Sequential phosphorylation of adjacent serine residues on the N-terminal region of cardiac troponin-I: structure-activity implications of ordered phosphorylation.

We have used NMR spectroscopy to monitor the phosphorylation of a peptide corresponding to the N-terminal region of human cardiac troponin-I (residues 17-30), encompassing the two adjacent serine residues of the dual phosphorylation site. An ordered incorporation of phosphate catalysed by PKA was observed, with phosphorylation of Ser-24 preceding that of Ser-23. Diphosphorylation induced a conformational transition in this region, involving the specific association of the Arg-22 and Ser-24P side-chains, and maximally stabilised when both phosphoserines were in the di-anionic form. The results suggest that the second phosphorylation at Ser-23 of cardiac troponin-I is of particular significance in the mechanism by which adrenaline regulates the calcium sensitivity of the myofibrillar actomyosin Mg-ATPase.