Takezawa Y, Kim D S, Ogino M, Sugimoto Y, Kobayashi T, Arata T, Wakabayashi K
Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan.
Biophys J. 1999 Apr;76(4):1770-83. doi: 10.1016/S0006-3495(99)77338-8.
The effects of the applied stretch and MgADP binding on the structure of the actomyosin cross-bridges in rabbit and/or frog skeletal muscle fibers in the rigor state have been investigated with improved resolution by x-ray diffraction using synchrotron radiation. The results showed a remarkable structural similarity between cross-bridge states induced by stretch and MgADP binding. The intensities of the 14.4- and 7.2-nm meridional reflections increased by approximately 23 and 47%, respectively, when 1 mM MgADP was added to the rigor rabbit muscle fibers in the presence of ATP-depletion backup system and an inhibitor for muscle adenylate kinase or by approximately 33 and 17%, respectively, when rigor frog muscle was stretched by approximately 4.5% of the initial muscle length. In addition, both MgADP binding and stretch induced a small but genuine intensity decrease in the region close to the meridian of the 5.9-nm layer line while retaining the intensity profile of its outer portion. No appreciable influence was observed in the intensities of the higher order meridional reflections of the 14.4-nm repeat and the other actin-based reflections as well as the equatorial reflections, indicating a lack of detachment of cross-bridges in both cases. The changes in the axial spacings of the actin-based and the 14.4-nm-based reflections were observed and associated with the tension change. These results indicate that stretch and ADP binding mediate similar structural changes, being in the correct direction to those expected for that the conformational changes are induced in the outer portion distant from the catalytic domain of attached cross-bridges. Modeling of conformational changes of the attached myosin head suggested a small but significant movement (about 10-20 degrees) in the light chain-binding domain of the head toward the M-line of the sarcomere. Both chemical (ADP binding) and mechanical (stretch) intervensions can reverse the contractile cycle by causing a backward movement of this domain of attached myosin heads in the rigor state.
利用同步辐射X射线衍射技术，以更高的分辨率研究了施加的拉伸和MgADP结合对处于僵直状态的兔和/或蛙骨骼肌纤维中肌动球蛋白横桥结构的影响。结果表明，拉伸和MgADP结合诱导的横桥状态之间存在显著的结构相似性。当在ATP耗尽备用系统和肌肉腺苷酸激酶抑制剂存在的情况下，向僵直的兔肌肉纤维中添加1 mM MgADP时，14.4 nm和7.2 nm子午向反射的强度分别增加了约23%和47%；当将僵直的蛙肌肉拉伸至初始肌肉长度的约4.5%时，这两个反射强度分别增加了约33%和17%。此外，MgADP结合和拉伸均导致靠近5.9 nm层线子午线区域的强度小幅但真实地下降，同时保留其外部部分的强度分布。在14.4 nm重复的高阶子午向反射以及其他基于肌动蛋白的反射和赤道反射的强度方面未观察到明显影响，表明在这两种情况下横桥均未脱离。观察到基于肌动蛋白和基于14.4 nm的反射的轴向间距变化，并将其与张力变化相关联。这些结果表明，拉伸和ADP结合介导了相似的结构变化，其方向与预期一致，即附着的横桥远离催化结构域的外部部分发生构象变化。对附着的肌球蛋白头部构象变化的建模表明，头部轻链结合结构域向肌节的M线有小幅但显著的移动（约10 - 20度）。化学干预（ADP结合）和机械干预（拉伸）均可通过使处于僵直状态的附着肌球蛋白头部的该结构域向后移动来逆转收缩周期。
