Myosin Structural (Rotational) Transitions In Both Catalytic And Light-Chain Domains Are Induced By Strong Binding To Actin

David D. Thomas, Josh E. Baker, Ingrid Brust-Mascher, Sampath Ramachandran, John J. Matta, Jack Grinband, and Edmund C. Howard

University of Minnesota, Minneapolis, MN 55455

We have used electron paramagnetic resonance (EPR) to investigate the effect of actin on structural transitions in the catalytic (CD) and light-chain (LCD) domains of myosin, in solution and in contracting muscle fibers. The CD was investigated by labeling Cys 707 (SH1) of rabbit S1 with a maleimide spin label (MSL), which monitors global orientation and dyamics of the CD, and an iodoacetamide spin label (IASL), which monitors internal dynamics within the CD. LCD global orientation was investigated by replacing the native regulatory light chain (RLC) in scallop muscle with gizzard RLC labeled with fluorodinitroaniline spin label (FDNASL) at Cys 108. EPR spectra of the LCD were resolved into two components having distinct probe orientations of 44° and 75° relative to the fiber axis. Changing the physiological state of the muscle fiber resulted in a change in the distribution between these two components. In the absence of ATP (rigor), the 44° component is predominant. In the presence of ATP (relaxation), the 44° and 75° components are equally represented. The addition of Ca (contraction) shifts 17% of the heads from the 75° component to the 44° component. Thus, force generation involves a shift in the distribution between these two states of LCD orientation, implying a 31° rotation of these LCD domains. These results constitute the first direct evidence for a large rotation between two distinct myosin head angles in muscle fibers. Transitions among weak-binding states (e.g., ATP hydrolysis) or among strong-binding states (e.g., ADP release) had no effect, so we conclude that the transition from weak to strong actin binding induces the observed LCD rotation. Similarly the global orientation and s dynamics of the CD depends primarily on the weak-to-strong transition: dynamic disorder in all weak-binding states, rigor-like order in strong-binding states. Similarly, the internal ns dynamics of the CD are restricted by actin in strong-binding states but not in weak-binding states.

SUMMARY: Strong binding to actin induces a disorder-to-order transition of the catalytic domain in all of the heads, and a 31° rotation of the light-chain domain in about half of the heads.

Back to participants