Ca2+ and Mg2+-dependent complex formation of tropomyosin with phosphotroponin (P1TI2C) or dephosphotroponin (TI2C)

Abstract

The reduced viscosity of troponin and dephosphotroponin is independent of the protein concentration in both states, either metal-free or with troponin C saturated with Ca2+ or Mg2+; that of tropomyosin increases linearly as function of the protein concentration, indicating aggregation. Addition of troponin to tropomyosin increases the reduced viscosity over the expected value being maximal at a 1:1 molar ratio of both proteins. The reduced viscosity of a 1:1 molar mixture of phosphotroponin-Mg4 or dephosphotroponin-Mg3 increases in two phases as function of the total protein concentration, indicating the formation of two kinds of troponin-tropomyosin complexes. In the first phase, troponin and tropomyosin form a non-aggregating 1:1 complex, which is characterized by a value of 0.45 dl/g for the intrinsic viscosity and a sedimentation coefficient of 3.6 S. Employing these two values a molecular weight of 150 000 can be calculated, which is in the range of the sum of molecular weights for troponin and tropomyosin (156 000). In the second phase the troponin-tropomyosin complex aggregates further, a process described by:n (troponin-tropomyosin) leads to (troponin-tropomyosin)n. This further aggregation occurs upon saturation of the Ca2+-specific sites in troponin C. A model is discussed which explains the shortening of 1.5 nm per tropomyosin molecule upon the shift of tropomyosin from the periphery into the groove of the actin filament by tropomyosin aggregation.