Energetic implications for protein phosphorylation. Conformational stability of HPr variants that mimic phosphorylated forms.

The HPr protein from Bacillus subtilis is a key protein in the phosphoenolpyruvate-sugar transport system. HPr has two biological phosphorylation sites. The active site histidine is transiently phosphorylated in the phosphotransferase reaction while phosphorylation of serine 46 diminishes the activity of HPr. Here, we use protein engineering and equilibrium protein folding experiments to determine if the two phosphorylation events are energetically coupled. Our approach is to use structural mimics of the two phosphorylated forms of HPr, where histidine 15 is replaced by a negatively charged glutamate and serine 46 is changed to an aspartate, both alone and in combination. The thermodynamic analysis of the differences in conformational stability between the single and double mutants shows that the two phosphorylation sites are not energetically coupled in the HPr protein. We also show that single mutants of the active site histidine residue can have dramatic effects on the conformational stability of HPr. Combined with structural information, the method employed here will be of general use in unraveling the biological effects of phosphorylation on protein activity.