The kinetic pathway of folding of barnase.
To search for folding intermediates, we have examined the folding and unfolding kinetics of wild-type barnase and four representative mutants under a wide range of conditions that span two-state and multi-state kinetics. The choice of mutants and conditions provided in-built controls for artifacts that might distort the interpretation of kinetics, such as the non-linearity of kinetic and equilibrium data with concentration of denaturant. We measured unfolding rate constants over a complete range of denaturant concentration by using by 1H/2H-exchange kinetics under conditions that favour folding, conventional stopped-flow methods at higher denaturant concentrations and continuous flow. Under conditions that favour multi-state kinetics, plots of the rate constants for unfolding against denaturant concentration fitted quantitatively to the equation for three-state kinetics, with a sigmoid component for a change of rate determining step, as did the refolding kinetics. The position of the transition state on the reaction pathway, as measured by solvent exposure (the Tanford beta value) also moved with denaturant concentration, fitting quantitatively to the same equations with a change of rate determining step. The sigmoid behaviour disappeared under conditions that favoured two-state kinetics. Those data combined with direct structural observations and simulation support a minimal reaction pathway for the folding of barnase that involves two detectable folding intermediates. The first intermediate, I(1), is the denatured state under physiological conditions, D(Phys), which has native-like topology, is lower in energy than the random-flight denatured state U and is suggested by molecular dynamics simulation of unfolding to be on-pathway. The second intermediate, I(2), is high energy, and is proven by the change in rate determining step in the unfolding kinetics to be on-pathway. The change in rate determining step in unfolding with structure or environment reflects the change in partitioning of this intermediate to products or starting materials.