Interaction of tropomyosin-troponin with actin filaments

Abstract

The assembly of actin filaments with tropomyosin-troponin was investigated by means of light scattering. Binding curves of tropomyosin-troponin [consisting of all 3 subunits (holotroponin)] and of tropomyosin-troponin-T-I to actin filaments were analyzed by separating the affinity of tropomyosin-troponin for actin filaments and the affinity for the end-to-end contact of tropomyosin molecules. Under the experimental conditions (42.4.degree. C, 300 mM KCl), tropomyosin-holotroponin in the absence of Ca and tropomyosin-troponin-T-I had similar affinities for actin filaments whereas tropomyosin-holotroponin in the presence of Ca bound more weakly. Tropomyosin-holotroponin and tropomyosin-troponin-T-I bound .apprx. 200- to 300-fold more strongly to binding sites with adjacent tropomyosin-troponin units than to isolated sites on actin filaments. The equilibrium constant for isolated association with actin filaments was > 2-fold higher for tropomyosin-holotroponin in the absence of Ca (15,400 M-1) and tropomyosin-troponin-T-I (17,500 M-1) than for tropomyosin-holotroponin in the presence of Ca (6600 M-1). Binding curves of mixtures of tropomyosin-holotroponin in the presence of Ca and of tropomyosin-troponin-T-I were measured and analyzed on the basis of a model of cooperative binding of 2 types of large ligands to a 1-dimensional homogeneous lattice. The results provided information on the strength of the end-to-end contacts of tropomyosin-troponin units in different positions on an actin filament. A tropomyosin-troponin unit binds adjacently to another unit in a different position on an actin filament .apprx. 2-fold more weakly than adjacent to a unit in the same position. With the aid of these results, it was possible to obtain information on the equilibrium distribution of tropomyosin-troponin in the 2 positions on actin filaments. Generation of a sequence of tropomyosin-troponin units in a different position on actin filaments was 4-fold less favored than elongation of an existing sequence (cooperativity parameter .sigma. = 1/4). Shifting of tropomyosin-troponin on actin filaments appears to be accompanied by small free-energy changes in the various interactions of the components of actin-tropomyosin-troponin filaments and not to be an all-or-none reaction.