Increased backbone flexibility in threonine45-phosphorylated hirudin upon pH change.

Protein phosphorylation on serine/threonine side chains represents a major regulatory event in the posttranslational control of protein functionality, where it is thought to operate at the level of structural changes in the polypeptide chain. However, key questions about molecular aspects of phosphate ester induced conformational alterations remain open. Among these concerns are the radius of action of the phosphate ester group, its effective ionic state, and its interplay with distinct bonds of the polypeptide chain. Primarily to define short-range effects upon threonine phosphorylation, the native 65 amino acid protein hirudin, conformationally restrained by a proline flanking the pThr45 site and three intramolecular disulfide bonds, was structurally characterized in both the phosphorylated and the unphosphorylated state in solution. Circular dichroism and hydrogen exchange experiments (MALDI-TOF) showed that structural changes were caused by Thr45-Pro46 phosphorylation only when the phosphate ester gro...