Troponin C (TnC) is implicated in the initiation of myocyte contraction via binding of cytosolic Caxc2xb2xe2x81xba and subsequent recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of engineered TnC variants that increase or decrease Caxc2xb2xe2x81xba sensitivity, in order to understand the structural basis of their impact on TnC function. We will use the distinction for mutants that are associated with increased Caxc2xb2xe2x81xba affinity and for those mutants with reduced affinity. Our studies demonstrate that for GOF mutants V44Q and L48Q, the structure of the physiologically-active site II Caxc2xb2xe2x81xba binding site in the Caxc2xb2xe2x81xba-free (apo) state closely resembled the Caxc2xb2xe2x81xba-bound (holo) state. In contrast, site II is very labile for LOF mutants E40A and V79Q in the apo form and bears little resemblance with the holo conformation. We hypothesize that these phenomena contribute to the increased association rate, k(on), for the GOF mutants relative to LOF. Furthermore, we observe significant positive and negative positional correlations between helices in the GOF holo mutants that are not found in the LOF mutants. We anticipate these correlations may contribute either directly to Caxc2xb2xe2x81xba affinity or indirectly through TnI association. Our observations based on the structure and dynamics of mutant TnC provide rationale for binding trends observed in GOF and LOF mutants and will guide the development of inotropic drugs that target TnC.