Salt Effects on Ionization Equilibria of Histidines in Myoglobin

Abstract

The salt dependence of histidine pK(a) values in sperm whale and horse myoglobin and in histidine-containing peptides was measured by (1)H-NMR spectroscopy. Structure-based pK(a) calculations were performed with continuum methods to test their ability to capture the effects of solution conditions on pK(a) values. The measured pK(a) of most histidines, whether in the protein or in model compounds, increased by 0.3 pH units or more between 0.02 M and 1.5 M NaCl. In myoglobin two histidines (His(48) and His(36)) exhibited a shallower dependence than the average, and one (His(113)) showed a steeper dependence. The (1)H-NMR data suggested that the salt dependence of histidine pK(a) values in the protein was determined primarily by the preferential stabilization of the charged form of histidine with increasing salt concentrations rather than by screening of electrostatic interactions. The magnitude and salt dependence of interactions between ionizable groups were exaggerated in pK(a) calculations with the finite-difference Poisson-Boltzmann method applied to a static structure, even when the protein interior was treated with arbitrarily high dielectric constants. Improvements in continuum methods for calculating salt effects on pK(a) values will require explicit consideration of the salt dependence of model compound pK(a) values used for reference in the calculations.

The pK a of His-24 in the folding transition state of apomyoglobin

“…The average pK a of the histidine residues in the transition state (6.7) is close to the average pK a in the molten globule (6.8; Table 1), and both pK a values are determined by procedures in which only a single adjustable parameter is determined. The average histidine pK a value determined for the molten globule is reasonably close to values measured in small peptides [6.8-6.9 at 25°C (17) or 7.2-7.3 at 4.5°C], considering that histidine pK a values are affected significantly by neighboring charged residues (17,18). In native apoMb, the range of pK a values covers 3 pH units even when His-24 is excluded (see Table 1).…”

“…The effect of net charge on the pK a values of the molten globule intermediate should be small at the pK a of the histidine residues in I because the net proton charge is small: there are 15 basic groups, including histidines, and 13 acidic groups in the A[B]GH subdomain of apoMb. On the other hand, the pK a values of His residues in holomyoglobin are affected by neighboring charged residues (17,18), and they increase measurably with NaCl concentration (18).…”

Accurate, conformation-dependent predictions of solvent effects on protein ionization constants

“…The rmsd and the maximum error to the experimental values were 0.14 and 0.2 pH unit, respectively. The sensitivity of pKas to the change in bulk ionic strength often reflects the nature of the electrostatic interactions stabilizing a particular ionized site (30,31). These results suggest that FDPB MF predicts quantitatively the complex balance of electrostatic interactions that stabilize charged residues.…”

Role of the Charge–Charge Interactions in Defining Stability and Halophilicity of the CspB Proteins

“…Experimental and theoretical studies suggest that long-range and short-range charge-charge interactions are affected differently upon increase in the ionic strength: salt efficiently screens long-range interactions, while short-range interactions start to weaken at 1 M ionic strength but may persist even at 1.5 M ionic strength. 43,56,57 Thus thermodynamic analysis of the halophilicity of proteins upon substitutions in the charged residues can distinguish between the effects on the long-range charge-charge interactions on one hand, and the effects on the short range charge-charge interactions or interactions other then charge-charge, on the other. Proteins can be classified as halophilic ("salt-loving") and halophobic ("salt-hating") with respect to the effects of the ionic strength on protein thermodynamics, and in particular, thermodynamic stability.…”