ORIENTATION OF PARAMAGNETIC PROBES ATTACHED TO GIZZARD REGULATORY LIGHT-CHAIN BOUND TO MYOSIN HEADS IN RABBIT SKELETAL-MUSCLE

The orientation of the myosin neck was monitored using electron parama gnetic resonance (EPR) spectroscopy. Gizzard regulatory light chain wa s labeled with a nitroxide spin probe and exchanged for the native sub unit, located in the myosin neck, in rabbit psoas muscle fibers. The E PR spectra of rigor fibers indicated a substantial degree of probe imm obilization and showed a strong dependence on the orientation of the f iber axis relative to the magnetic field, indicating that the neck was ordered in this state. Spectra of relaxed fibers at 24 degrees C show ed that the neck was disordered, but the spectra of relaxed fibers at 4 degrees C indicated that the neck was partially ordered. Active fibe rs at the two temperatures produced spectra identical to relaxed fiber s, indicating that Ilo novel angles could be seen in the neck during t he powerstroke. Proteolytic fragments of myosin, S1 and HMM, were exch anged with labeled light chains and bound to thin filaments in unlabel ed fibers. The distribution of probe orientations for HMM was identica l to that of labeled rigor fibers, while S1 showed a slightly differen t distribution, suggesting that the neck is distorted (by a few degree s) by the interactions of the two heads of myosin when bound to actin.