X-ray diffraction studies on the structural changes of rigor muscles induced by binding of phosphate analogs in the presence of MgADP

Abstract

To clarify the structure of the ATP hydrolysis intermediates (ADP.P i bound state) formed by actomyosin crossbridges, the effects of various phosphate analogs in the presence of MgADP on the structures of the thin and thick filaments in glycerinated rabbit psoas muscle fibers in the rigor state have been investigated by X-ray diffraction with a short exposure time using synchrotron radiation. When MgADP and phosphate analogs such as metallofluorides (BeF x=3,4 and AlF 4) and vanadate (VO 4(V i)) were added to rigor fibers in the presence of the ATP-depletion backup system, the intensities of the actin-based layer lines were markedly weakened. The greatest effect (∼50% decrease in intensity) was observed in the presence of BeF x among the analogs examined. The intensity distribution of the 5.9 nm actin-based layer line shifted towards that observed in the Ca 2+-activated fibers, while the first actin layer line at ∼1/36.7 nm −1 retained a rigor-like profile with an intensity weakened by ∼50%. The intensity of the equatorial 10 reflection increased while that of the 11 reflection changed little, resulting in only a small increase (∼1.7 fold) in the intensity ratio of the 10 to the 11 reflection. No resting-like pattern appeared upon the addition of MgADP and BeF x . These results indicate that a substantial fraction (∼40%) of the myosin heads dissociate from actin but the detached heads remain in the vicinity of the actin filaments when MgADP and BeF x bind. The states produced by binding phosphate analogs to a rigor muscle differ from the resting-like state produced by adding them to a contracting muscle (Takemori et al., J. Biochem. (Tokyo) 117 (1995) 603–608). Our conclusion put forward to explain the data is that one of the two heads of a crossbridge is detached and the other retains a rigor-like attachment.