CHEMICAL DENATURATION - POTENTIAL IMPACT OF UNDETECTED INTERMEDIATES IN THE FREE-ENERGY OF UNFOLDING AND M-VALUES OBTAINED FROM A 2-STATE ASSUMPTION

The chemical unfolding transition of a protein was simulated, includin g the presence of an intermediate (I) in equilibrium with the native ( N) and unfolded (U) states. The calculations included free energies of unfolding, Delta G(u)(w), in the range of 1.4 kcal/mol to 10 kcal/mol and three different global m-values. The simulations included a broad range of equilibrium constants for the N <-> I process. The dependenc e of the N <-> I equilibrium on the concentration of denaturant was al so included in the simulations. Apparent Delta G(u)(w) and m-values we re obtained from the simulated unfolding transitions by fitting the da ta to a two-state unfolding process. The potential-errors were calcula ted for two typical experimental situations: 1) the unfolding is monit ored by a physical property that does not distinguish between native a nd intermediate states (case 1), and 2) the physical property does not distinguish between intermediate and unfolded states (case II). The r esults obtained indicated that in the presence of an intermediate, and in both experimental situations, the free energy of unfolding and the m-values could be largely underestimated. The errors in Delta G(u)(w) and m-values do not depend on the m-values that characterize the glob al N <-> U transition. They are dependent on the equilibrium constant for the N <-> I transition and its characteristic m(1)-value. The exte nt of the underestimation increases for higher energies of unfolding. Including no random error in the simulations, it was estimated that th e underestimation in Delta G(u)(w) could range between 25% and 35% for unfolding transitions of 3-10 kcal/mol (case I). In case II, the unde restimation in Delta G(u)(w) could be even larger than in case I. In t he same energy range, a 50% error in the m-value could also take place . The fact that most of the mutant proteins are characterized by both a lower m-value and a lower stability than the wild-type protein sugge sts that in some cases the results could have been underestimated due to the application of the two-state assumption.