PHOSPHORYLATION OF SERINE-46 IN HPR, A KEY REGULATORY PROTEIN IN BACTERIA, RESULTS IN STABILIZATION OF ITS SOLUTION STRUCTURE

The serine-phosphorylated form of histidine-containing protein (HPr), a component of the phosphoenolpyruvate:sugar phosphotransferase system from Bacillus subtilis, has been characterized by NMR spectroscopy an d solvent denaturation studies. The results indicate that phosphorylat ion of Ser 46, the N-cap of alpha-helix-B, does not cause a conformati onal change but rather stabilizes the helix. Amide proton exchange rat es in helix-B are decreased and phosphorylation stabilizes the protein to solvent and thermal denaluration, with a Delta Delta G of 0.7-0.8 kcal mol(-1). A mutant in which Ser 46 is replaced by aspartic acid sh ows a similar stabilization, indicating that an electrostatic interact ion between the negatively charged groups and the helix macrodipole co ntributes significantly to the stabilization.