Differential salt-induced stabilization of structure in the initial folding intermediate ensemble of barstar

Abstract

The effects of two salts, KCl and MgCl₂, on the stability and folding kinetics of barstar have been studied at pH 8. Equilibrium urea unfolding curves were used to show that the free energy of unfolding, ΔG_UN, of barstar increased from a value of 4.7 kcal mol⁻¹ in the absence of salt to a value of 6.9 kcal mol⁻¹ in the presence of 1 M KCl or 1 M MgCl₂. For both salts, ΔG_UN increases linearly with an increase in concentration of salt from 0 M to 1 M, suggesting that stabilization of the native state occurs primarily through a Hofmeister effect. Refolding kinetics were studied in detail in the presence of 1 M KCl as well as in the presence of 1 M MgCl₂, and it is shown that the basic folding mechanism is not altered upon addition of salt. The major effects on the refolding kinetics can be attributed to the stabilization of the initial burst phase ensemble, IE, by salt. Stabilization of structure in I_E by KCl causes the fluorescence properties of I_E to change, so that there is an initial burst phase change in fluorescence at 320 nm, during refolding. The structure in I_E is stabilized by MgCl₂, but no burst phase change in fluorescence at 320 nm is observed during refolding. The fluorescence emission spectra of I_E show that when refolding is initiated in 1 M KCl, the three tryptophan residues in IE are less solvent exposed than when folding is initiated in 1 M MgCl₂. Stabilization of I_E leads to an acceleration in the rate of the fast observable phase of folding by both salts, suggesting that structure of the transition state resembles that of I_E. The stabilization of I_E by salts can be accounted for largely by the same mechanism that accounts for the stabilization of the native state of the protein, namely through the Hofmeister effect. The salts do not affect the rates of the slower phases of folding, indicating that the late intermediate ensemble, I_L, is not stabilized by salts. Stabilization of the native state results in deceleration of the fast unfolding rate, which has virtually no dependence on the concentration of KCl or MgCl₂ at high concentrations. The observation that the salt-induced stabilization of structure in I_E is accompanied by an acceleration in the fast folding rate, suggests that I_E is likely to be a productive on-pathway intermediate.