Ionization equilibria and electrostatic effects in native and denatured staphylococcal nuclease

Ionization equilibria in a globular protein, staphylococcal nuclease (SNase), were examined by 1H-NMR spectroscopy and potentiometric H+ titration in order to quantify the strengths of electrostatic interactions among surface ionizable groups and to understand the contributions of these interactions to protein stability. The pKa values of histidines in staphylococcal nuclease are highly salt-sensitive, increasing by as much as a full pKa, unit between salt concentrations of 10 mM and 1.5 M. Continuum calculations captured quantitatively the dependence of pKa values on the concentration of KCl. According to the calculations, the dramatic salt-dependence reflects the attenuation of a large number of weak, repulsive interactions among charges separated by >10 A, which in aggregate produce a substantial effect. The magnitude, distance-dependence, and salt-sensitivity of individual pairwise electrostatic interactions were also assessed by analysis of pK a, values of histidines in charge-reversal and charge-neutralization mutants. Interactions are generally weak (≤0.6 kcal/mol at 5 A) and strongly distance-dependent. A simple Coulomb's law expression with a Debye-Huckel term to account for screening by the ionic strength captured the distance and ionic strength-dependence of charge-charge interactions as accurately as, and in the case of short-range interactions, more accurately than structure-based semi-macroscopic methods. The data demonstrate that in proteins with substantial charge imbalance, or under conditions of pH that promote charge imbalance, a large number of weak electrostatic interactions, in aggregate, can significantly affect the electrostatic properties of proteins.;The electrostatic character of the denatured ensemble of SNase was examined as well. The pKa, values of histidines in SNase at multiple concentrations of urea and GdnHCl were measured by 1H-NMR. Potentiometric titrations of SNase in GdnHCl and at varying concentrations of salt were also performed. Additionally, two models for denatured SNase were examined including a destabilized single-site mutant of SNase which resembles chemically denatured SNase spectroscopically and dimensionally, and short peptides derived from the primary sequence neighborhoods surrounding each of four SNase histidines which serve as models for fully unfolded protein. These models of denatured SNase indicate that despite the apparent compactness and native-like topology of denatured SNase, all native-like tertiary electrostatic interactions are neutralized in the denatured state; local sequence effects, however, persist.