Figure 5.

A cyclic representation of the FRET distance between actin C374 and cTnI C167C in cardiac thin filaments (TF) determined in three states. The initial complex is TF•Mg²⁺ in the Mg²⁺ state in which the distance is 40.8 Å. In the clockwise direction, Ca²⁺ binding to TF•Mg²⁺ to form the second complex TF•Mg²⁺•Ca²⁺ increases the distance by 9.3 Å, and the subsequent binding of S1 to form the final complex TF•S1•Mg²⁺•Ca²⁺ (right vertical pathway) lengthens the distance by an additional 10.6 Å. The sum of the two increases in these two clockwise steps is 9.3 + 10.6 = 19.9 Å. In the counter clockwise pathway, S1 first binds to TF•Mg²⁺ to form TF•S1•Mg²⁺, increasing the distance by 13.5 Å. Ca²⁺ binding to form the final complex TF•S1•Mg²⁺•Ca²⁺ (lower pathway) lengthens the distance by another 6.4 Å. The sum of these two increases in the counter clockwise direction is 13.5 + 6.4 = 19.9 Å. The difference in the distances between TF•Mg²⁺ (initial state) and TF•S1•Mg²⁺•Ca²⁺(final state) is 19.9 Å (60.7 – 40.8), independent of how the final state is reached, whether along the clockwise or the counter clockwise direction.