In cardiac muscle, troponin (Tn) and tropomyosin inhibit actin and myosin interactions through the steric blocking of myosin binding to F-actin. Ca binding to Tn C modulates this inhibition. Thin filaments become activated upon Ca binding, which enables strong binding of myosin with a concomitant release of ATP hydrolysis products and level arm swinging responsible for force generation. Despite this level of description, the current cross-bridge cycle model does not fully define the structural events that take place within Tn during combinatorial myosin and Ca interventions. Here, we studied conformational changes within Tn bound to F-actin and tropomyosin by fluorescence lifetime imaging combined with Förster resonance energy transfer. Fluorescent dye molecules covalently bound to the Tn C C-lobe and Tn I C-terminal domain report Ca- and myosin-induced activation of Tn. Reconstituted thin filaments were deposited on a myosin-coated surface similar to an in vitro motility assay setup without filament sliding involved. Under all the tested conditions, Ca was responsible for the most significant changes in Tn activation. Rigor myosin activated Tn at subsaturated Ca conditions but not to the degree seen in thin filaments with Ca. ATP-γ-S did not affect Tn activation significantly; however, blebbistatin induced significant activation at subsaturating Ca levels. The relation between the extent of Tn activation and its conformational flexibility suggests that active/inactive Tn states coexist in different proportions that depend on the combination of effectors. These results satisfy an allosteric activation model of the thin filament as a function of Ca and the myosin catalytic cycle state.

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