Interaction of tropomyosin-troponin with actin filaments.
The assembly of actin filaments with tropomyosin-troponin was investigated by means of light scattering. Binding curves of tropomyosin-troponin [consisting of all three 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 degrees C, 300 mM KCl), tropomyosin-holotroponin in the absence of calcium and tropomyosin-troponin-T-I had similar affinities for actin filaments whereas tropomyosin-holotroponin in the presence of calcium was found to bind more weakly. Tropomyosin-holotroponin and tropomyosin-troponin-T-I bound about 200-300-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 more than 2-fold higher for tropomyosin-holotroponin in the absence of calcium (15 400 M-1) and tropomyosin-troponin-T-I (17 500 M-1) than for tropomyosin-holotroponin in the presence of calcium (6600 M-1). Binding curves of mixtures of tropomyosin-holotroponin in the presence of calcium and of tropomyosin-troponin-T-I were measured and analyzed on the basis of a model of cooperative binding of two types of large ligands to a one-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. It was found that a tropomyosin-troponin unit binds adjacently to another unit in a different position on an actin filament about 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 of the equilibrium distribution of tropomyosin-troponin in the two positions on actin filaments. Generation of a sequence of tropomyosin-troponin units in a different position on actin filaments was found to be 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
D000199:Actins / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008027:Light; D008433:Mathematics; D009124:Muscle Proteins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012542:Scattering, Radiation; D013696:Temperature; D014335:Tropomyosin / Q000378:metabolism*; D014336:Troponin / Q000378:metabolism*
D000199:Actins; D009124:Muscle Proteins; D014335:Tropomyosin; D014336:Troponin; D000251:Adenosine Triphosphatases; D002118:Calcium