Strikingly, the effect of phosphorylation on these metrics is in the opposite direction to the wild-type. For instance, the A/B metric, phosphorylation of G159D increased the mean angle by 1.9° and decreased the proportion below 110° (open state threshold) from 99% to 93%. Phosphorylation of G159D also shifts the inter-domain angle distribution towards a lower mean angle and increased RMSF across the whole system, particularly troponin C indicating a global as well as the specific effect of the point mutation. The findings that the wild-type response to phosphorylation, observed in our MD simulations, is disrupted by the G159D mutation correlates with the observation that the G159D mutation abolishes the shift in Ca 2+ -sensitivity due to PKA phosphorylation of Ser22 and 23 . It is notable that the changes in the interdomain interface region occur without changes to the NcTnI-NcTnC interaction indicating an conformational uncoupling that mirrors the functional uncoupling caused by the mutation ( Figure ). These results support our proposal that the MD simulations faithfully report relevant changes induced by phosphorylation and mutation.