Equilibrium distribution of skeletal actin–tropomyosin–troponin states, determined by pyrene–tropomyosin fluorescence

Actin–tropomyosin–troponin has three structural states, but the functional properties of regulation can be explained with models having two functional states. As a step towards assigning functional properties to all the structural states, we examined fluorescent probes that monitor changes in troponin and tropomyosin. Tropomyosin labeled with pyrene–iodoacetamide is thought to reflect the transition to the most active state, whereas N ‐((2‐iodoacetoxy)ethyl)‐ N ‐methyl)‐amino‐7‐nitrobenz‐2‐oxa‐1,3‐diazole‐labeled troponin I is thought to monitor the transition to any state other than the inactive state. The fraction of actin in an active state determined from pyrene excimer fluoresecence agreed with that calculated from light‐scattering measurements of myosin subfragment 1 (S1)–ADP to regulated actin in both the presence and absence of Ca 2+ over a range of ionic strength conditions. The only exceptions were conditions where the binding of S1–ADP to actin was too strong to measure accurately. Pyrene–tropomyosin excimer fluorescence was Ca 2+ dependent and so reflected the change in population caused by both Ca 2+ binding and S1–ADP binding. Pyrene labeling of tropomyosin did not cause a large perturbation of the transition among states of regulated actin. Using pyrene–tropomyosin fluorescence we were able to extend the ionic strength dependence of the parameters describing the co‐operativity of binding of S1–ADP to actin as low as 0.1  m . The probes on tropomyosin and troponin I had different responses to Ca 2+ and S1–ADP binding. These different sensitivities can be explained by an intermediate between the inactive and active states of regulated actin.