NH2-TERMINAL TRUNCATION OF SKELETAL-MUSCLE TROPONIN-T DOES NOT ALTER THE CA2+ SENSITIVITY OF THIN FILAMENT ASSEMBLY

To investigate how Ca2+ binding to troponin C regulates muscle contraction, the Ca2+-sensitive properties of thin filament assembly were studied as the tropomyosin binding, NH2-terminal region of troponin T was progressively shortened. Troponin complexes were prepared that contained skeletal muscle troponin C, troponin I, and either intact troponin T (TnT) (residues 1-259) or fragment TnT-(70-259), TnT-(151-259), or TnT-(159-259). In the absence of Ca2+ their respective affinities for pyrene-labeled tropomyosin were 2.3 x 10(7) M(-1), 1.2 x 10(7) M(-1), 1.9 x 10(5) M(-1), and 1.9 x 10(5) M(-1). Ca2+ had only a small effect on these affinities: 1.1 x 10(7) M-1 for whole troponin, 2 x 10(5) M(-1) for troponin-(151-259), and 2.8 x 105 M-1 for troponin-(159-259). Forms of troponin that bound weakly to tropomyosin in the absence of actin increased the actin affinity of tropomyosin only 2-3-fold, even in the absence of Ca2+; weak. binding of troponin to tropomyosin correlated with weak effects on tropomyosin-actin binding. In contrast, whole troponin had an approximately 500-fold effect on tropomyosin binding to actin, regardless of whether Ca2+ was present. The small effect of Ca2+ on the energetics of thin filament assembly is not attributable to the amino-terminal region of troponin T. The results suggest that Ca2+ causes the interaction between actin and the globular region of troponin to switch between two energetically similar states.