Roopnarine O, Thomas D D
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455, USA.
Biophys J. 1995 Apr;68(4):1461-71. doi: 10.1016/S0006-3495(95)80319-X.
We have used electron paramagnetic resonance (EPR) spectroscopy to study the orientation and rotational motions of spin-labeled myosin heads during steady-state relaxation and contraction of skinned rabbit psoas muscle fibers. Using an indane-dione spin label, we obtained EPR spectra corresponding specifically to probes attached to Cys 707 (SH1) on the catalytic domain of myosin heads. The probe is rigidly immobilized, so that it reports the global rotation of the myosin head, and the probe's principal axis is aligned almost parallel with the fiber axis in rigor, making it directly sensitive to axial rotation of the head. Numerical simulations of EPR spectra showed that the labeled heads are highly oriented in rigor, but in relaxation they have at least 90 degrees (Gaussian full width) of axial disorder, centered at an angle approximately equal to that in rigor. Spectra obtained in isometric contraction are fit quite well by assuming that 79 +/- 2% of the myosin heads are disordered as in relaxation, whereas the remaining 21 +/- 2% have the same orientation as in rigor. Computer-simulated spectra confirm that there is no significant population (> 5%) of heads having a distinct orientation substantially different (> 10 degrees) from that in rigor, and even the large disordered population of heads has a mean orientation that is similar to that in rigor. Because this spin label reports axial head rotations directly, these results suggest strongly that the catalytic domain of myosin does not undergo a transition between two distinct axial orientations during force generation. Saturation transfer EPR shows that the rotational disorder is dynamic on the microsecond time scale in both relaxation and contraction. These results are consistent with models of contraction involving 1) a transition from a dynamically disordered preforce state to an ordered (rigorlike) force-generating state and/or 2) domain movements within the myosin head that do not change the axial orientation of the SH1-containing catalytic domain relative to actin.
我们利用电子顺磁共振（EPR）光谱技术，研究了去表皮兔腰大肌纤维在稳态松弛和收缩过程中，自旋标记肌球蛋白头部的取向和旋转运动。使用茚二酮自旋标记，我们获得了与附着在肌球蛋白头部催化结构域上Cys 707（SH1）的探针特异性对应的EPR光谱。该探针被牢固固定，因此它报告肌球蛋白头部的整体旋转，并且在僵直状态下探针的主轴几乎与纤维轴平行，使其对头部的轴向旋转直接敏感。EPR光谱的数值模拟表明，标记的头部在僵直状态下高度取向，但在松弛状态下它们具有至少90度（高斯全宽）的轴向无序，中心角度近似于僵直状态下的角度。通过假设79±2%的肌球蛋白头部如在松弛状态下一样无序，而其余21±2%具有与僵直状态下相同的取向，等长收缩时获得的光谱拟合得相当好。计算机模拟光谱证实，不存在显著比例（>5%）的头部具有与僵直状态下明显不同（>10度）的独特取向，甚至大量无序的头部群体也具有与僵直状态下相似的平均取向。由于这种自旋标记直接报告头部的轴向旋转，这些结果强烈表明肌球蛋白的催化结构域在力产生过程中不会在两个不同的轴向取向之间发生转变。饱和转移EPR表明，在松弛和收缩过程中，旋转无序在微秒时间尺度上是动态的。这些结果与收缩模型一致，该模型涉及1）从动态无序的预力状态到有序（类似僵直）的力产生状态的转变和/或2）肌球蛋白头部内的结构域运动，这些运动不会改变含SH1的催化结构域相对于肌动蛋白的轴向取向。
