altered stabilities of Ubq/UIM interface and/or stabilities of individual Ubq- and UIM-domains. All variants show increased cooperativity and one variant, E43N_Ubq–UIM, appears to behave very close to an equilibrium two-state.