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

Mayank M Patel, Franco Tzul, George I Makhatadze

Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12065, USA.

Biophysical chemistry 2011 Nov

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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. Copyright © 2011 Elsevier B.V. All rights reserved.

Citation

Mayank M Patel, Franco Tzul, George I Makhatadze. Equilibrium and kinetic studies of protein cooperativity using urea-induced folding/unfolding of a Ubq-UIM fusion protein. Biophysical chemistry. 2011 Nov;159(1):58-65