MICROSECOND ROTATIONAL-DYNAMICS OF F-ACTIN IN ACTOS1 FILAMENTS DURINGATP HYDROLYSIS

Rabbit skeletal muscle F-actin labeled at Cys374 with the triplet prob e erythrosin-5-iodoacetamide had a steady-state phosphorescence anisot ropy ((r) over bar(p)) of 0.090 +/- 0.005 at 20 degrees C in 100 mM KC l, pH 7.0, buffer. Titration with skeletal muscle S1 fragment increase d (r) over bar(p) to 0.138 +/- 0.006 at a mole ratio of 1:1. In the pr esence of ATP, the anisotropy of the actoS1 complex initially decrease d to 0.050 +/- 0.005, a value significantly smaller than the anisotrop y of pure F-actin; (r) over bar(p) subsequently increased to 0.126 +/- 0.002. The time course of the increase matched that expected from the measured actin-activated ATPase of S1. The plateau value at long time , 0.126, was identical to that of actoS1 in the presence of exogenous ADP or ADP plus phosphate. Characterization of the spectroscopic prope rties of the erythrosin probe indicated that the changes in (r) over b ar(p) were not due to changes in fast probe motions on the surface of the filament or the phosphorescence emission lifetime, or in the orien tation of the probe on the surface of F-actin, suggesting that they re flect large-scale changes in the microsecond rotational dynamics of ac tin. ATP hydrolysis by actoS1 thus appeared to induce rotational motio ns of or within F-actin on the phosphorescence time scale (approximate to 300 mu s). Although the precise physical origin of the induced rot ational motions is unknown, this study provides direct evidence that l arge-scale conformational fluctuations of the actin filament are assoc iated with the force-generating event in actomyosin.