Examining first the effects of the G159D mutation on the cTnC which are phosphorylation independent, the most significant change from the WT is the formation of a strong ionic and hydrogen bond interaction between Arg83, at the bottom of the D-helix and the Asp159, the end of the H-helix (~80% of the simulations). Arg83 also forms a strong ionic interaction with the N-helix of cTnC which is phosphorylation dependent in the WT. (Figure , supplement Figure ).