Narita A, Yasunaga T, Ishikawa T, Mayanagi K, Wakabayashi T
Department of Physics, School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.
J Mol Biol. 2001 Apr 27;308(2):241-61. doi: 10.1006/jmbi.2001.4598.
Muscle contraction is regulated by the intracellular Ca(2+ )concentration. In vertebrate striated muscle, troponin and tropomyosin on actin filaments comprise a Ca(2+)-sensitive switch that controls contraction. Ca(2+ )binds to troponin and triggers a series of changes in actin-containing filaments that lead to cyclic interactions with myosin that generate contraction. However, the precise location of troponin relative to actin and tropomyosin and how its structure changes with Ca(2+ )have been not determined. To understand the regulatory mechanism, we visualized the location of troponin by determining the three-dimensional structure of thin filaments from electron cryo-micrographs without imposing helical symmetry to approximately 35 A resolution. With Ca(2+), the globular domain of troponin was gourd-shaped and was located over the inner domain of actin. Without Ca(2+), the main body of troponin was shifted by approximately 30 A towards the outer domain and bifurcated, with a horizontal branch (troponin arm) covering the N and C-terminal regions of actin. The C-terminal one-third of tropomyosin shifted towards the outer domain of actin by approximately 35 A supporting the steric blocking model, however it is surprising that the N-terminal half of tropomyosin shifted less than approximately 12 A. Therefore tropomyosin shifted differentially without Ca(2+). With Ca(2+), tropomyosin was located entirely over the inner domain thereby allowing greater access of myosin for force generation. The interpretation of three-dimensional maps was facilitated by determining the three-dimensional positions of fluorophores labelled on specific sites of troponin or tropomyosin by applying probabilistic distance geometry to data from fluorescence resonance energy transfer measurements.
肌肉收缩受细胞内钙离子浓度调节。在脊椎动物的横纹肌中，肌动蛋白丝上的肌钙蛋白和原肌球蛋白构成一个控制收缩的钙离子敏感开关。钙离子与肌钙蛋白结合，引发含肌动蛋白丝的一系列变化，导致与肌球蛋白的循环相互作用从而产生收缩。然而，肌钙蛋白相对于肌动蛋白和原肌球蛋白的确切位置以及其结构如何随钙离子变化尚未确定。为了解调节机制，我们通过从电子冷冻显微照片确定细肌丝的三维结构来可视化肌钙蛋白的位置，且未施加螺旋对称性，分辨率约为35埃。有钙离子时，肌钙蛋白的球状结构域呈葫芦形，位于肌动蛋白的内部结构域上方。无钙离子时，肌钙蛋白的主体向外部结构域移动约30埃并分叉，有一个水平分支（肌钙蛋白臂）覆盖肌动蛋白的N端和C端区域。原肌球蛋白的C端三分之一向肌动蛋白的外部结构域移动约35埃，支持空间位阻模型，但令人惊讶的是原肌球蛋白的N端一半移动不到约12埃。因此，无钙离子时原肌球蛋白的移动存在差异。有钙离子时，原肌球蛋白完全位于内部结构域上方，从而使肌球蛋白有更多机会产生力。通过将概率距离几何应用于荧光共振能量转移测量数据来确定标记在肌钙蛋白或原肌球蛋白特定位点上的荧光团的三维位置，有助于对三维图谱的解读。
