Global Analysis of the Acid-Induced and Urea-Induced Unfolding of Staphylococcal Nuclease and Two of Its Variants

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Abstract
We have studied the equilibrium unfolding of staphylococcal nuclease and two of its variants, V66W and V66W‘, over two perturbation axes (acid-induced unfolding as a function of urea concentration and urea-induced unfolding as a function of pH). The transitions were monitored by simultaneous measurements of circular dichroism and fluorescence. With this multidimensional array of data (2 perturbation axes and 2 signals), we present a strategy of performing a global analysis, over as many as 12 individual data sets, to test various models for the unfolding process, to determine with greater confidence the pertinent thermodynamic parameters, and to characterize unfolding intermediates. For example, wild-type nuclease shows a cooperative two-state transition with either urea or pH as denaturant, but the global fits are improved when the model is expanded to include a pH dependence of the urea m value or when two distinct classes of protonic groups are considered. The best fit for wild-type nuclease is with ΔG°0,UN = 6.4 kcal/mol at pH 7, with the acid-induced unfolding being triggered by protonation of three to five carboxylate groups (with possible contribution from His121), and with the urea m = 2.5 kcal mol-1 M-1. V66W‘ lacks the last 13 amino acids on the C-terminus, has a tryptophan at position 66, has a predominantly β-sheet structure, and is less stable than the wild type. For V66W‘, ΔG°0,UN = 1.6 kcal/mol, m = 1.2 kcal mol-1 M-1, and there are two or three groups responsible for acid unfolding. V66W, a full-length mutant with two tryptophan residues, unfolds via a three-state mechanism: native ⇌ intermediate ⇌ unfolded. It appears that its β-barrel subdomain retains structure in the intermediate state. Assuming that the unfolding of V66W‘ and the β-barrel subdomain of V66W can be described by the same thermodynamic parameters, a global analysis enabled a description of the α subdomain of V66W with ΔG°0,IN = 2.7 kcal/mol, mIN = 1.1 kcal mol-1 M-1, and with the acid unfolding being triggered by protonation of a single group. This group has a pKa around 6 in the unfolded state, suggesting that the state of protonation of a histidine residue may contribute significantly to the stability of V66W.