Phosphorylation significantly modulates cTnI-cTnC, cTnI-cTnI and cTnI-cTnT interactions. In the unphosphorylated troponin the major interaction between NcTnC and NcTnI is cTnC Asp33, in the EF hand loop I, with cTnI Arg19, 20 and 21 (blue circle, Figure ). This is significant as Asp33 is one of the few cardiac specific variants in cTnC (Gly33 in skeletal muscle cTnC). Upon phosphorylation, there is a cumulative loss of these interactions. This is accompanied by altered NcTnC-NcTnI interactions with increased cTnC Asp25 to cTnI Arg12 interaction and the cTnC Glu32 to cTnI Arg12 interaction, both cTnC residues being at the end of the A helix.