The cooperativity of burst phase reactions explored

The denaturant-dependence of the major, observable relaxation rates for fol ding (k(obs)) of ribonuclease HI from Escherichia coli (RNase H) and phage T4 lysozyme (T4L) reveal that, for both proteins, folding begins with the r apid and transient accumulation of intermediate species in a "burst phase" which precedes the rate-limiting formation of the native state; this is evi denced by a "rollover" in the folding limb of the rate profiles (k(obs) ver sus denaturant, or chevron plot). These rate profiles are most simply descr ibed by a three-state mechanism (unfolded-to-intermediate-to-native), which implies that the burst phase represents a transition between two distinct thermodynamic states. It is shown here that the equilibrium properties of t hese burst phase reactions can be equally well modeled by a mechanism invol ving a continuum of states where the free energy of each state is linearly related to its m-value (the parameter describing the linear relationship be tween free energy and denaturant). A numerical model is also developed to d escribe the time evolution of such a system, which exhibits nearly perfect exponential behavior. Both models emphasize how a continuum of states opera ting under a Linear free energy relationship may behave like a two state sy stem. Such a scheme finds experimental justification from an interpretation of recent native state hydrogen exchange data. The analytical model descri bed for a continuum can account for the observed kinetic profiles of severa l other model proteins. The results, however, appear context specific, sugg esting that burst phase reactions are not entirely random and non-specific. The results reported in this study have important implications for the con cept of cooperativity in protein folding reactions. (C) 1999 Academic Press .