Protein stability [determination] problems

Human health depends on the correct folding of proteins, for misfolding and aggregation lead to diseases. An unfolded (denatured) protein can refold to its original folded state. How does this occur is known as the protein folding problem. One of several related questions to this problem is that how much more stable is the folded state than the unfolded state. There are several measures of protein stability. In this article, protein stability is given a thermodynamic definition and is measured by Gibbs free energy change ( $$ ) associated with the equilibrium, native (N) conformation ↔ denatured (D) conformation under the physiological condition usually taken as dilute buffer (or water) at 25 °C. We show that this thermodynamic quantity ( $$ ), where subscript D represents transition between N and D states, and superscript 0 (zero) represents the fact that the transition occurs in the absence of denaturant, can be neither measured nor predicted under physiological conditions. However, $$ can be measured in the presence of strong chemical denaturants such as guanidinium chloride and urea which are shown to destroy all noncovalent interactions responsible for maintaining the folded structure. A problem with this measurement is that the estimate of $$ comes from the analysis of the plot of $$ versus denaturant concentration, which requires a long extrapolation of values of $$ , and all the three methods of extrapolation give three different values of $$ for a protein. Thus, our confidence in the authentic value of $$ is eroded. Another problem with this in vitro measurement of $$ is that it is done on the pure protein sample in dilute buffer which is a very large extrapolation of the in vivo conditions, for the crowding effect on protein stability is ignored.