Two-state folding of horse ferrocytochrome c : Analyses of linear free energy relationship, chevron curvature, and stopped-flow burst relaxation kinetics

Ferrocytchrome c is a classic two-state fast folder. This assurance comes from extensive equilibrium and kinetic folding studies carried out under strictly anaerobic conditions at 22 °C. Conventional guanidine hydrochloride (GdnHCl)-induced unfolding transitions monitored by the use of a sizable set of optical probes do not reveal the accumulation of any intermediate to a detectable level. The GdnHCl dependence of unfolding free energy (ΔGD) is linear over the full range of the denaturant concentration. The GdnHCl folding chevron is characterized by curvatures in both folding and unfolding limbs. However, refolding rates as a function of urea in the presence of different concentrations of GdnHCl yield values (the kinetic m-value) that are quantitatively identical. This result, analyzed in terms of the denaturant dependence of the difference in the extent of solvent exposure between a relatively fixed transition state and the preceding state involved in the transition, suggests that the chevron curvature i...