Reconditi Massimo, Linari Marco, Lucii Leonardo, Stewart Alex, Sun Yin-Biao, Narayanan Theyencheri, Irving Tom, Piazzesi Gabriella, Irving Malcolm, Lombardi Vincenzo
Laboratorio di Fisiologia, DBAG, Università di Firenze, 50019 Sesto Fiorentino, Italy.
Ann N Y Acad Sci. 2005 Jun;1047:232-47. doi: 10.1196/annals.1341.021.
Force and shortening in striated muscle are driven by a structural working stroke in the globular portion of the myosin molecules-the myosin head-that cross-links the myosin-containing filaments and the actin-containing filaments. We use time-resolved X-ray diffraction in single fibers from frog skeletal muscle to link the conformational changes in the myosin head determined at atomic resolution in crystallographic studies with the kinetic and mechanical features of the molecular motor in the preserved sarcomeric structure. Our approach exploits the improved brightness and collimation of the X-ray beams of the third generation synchrotrons by using X-ray interference between the two arrays of myosin heads in each bipolar myosin filament to measure with A sensitivity the axial motions of myosin heads in situ during the synchronous execution of the working stroke elicited by rapid decreases in length or load imposed during an active isometric contraction. Changes in the intensity and interference-fine structure of the axial X-ray reflections following the mechanical perturbation allowed to establish the average conformation of the myosin heads during the active isometric contraction and the extent of tilt during the elastic response and during the subsequent working stroke. The myosin working stroke is 12 nm at low loads, which is consistent with crystallographic studies, while it is smaller and slower at higher loads. The load dependence of the size and speed of the myosin working stroke is the molecular determinant of the macroscopic performance and efficiency of muscle.
横纹肌中的力和缩短是由肌球蛋白分子球状部分（即肌球蛋白头部）的结构性工作冲程驱动的，肌球蛋白头部将含肌球蛋白的细丝和含肌动蛋白的细丝交联在一起。我们利用来自青蛙骨骼肌单纤维的时间分辨X射线衍射，将晶体学研究中在原子分辨率下确定的肌球蛋白头部构象变化与保留的肌节结构中分子马达的动力学和机械特征联系起来。我们的方法利用了第三代同步加速器X射线束改善的亮度和准直性，通过利用每个双极肌球蛋白细丝中两排肌球蛋白头部之间的X射线干涉，以高灵敏度测量在主动等长收缩期间因长度或负荷快速降低引发的工作冲程同步执行过程中原位肌球蛋白头部的轴向运动。机械扰动后轴向X射线反射的强度和干涉精细结构的变化，使我们能够确定主动等长收缩期间肌球蛋白头部的平均构象，以及弹性反应期间和随后工作冲程期间的倾斜程度。在低负荷下，肌球蛋白的工作冲程为12纳米，这与晶体学研究结果一致，而在高负荷下，工作冲程更小且更慢。肌球蛋白工作冲程的大小和速度对负荷的依赖性是肌肉宏观性能和效率的分子决定因素。
