pH Dependence of Stability of Staphylococcal Nuclease:  Evidence of Substantial Electrostatic Interactions in the Denatured State

Abstract

The pH dependence of stability of staphylococcal nuclease was studied with two independent equilibrium thermodynamic approaches. First, by measurement of stability in the pH range 9 to 3.5 by fluorescence-monitored denaturation with urea (Delta), GdnHCl (Delta), and heat (Delta). Second, by numerical integration of H(+) titration curves (Delta) measured potentiometrically under native (100 mM KCl) and unfolding (6.0 M GdnHCl) conditions. The pH dependence of stability described by Delta, Delta, and Delta was comparable but significantly different from the one described by Delta. The decrease in Delta between pH 9 and pH 4 was 4 kcal/mol greater than the decrease in Delta, Delta, and Delta in the same pH range. In 6 M GdnHCl, all the ionizable groups titrated with the pK(a) values of model compounds. Therefore, Delta represents the free energy difference between the native state (N) and an ensemble of unstructured, or expanded, and highly screened conformations. In contrast, the shallower pH dependence of stability described by Delta and by Delta between pH 9 and 5 was consistent with the titration of histidines with depressed, nativelike pK(a) values in the denatured state (D). These depressed pK(a) values likely reflect long-range electrostatic interactions with the other 29 basic groups and are a consequence of the compact character of the D state. The steep change in Delta and Delta at pH < 5 suggests that near pH 5 the structural and thermodynamic character of the D state shifts toward a state in which acidic residues titrate with normal pK(a) values, presumably because the electrostatic interactions with basic residues are lost, maybe as a consequence of an expansion.

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

“…The curve can compared to the one generated using experimentally determined ΔG 0 values. Again, any deviation between the two plots demonstrates the presence of electrostatic interactions in the DSE above and beyond those expected for a fully unfolded state [1].…”

“…They also offer a relatively simple method for probing electrostatic interactions in the DSE [1,6,17,[23][24][25][26][27]. The approach exploits the linkage relationship that connects pH dependent changes in ΔG o with the difference in the number of protons bound to the native state and to the DSE [23]: (1) ΔQ in equation 1 is equal to the difference in the number of protons bound to the native state (Q N ) and to the DSE (Q D ) and is a function of pH. ΔG° is the apparent free energy of unfolding.…”

“…There has been a flurry of interest in the properties of the DSE in the last ten to fifteen years and it is clear that the DSE ensemble of real proteins is very different from the classic random coil especially under native or near native conditions. Considerable effort has been invested in characterizing residual structure in the DSE [1][2][3][4][5][6][7][8][9][10]. Well documented examples include hydrophobic clusters as well as small units of hydrogen bonded secondary structure, particularly helices or turns [1,2,4,5,7,9,10].…”

“…For example, it is often assumed that electrostatic interactions play a minor role in the DSE since charged residues are thought to be well solvated in the DSE and interactions amongst them are expected to screened by the high dielectric medium. However a number of investigations have shown that both favorable and unfavorable electrostatic interactions can be formed in the DSE and importantly have demonstrated that they can make a significant contribution to the energetics of the DSE [1,6,7]. The focus of this article is on the role of electrostatic interactions in the DSE and upon the experimental methods that can be employed to characterize them.…”

“…Our understanding of protein folding and stability depends in large part on mutational studies, thus careful assessment of the effects of mutations upon the DSE is essential. Indeed, the characterization of DSE interactions has emerged as a major research topic in protein engineering and protein folding [1][2][3][4][5][6][7]. A key challenge is to move beyond just characterizing the structural propensities of the DSE and to instead develop an understanding of how modulating DSE interactions can influence protein stability and protein folding [8].…”