Microsecond rotational dynamics of F-actin in ActoS1 filaments during ATP hydrolysis.
Rabbit skeletal muscle F-actin labeled at Cys374 with the triplet probe erythrosin-5-iodoacetamide had a steady-state phosphorescence anisotropy (rp) of 0.090 +/- 0.005 at 20 degrees C in 100 mM KCl, pH 7.0, buffer. Titration with skeletal muscle S1 fragment increased rp to 0.138 +/- 0.006 at a mole ratio of 1:1. In the presence of ATP, the anisotropy of the actoS1 complex initially decreased to 0.050 +/- 0.005, a value significantly smaller than the anisotropy of pure F-actin; rp 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 properties of the erythrosin probe indicated that the changes in rp were not due to changes in fast probe motions on the surface of the filament or the phosphorescence emission lifetime, or in the orientation of the probe on the surface of F-actin, suggesting that they reflect large-scale changes in the microsecond rotational dynamics of actin. ATP hydrolysis by actoS1 thus appeared to induce rotational motions of or within F-actin on the phosphorescence time scale (approximately 300 microseconds). Although the precise physical origin of the induced rotational motions is unknown, this study provides direct evidence that large-scale conformational fluctuations of the actin filament are associated with the force-generating event in actomyosin.
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Department of Food Science, Rutgers University, New Brunswick, New Jersey.
Ludescher
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RD
D000199:Actins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D005454:Fluorescence Polarization; D007700:Kinetics; D008163:Luminescent Measurements; D008433:Mathematics; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism*; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D000199:Actins; D015879:Myosin Subfragments; D009218:Myosins