The dynamics of the interaction between myosin subfragment 1 and pyrene-labelled thin filaments, from rabbit skeletal muscle.

We have used actin labelled in Cys-374 with N-(1-pyrenyl)iodoacetamide to monitor the dynamics and equilibria of the interaction between myosin subfragment 1 and the actin-troponin-tropomyosin complex in the presence of calcium. These results are compared with those obtained for pure actin and myosin subfragment 1. The sensitivity of this fluorescent label allowed us to measure the binding affinity of myosin subfragment 1 for actin directly by fluorescence titration. The affinity of subfragment 1 for actin is increased sixfold by troponin-tropomyosin in the presence of calcium. Kinetic studies of the interaction of subfragment 1 and actin have revealed an isomerization of the actin-subfragment 1 complex from a state in which actin is weakly bound (Ka = 5.9 X 10(4) M-1) to a more tightly bound complex (Ka = 1.7 X 10(7) M-1) (Coates, Criddle & Geeves (1985) Biochem. J. 232, 351). Results in the presence of troponin-tropomyosin show the same isomerization. The sixfold increase in affinity of subfragment 1 for actin is shown to be due to a decrease in the rate of dissociation of actin from the weakly bound complex.