PATH AND EXTENT OF CROSS-BRIDGE ROTATION DURING MUSCLE-CONTRACTION

The angular distribution of myosin cross-bridges in muscle fibers was investigated in four physiological states using a multiple probe analy sis of varied extrinsic probes of the cross-bridge [Burghardt & Ajtai (1994) Biochemistry (preceding paper in this issue)]. The analysis com bines data of complementary techniques from different probes giving th e highest possible angular resolution. Four extrinsic probes of the fa st reactive sulfhydryl (SH1) on myosin subfragment 1 (S1) were employe d. Electron paramagnetic resonance (EPR) spectra from paramagnetic pro bes, deuterium- and N-15-substituted for greater sensitivity to orient ation, on S1 were measured when the protein was freely tumbling in sol ution and when it was decorating muscle fibers. The EPR spectra from l abeled S1 tumbling in solution were measured at X- and Q-band microwav e frequencies to uniquely specify the orientation of the probe relativ e to the S1 principal hydrodynamic frame. The EPR spectra from labeled S1 decorating muscle fibers in rigor and in the presence of MgADP wer e measured at X-band and used in the multiple probe analysis of cross- bridge orientation. The time-resolved fluorescence anisotropy decay (T RFAD) of fluorescent probes on S1 was measured when the protein was fr eely tumbling in solution, and fluorescence polarization (FP) intensit ies from fluorescent probes modifying SH1 in intact muscle fibers were measured for fibers in rigor, in the presence of MgADP, in isometric contraction, and in relaxation at low ionic strength. The TRFAD measur ements limit the range of possible orientations of the probe relative to the S1 principal hydrodynamic frame. The FP intensity measurements were used in the multiple probe analysis of cross-bridge orientation. The combination of the EPR and FP data determined a highly resolved cr oss-bridge angular distribution in rigor, in the presence of MgADP, in isometric contraction, and in relaxation at low ionic strength. These findings confirm earlier observations of a rigid body rotation of the SH1 region in the myosin head group upon physiological state changes and indicate the path and extent of cross-bridge rotation during contr action. The rotation of the cross-bridge is visualized with computer-g enerated space-filling models of actomysin in six states of the contra ction cycle.