Barnett V A, Thomas D D
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Biophys J. 1989 Sep;56(3):517-23. doi: 10.1016/S0006-3495(89)82698-0.
We have used saturation transfer electron paramagnetic resonance (ST-EPR) to detect the microsecond rotational motions of spin-labeled myosin heads in bundles of skinned muscle fibers, under conditions of rigor, relaxation, and isometric contraction. Experiments were performed on fiber bundles perfused continuously with an ATP-regenerating system. Conditions were identical to those we have used in previous studies of myosin head orientation, except that the fibers were perpendicular to the magnetic field, making the spectra primarily sensitive to rotational motion rather than to the orientational distribution. In rigor, the high intensity of the ST-EPR signal indicates the absence of microsecond rotational motion, showing that heads are all rigidly bound to actin. However, in both relaxation and contraction, considerable microsecond rotational motion is observed, implying that the previously reported orientational disorder under these conditions is dynamic, not static, on the microsecond time scale. The behavior in relaxation is essentially the same as that observed when myosin heads are detached from actin in the absence of ATP (Barnett and Thomas, 1984), corresponding to an effective rotational correlation time of approximately 10 microseconds. Slightly less mobility is observed during contraction. One possible interpretation is that in contraction all heads have the same mobility, corresponding to a correlation time of approximately 25 microseconds. Alternatively, more than one motional population may be present. For example, assuming that the spectrum in contraction is a linear combination of those in relaxation (mobile) and rigor (immobile), we obtained a good fit with a mole fraction of 78-88% of the heads in the mobile state. These results are consistent with previous STEPR studies on contracting myofibrils(Thomas et al., 1980). Thus most myosin heads undergo microsecond rotational motions most of the time during isometric contraction, at least in the probed region of the myosin head.These motions could arise primarily from the free rotations of heads detached from actin. However, if most of these heads are attached to actin during contraction, as suggested by stiffness measurements, this result provides support for the hypothesis that sub-millisecond rotational motions of actin-attached myosin heads play an important role in force generation.
我们利用饱和转移电子顺磁共振（ST-EPR）技术，在僵直、松弛和等长收缩条件下，检测了皮肤肌肉纤维束中自旋标记肌球蛋白头部的微秒级旋转运动。实验是在连续灌注ATP再生系统的纤维束上进行的。实验条件与我们之前研究肌球蛋白头部取向时所使用的条件相同，只是纤维与磁场垂直，使得光谱主要对旋转运动敏感，而不是对取向分布敏感。在僵直状态下，ST-EPR信号的高强度表明不存在微秒级旋转运动，这表明头部都牢固地结合在肌动蛋白上。然而，在松弛和收缩状态下，都观察到了相当程度的微秒级旋转运动，这意味着在这些条件下先前报道的取向无序在微秒时间尺度上是动态的，而非静态的。松弛状态下的行为与在无ATP时肌球蛋白头部从肌动蛋白上脱离时观察到的行为基本相同（Barnett和Thomas，1984），对应的有效旋转相关时间约为10微秒。在收缩过程中观察到的流动性略低。一种可能的解释是，在收缩过程中所有头部具有相同的流动性，对应的相关时间约为25微秒。或者，可能存在不止一种运动群体。例如，假设收缩状态下的光谱是松弛（可移动）和僵直（不可移动）状态下光谱的线性组合，我们得到了一个很好的拟合结果，其中处于可移动状态的头部摩尔分数为78 - 88%。这些结果与之前关于收缩肌原纤维的STEPR研究结果一致（Thomas等人，1980）。因此，在等长收缩过程中，至少在肌球蛋白头部的探测区域，大多数肌球蛋白头部大部分时间都经历微秒级旋转运动。这些运动可能主要源于从肌动蛋白上脱离的头部的自由旋转。然而，如果如刚度测量所表明的那样，这些头部中的大多数在收缩过程中附着在肌动蛋白上，那么这一结果为附着在肌动蛋白上的肌球蛋白头部的亚毫秒级旋转运动在力产生中起重要作用这一假设提供了支持。
