Asymmetric myosin binding to the thin filament as revealed by a fluorescent nanocircuit.

The interplay between myosin, actin, and striated muscle regulatory proteins involves complex cooperative interactions that propagate along the thin filament. A repeating unit of the tropomyosin dimer, troponin heterotrimer, and the actin protofilament heptamer is sometimes assumed to be able to bind myosin at any of its seven actins when activated even though the regulatory proteins are asymmetrically positioned along this repeating unit. Analysis of the impact of this asymmetry on actin and myosin interactions by sensitized emission luminescence resonance energy transfer spectroscopy and a unique fluorescent nanocircuit design reveals that the troponin affects the structure and function of myosin heads bound nearby in a different manner than myosin heads bound further away from the troponin. To test this hypothesis, a fluorescent nanocircuit reported the position of the myosin lever arm only when the myosin was bound adjacent to the troponin, or in controls, only when the myosin was bound distant from the troponin. Confirming the hypothesis, the myosin lever arm is predominantly in the pre powerstroke orientation when bound near troponin, but is predominantly in the post powerstroke orientation when bound distant from troponin. These data are consistent with the hypothesis that troponin is responsible for the formation of myosin binding target zones along the thin filament.