Structural changes in troponin in response to Ca2+ and myosin binding to thin filaments during activation of skeletal muscle

Contraction of skeletal and cardiac muscle is regulated by Ca2+-dependent structural changes in troponin that control the interaction between myosin and actin. We measured the orientations of troponin domains in skeletal muscle fibers using polarized fluorescence from bifunctional rhodamine probes on the C and E helices of troponin C. The C helix, in the regulatory head domain, tilts by approximate to 30 degrees when muscle is activated in physiological conditions, with a Ca2+-sensitivity similar to that of active force. Complete inhibition of active force did not affect C-helix orientation, and binding of rigor myosin heads did not affect its orientation at saturating [Ca2+]. The E helix, in the IT arm of troponin, tilted by approximate to 10 degrees on activation, and this was reduced to only 3 degrees when active force was inhibited. Binding of rigor myosin heads produced a larger tilt of the E helix. Thus, in situ, the regulatory head acts as a pure Ca2+-sensor, whereas the IT arm is primarily sensitive to myosin head binding. The polarized fluorescence data from active muscle are consistent with an in vitro structure of the troponin core complex in which the D and E helices of troponin C are collinear. The present data were used to orient this structure in the fiber and suggest that the IT arm is at approximate to 30 degrees to the filament axis in active muscle. In relaxed muscle, the IT arm tilts to approximate to 40 degrees but the D/E helix linker melts, allowing the regulatory head to tilt through a larger angle.