Increased backbone flexibility in threonine(45)-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 p olypeptide chain. However, key questions about molecular aspects of phospha te ester induced conformational alterations remain open. Among these concer ns are the radius of action of the phosphate ester group, its effective ion ic state, and its interplay with distinct bonds of the polypeptide chain. P rimarily to define short-range effects upon threonine phosphorylation, the native 65 amino acid protein hirudin, conformationally restrained by a prol ine flanking the pThr(45) site and three intramolecular disulfide bonds, wa s structurally characterized in both the phosphorylated and the unphosphory lated state in solution. Circular dichroism and hydrogen exchange experimen ts (MALDI-TOF) showed that structural changes were caused by Thr(45)-Pro(46 ) phosphorylation only when the phosphate ester group was in its dianionic state. The spatial arrangement of the amino acids, monitored by H-1 NMR spe ctroscopy, appears to be affected within a radius of about 10 A around the pThr(45)-O gammaH, with phosphorylation resulting in a loss of structure an d increased flexibility within a segment of at least seven amino acid resid ues. Thus, the transition from the monoanionic to the dianionic phosphate g roup over the pH range 5.2-8.5 represents a general phosphorylation-depende nt conformational switch operating at physiological pH values.