Laboratory of Physical Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biophys J. 1999 Nov;77(5):2665-76. doi: 10.1016/s0006-3495(99)77101-8.
The thick filaments of mammalian and avian skeletal muscle fibers are disordered at low temperature, but become increasingly ordered into an helical structure as the temperature is raised. Wray and colleagues (Schlichting, I., and J. Wray. 1986. J. Muscle Res. Cell Motil. 7:79; Wray, J., R. S. Goody, and K. Holmes. 1986. Adv. Exp. Med. Biol. 226:49-59) interpreted the transition as reflecting a coupling between nucleotide state and global conformation with M.ATP (disordered) being favored at 0 degrees C and M.ADP.P(i) (ordered) at 20 degrees C. However, hitherto this has been limited to a qualitative correlation and the biochemical state of the myosin heads required to obtain the helical array has not been unequivocally identified. In the present study we have critically tested whether the helical arrangement of the myosin heads requires the M.ADP.P(i) state. X-ray diffraction patterns were recorded from skinned rabbit psoas muscle fiber bundles stretched to non-overlap to avoid complications due to interaction with actin. The effect of temperature on the intensities of the myosin-based layer lines and on the phosphate burst of myosin hydrolyzing ATP in solution were examined under closely matched conditions. The results showed that the fraction of myosin mass in the helix closely followed that of the fraction of myosin in the M.ADP.P(i) state. Similar results were found by using a series of nucleoside triphosphates, including CTP and GTP. In addition, fibers treated by N-phenylmaleimide (Barnett, V. A., A. Ehrlich, and M. Schoenberg. 1992. Biophys. J. 61:358-367) so that the myosin was exclusively in the M.ATP state revealed no helical order. Diffraction patterns from muscle fibers in nucleotide-free and in ADP-containing solutions did not show helical structure. All these confirmed that in the presence of nucleotides, the M.NDP.P(i) state is required for helical order. We also found that the spacing of the third meridional reflection of the thick filament is linked to the helical order. The spacing in the ordered M.NDP.P(i) state is 143.4 A, but in the disordered state, it is 144. 2 A. This may be explained by the different interference functions for the myosin heads and the thick filament backbone.
哺乳动物和禽类骨骼肌纤维的粗丝在低温下是无序的，但随着温度的升高，它们会逐渐有序排列成螺旋结构。Wray 和同事（Schlichting，I.，和 J. Wray. 1986. J. Muscle Res. Cell Motil. 7:79；Wray，J.，R. S. Goody，和 K. Holmes. 1986. Adv. Exp. Med. Biol. 226:49-59）将这种转变解释为核苷酸状态和整体构象之间的一种耦合，其中 M.ATP（无序）在 0°C 时更为有利，而 M.ADP.P(i)（有序）在 20°C 时更为有利。然而，到目前为止，这仅限于定性关联，并且尚未明确确定获得螺旋排列所需的肌球蛋白头部的生化状态。在本研究中，我们批判性地测试了肌球蛋白头部的螺旋排列是否需要 M.ADP.P(i)状态。从拉伸至无重叠以避免与肌动蛋白相互作用引起的复杂化的兔腰大肌纤维束中记录了 X 射线衍射图谱。在紧密匹配的条件下，研究了温度对基于肌球蛋白的层线强度和溶液中肌球蛋白水解 ATP 的磷酸盐爆发的影响。结果表明，螺旋中肌球蛋白的质量分数与 M.ADP.P(i)状态下肌球蛋白的质量分数密切相关。使用一系列核苷酸三磷酸，包括 CTP 和 GTP，也得到了类似的结果。此外，用 N-苯马来酰亚胺（Barnett，V. A.，A. Ehrlich，和 M. Schoenberg. 1992. Biophys. J. 61:358-367）处理纤维，使肌球蛋白仅处于 M.ATP 状态，并未显示出螺旋有序性。无核苷酸和 ADP 溶液中的纤维的衍射图谱没有显示出螺旋结构。所有这些都证实，在存在核苷酸的情况下，M.NDP.P(i)状态是螺旋有序性所必需的。我们还发现，厚丝的第三子午线反射的间距与螺旋有序性有关。在有序的 M.NDP.P(i)状态下的间距为 143.4 A，但在无序状态下，间距为 144.2 A。这可以通过肌球蛋白头部和厚丝骨架的不同干涉函数来解释。
