P K Values of Histidine Residues in Ribonuclease Sa: Effect of Salt and Net Charge

Abstract

The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the p K values of histidine residues in proteins. The salt-dependence of p K values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to p K values of model histidine-containing peptides. The p K of His53 is elevated by two pH units (p K=8.61) in RNase Sa and by nearly one pH unit (p K=7.39) in 5K at low salt relative to the p K of histidine in the model peptides (p K=6.6). The p K for His53 remains elevated in 1.5 M NaCl (p K=7.89). The elevated p K for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water. The p K of His85 in RNase Sa and 5K is slightly below the model p K at low salt and merges with this value at 1.5 M NaCl. The p K of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt. The tautomeric states of the neutral histidine residues are changed by charge reversal. The histidine p K values in RNase Sa are always higher than the p K values in the 5K variant. These results emphasize that the net charge of the protein influences the p K values of the histidine residues. Structure-based p K calculations capture the salt-dependence relatively well but are unable to predict absolute histidine p K values.