Relationship between Equilibrium Amide Proton Exchange Behavior and the Folding Pathway of Barnase

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Abstract
We describe a three-part strategy for analyzing the relationship between equilibrium amide proton exchange behavior of barnase and its folding/unfolding pathway. First, the effects of mutation on stability and kinetics are compared to reveal which residues exchange by local breathing, which by local unfolding, and which by a mixture of the two mechanisms. Second, is to detect any change of mechanism between EX2 and EX1 from the pH dependence of exchange and its relationship to structure and kinetics. The third is to determine from which state exchange takes place for residues that nominally exchange by a global process: the fully unfolded state or the folding intermediate. Experiments were performed at values of pH and temperature around physiological and close to conditions under which the folding pathway of barnase has been studied in detail. A set of residues was found for which the rate constants for exchange change on mutation by exactly the same factor as does the equilibrium constant for unfolding. Further, the protection factor against exchange for these residues in wild-type barnase is very similar to the equilibrium constant for overall folding measured by differential scanning calorimetry and extrapolated to the identical reaction conditions. These residues clearly exchange by a global unfolding mechanism, and the protection factors are consistent with the denatured state of barnase being largely as unprotected as model peptides. The rate constants for exchange of a second set of residues are unaffected by distant mutations, and so these exchange by local breathing. The logarithms of the rate constants (log kex) increase linearly with pH for the locally exchanging residues, consistent with the kinetics of the EX2 mechanism at these values of pH. The pH dependence for the globally exchanging residues, however, indicates a switch away from EX2 between pH 6.7 and 7.9 at 37 degrees C. The state from which "global" exchange occurs was probed also by using mutants in which the folded state of each is destabilized by the same amount by mutation relative to the unfolded state but the destabilization of the folding intermediate varies considerably. Under EX2 conditions, the changes in kex for all these residues follow the overall destabilization, confirming that exchange occurs from the fully unfolded state, not from the folding intermediate. The common characteristic of the residues that exchange by global unfolding is that they are all buried within the protein.

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