Removing the first six amino acids of tropomyosin disrupts a binding site for troponin-T and alters its interaction with F-actin and myosin-MgATPase activation.
The amino-terminal region of tropomyosin plays a key regulatory role in its interaction with troponin-T and the activation of myosin-MgATPase.
The role of the amino-terminal region of alpha-striated muscle tropomyosin has been analyzed by reconstituting thin filaments with a version of the protein lacking the first six amino acids. While Omp T-digested tropomyosin (residues 7-284) does not bind significantly to F-actin at micromolar concentrations, an interaction is induced by skeletal troponin. At a moderate ionic strength (50 mM KCl), the apparent Kd values are 0.26 microM (with EGTA) and 1.6 microM (with Ca2+). At higher neutral salt (140 mM KCl), reconstitution is observed in the micromolar range only at high pCa (Kd = 1.3 microM). However, when chloride is replaced by acetate, the binding isotherms reach saturation under both extremes of Ca2+ apparent Kd values of 0.32 microM (with EGTA) and 2.6 microM (with Ca2+). The induction of binding of truncated tropomyosin to F-actin by troponin is attributable, in part, to troponin-I, but whereas the amino-terminal fragment of troponin-T (N-Tn-T, residues 1-158) enhances the effect of troponin-I in the case of other tropomyosin products specifically, unacetylated tropomyosin (residues 1-284), and carboxypeptidase-digested tropomyosin (residues 1-273) Heeley, D. H., et al. (1987) J. Biol. Chem. 262, 9971-9978, it is ineffective with regard to Omp T-digested tropomyosin, suggesting that cleavage has disrupted a binding site for this section of troponin-T. Thin filaments (with Ca2+) containing Omp T-digested tropomyosin activate the steady-state myosin-MgATPase to a greater extent than the integral system, consistent with the interaction between N-Tn-T and the amino-terminal region of tropomyosin having a regulatory function. At high pCa, the truncated system exhibits a less cooperative interaction with myosin-S1-ADP but the affinity for the ligand is stronger. In context with the current methodologies, the consequences of shortening tropomyosin at one end as opposed to the other are the reverse of each other.
Goonasekara et al. (Tue,) reported a other. Removal of the amino-terminal hexapeptide of tropomyosin vs. Integral system (unacetylated tropomyosin) was evaluated on Binding to F-actin and activation of steady-state myosin-MgATPase. Removing the first six amino acids of tropomyosin disrupts a binding site for troponin-T and alters its interaction with F-actin and myosin-MgATPase activation.
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