Pirani Alnoor, Xu Chen, Hatch Victoria, Craig Roger, Tobacman Larry S, Lehman William
Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA.
J Mol Biol. 2005 Feb 25;346(3):761-72. doi: 10.1016/j.jmb.2004.12.013. Epub 2005 Jan 11.
The movement of tropomyosin from actin's outer to its inner domain plays a key role in sterically regulating muscle contraction. This movement, from a low Ca2+ to a Ca2+-induced position has been directly demonstrated by electron microscopy and helical reconstruction. Solution studies, however, suggest that tropomyosin oscillates dynamically between these positions at all Ca2+ levels, and that it is the position of this equilibrium that is controlled by Ca2+. Helical reconstruction reveals only the average position of tropomyosin on the filament, and not information on the local dynamics of tropomyosin in any one Ca2+ state. We have therefore used single particle analysis to analyze short filament segments to reveal local variations in tropomyosin behavior. Segments of Ca2+-free and Ca2+ treated thin filaments were sorted by cross-correlation to low and high Ca2+ models of the thin filament. Most segments from each data set produced reconstructions matching those previously obtained by helical reconstruction, showing low and high Ca2+ tropomyosin positions for low and high Ca2+ filaments. However, approximately 20% of segments from Ca2+-free filaments fitted best to the high Ca2+ model, yielding a corresponding high Ca2+ reconstruction. Conversely, approximately 20% of segments from Ca2+-treated filaments fitted best to the low Ca2+ model and produced a low Ca2+ reconstruction. Hence, tropomyosin position on actin is not fixed in either Ca2+ state. These findings provide direct structural evidence for the equilibration of tropomyosin position in both high and low Ca2+ states, and for the concept that Ca2+ controls the position of this equilibrium. This flexibility in the localization of tropomyosin may provide a means of sterically regulating contraction at low energy cost.
原肌球蛋白从肌动蛋白的外部结构域向内部结构域的移动在空间上调节肌肉收缩中起关键作用。这种从低钙离子浓度状态到钙离子诱导状态的移动已通过电子显微镜和螺旋重建直接得到证实。然而，溶液研究表明，在所有钙离子浓度水平下，原肌球蛋白都在这些位置之间动态振荡，并且正是这种平衡的位置受钙离子控制。螺旋重建仅揭示了原肌球蛋白在细丝上的平均位置，而没有提供任何一种钙离子状态下原肌球蛋白局部动力学的信息。因此，我们使用单颗粒分析来分析短细丝片段，以揭示原肌球蛋白行为的局部变化。通过与细丝的低钙离子浓度和高钙离子浓度模型进行互相关，对无钙离子处理和钙离子处理的细肌丝片段进行分类。每个数据集中的大多数片段产生的重建结果与先前通过螺旋重建获得的结果匹配，显示出低钙离子浓度细丝和高钙离子浓度细丝的低钙离子浓度和高钙离子浓度原肌球蛋白位置。然而，来自无钙离子细丝的约20%的片段与高钙离子浓度模型拟合最佳，产生相应的高钙离子浓度重建结果。相反，来自钙离子处理细丝的约20%的片段与低钙离子浓度模型拟合最佳，并产生低钙离子浓度重建结果。因此，肌动蛋白上原肌球蛋白的位置在任何一种钙离子状态下都不是固定的。这些发现为高钙离子浓度和低钙离子浓度状态下原肌球蛋白位置的平衡以及钙离子控制这种平衡位置的概念提供了直接的结构证据。原肌球蛋白定位的这种灵活性可能提供一种以低能量成本在空间上调节收缩的方式。
