Allen T S, Sabido-David C, Ling N, Irving M, Goldman Y E
Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia 19104, USA.
Biophys J. 1995 Apr;68(4 Suppl):81S-84S; discussion 85S-86S.
Structural changes of the myosin heads were correlated with mechanical events in the cross-bridge cycle by measuring fluorescence polarization signals at high time resolution from rhodamine probes bound to myosin regulatory light chains in skeletal muscle fibers. Motions of the cross-bridges were partially synchronized either by applying quick length changes to the fibers during active contractions or by activating the fibers from rigor by photolysis of caged ATP in the presence of Ca2+. With fibers in rigor, the fluorescence polarization values indicate that the probe dipoles are quite well ordered and are directed away from the muscle fiber axis. After photorelease of ATP from caged ATP, changes in polarization signals are consistent with broadening of the distribution of probe orientations. The signal deflections occur when ATP binds to actomyosin or when the cross-bridges detach, but the orientational distribution changes surprisingly little during active force development. In contrast, when staircases of quick releases are applied to labeled fibers during active contractions, the fluorescence polarization signals suggest a concerted rotation of the probes. The results indicate that the light chain region of myosin tilts during the quick release and/or during the tension recovery phase within the next few ms.
通过在高时间分辨率下测量与骨骼肌纤维中肌球蛋白调节轻链结合的罗丹明探针的荧光偏振信号，将肌球蛋白头部的结构变化与横桥循环中的机械事件相关联。在主动收缩过程中，通过对纤维施加快速长度变化，或者在Ca2+存在下通过笼状ATP的光解使纤维从僵直状态激活，横桥的运动部分同步。对于处于僵直状态的纤维，荧光偏振值表明探针偶极排列相当有序，并且指向远离肌肉纤维轴的方向。从笼状ATP光释放ATP后，偏振信号的变化与探针取向分布的变宽一致。当ATP与肌动球蛋白结合或横桥分离时会出现信号偏转，但在主动力产生过程中取向分布变化惊人地小。相比之下，当在主动收缩过程中对标记纤维施加快速释放阶梯时，荧光偏振信号表明探针发生协同旋转。结果表明，肌球蛋白的轻链区域在快速释放期间和/或在接下来的几毫秒内的张力恢复阶段发生倾斜。
