MUSCLE CROSS-BRIDGES BOUND TO ACTIN ARE DISORDERED IN THE PRESENCE OF2,3-BUTANEDIONE MONOXIME

Electron paramagnetic resonance spectroscopy was used to monitor the o rientation of muscle cross-bridges attached to actin in a low force an d high stiffness state that may occur before force generation in the a ctomyosin cycle of interactions. 2,3-butanedione monoxime (BDM) has be en shown to act as an uncompetitive inhibitor of the myosin ATPase tha t stabilizes a myosin . ADP . P-i complex. Such a complex is thought t o attach to actin at the beginning of the powerstroke. Addition of 25 mM BDM decreases tension by 90%, although stiffness remains high, 40-5 0% of control, showing that cross-bridges are attached to actin but ge nerate little or no force. Active cross-bridge orientation was monitor ed via electron paramagnetic resonance spectroscopy of a maleimide spi n probe rigidly attached to cys-707 (SH-1) on the myosin head. A new l abeling procedure was used that showed improved specificity of labelin g. In 25 mM BDM, the probes have an almost isotropic angular distribut ion, indicating that cross-bridges are highly disordered. We conclude that in the pre-powerstroke state stabilized by BDM, cross-bridges are attached to actin, generating little force, with a large portion of t he catalytic domain of the myosin heads disordered.