The Stability of a Three-State Unfolding Protein

Abstract

The mechanism by which proteins fold to their unique native conformations from an initially disorganized form is one of the fundamental problems in molecular biology. In the study of proteins fold or refold standard Gibbs free energy, 0 2 (H O) G  is the single most important parameter for quantitating-protein stability and comparing stabilities of closely related proteins. Nearly all theoretical and experimental aspects of protein folding relate in some way to unfolding free energy changes, and much of the current work involving comparisons of mutant proteins is highly dependent on accurate evaluation of unfolding free energy measurements [1]. The history of evaluation of the quantity known as standard Gibbs free energy spans more than four decades, and at least three procedures involving strong solvent denaturation have been used in evaluation of this quantity. The three procedures are known as the transfer model of Tanford, the denaturant binding model, and the linear extrapolation method. Of the three methods employed in evaluation of 0 2 (H O) G  the so-called linear extrapolation method appears to enjoy the greatest acceptance. The virtue of the linear extrapolation method over the other two methods resides in the perception that it is more reliable since it gives 0 2 (H O) G  values that appear to be independent of whether urea or guanidinium hydrochloride is used as denaturant [2]. The linear extrapolation method is the most frequently used method of determining protein unfolding free energy changes induced by urea or guanidine hydrochloride (GdnHCl). It is based upon the premise that unfolding free energy changes are linearly dependent on denaturant concentration and that extrapolation to zero denaturant concentration gives 0 2 (H O) G  , the unfolding free energy change in the absence of denaturant. When only the native and unfolded forms of protein are present in significant concentrations at equilibrium, K F U  , the unfolded process corresponds to one transition, i.e. two-state model. The 0 2 (H O) G  can be obtained from 0 2 (H O) [ ] G G m D     , where m reflects the sensitivity of the transition to denaturant concentration [D] by using the linear extrapolation method [3]. When the native, partially unfolded (stable intermediate) and unfolded forms of protein are present in significant concentrations at equilibrium, IU FI   K K F I U , the unfolded process corresponds to two transitions, i.e. three-state model. Moreover, the 0 total 2 (H O) G  is