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

Abstract

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.