Cold Denaturation in a Small Protein Domain

Abstract

Small, stable protein domains have become increasingly important as models for protein folding. However, one of the general thermodynamic characteristics of protein structures - cold denaturation - has not been observed for such model domains. We have investigated the thermal unfolding of a small 45 residue α-helical UBA domain using CD and fluorescence spectroscopy. In addition to a relatively high thermal stability (Tm ∼ 330K), we have also detected unfolding at cold temperatures, whose onset begins around 285K in the absence of denaturant. To further probe the cold denaturation, urea was used to destabilize the protein and therefore shift the onset of the cold denaturation to higher temperatures. All experimental data could be explained using a simple thermodynamic model, which assumes linear dependence of the unfolding free energy (ΔG) on the denaturant concentration. The model yields a large positive heat capacity change upon unfolding, which is traditionally associated with solvent exposure of hydrophobic groups. This small UBA domain therefore provides a valuable model for studying the still controversial phenomenon of cold denaturation and for understanding folding of larger proteins, which exhibit cold denaturation behavior.