Photolysis of a photolabile precursor of ATP (caged ATP) induces microsecond rotational motions of myosin heads bound to actin.
To test the proposal that ATPase activity is coupled to the rotation of muscle cross-bridges (myosin heads attached to actin), we have used saturation-transfer EPR to detect the rotational motion of spin-labeled myosin heads (subfragment 1; S1) bound to actin following the photolysis of caged ATP (a photoactivatable analog of ATP). In order to ensure that most of the heads were bound to actin in the presence of ATP, solutions contained high (200 microns) actin concentrations and were of low (36 mM) ionic strength. Sedimentation measurements indicated that 52 +/- 2% of the spin-labeled heads were attached in the steady state of ATP hydrolysis during EPR measurements. Five millimolar caged ATP was added to the actin-S1 solution in an EPR cell in the dark, with no effect on the intense saturation-transfer EPR signal, implying a rigid actin-S1 complex. A laser pulse produced 1 mM ATP, which decreased the signal rapidly to a brief steady-state level that indicated only slightly less rotational mobility than that of free heads. After correcting for the fraction of free heads, we conclude that the bound heads have an effective rotational correlation time of 1.0 +/- 0.3 microseconds, which is about 100 times shorter (faster) than that in the absence of ATP. To our knowledge, this is the first direct evidence that myosin heads undergo rotational motion when bound to actin during the ATPase cycle. It is likely that similar cross-bridge rotations occur during muscle contraction.
Berger
C L
CL
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Svensson
E C
EC
Thomas
D D
DD
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000031:analogs & derivatives; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D009994:Osmolar Concentration; D010782:Photolysis; D011485:Protein Binding; D011487:Protein Conformation; D012399:Rotation; D013997:Time Factors
D000199:Actins; D015879:Myosin Subfragments; C029273:P(3)-1-(2-nitro)phenylethyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D009218:Myosins