Fluorescence resonance energy transfer between points on actin and the C-terminal region of tropomyosin in skeletal muscle thin filaments
Fluorescence resonance energy transfer between points on actin and the C-terminal region of tropomyosin in skeletal muscle thin filaments
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DOI：
10.1093/jb/mvh090
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发表时间：
2004-07-01
影响因子：
2.7
通讯作者：
Wakabayashi, T
中科院分区：
文献类型：
--
作者：
Miki, M;Hai, H;Wakabayashi, T
原肌球蛋白（位置87和190）和肌动蛋白（Gln-41、Lys-61、Cys-374和atp结合位点）之间的荧光共振能量转移显示，随着Ca2+浓度的变化，原肌球蛋白相对于肌动蛋白在细丝上的位置没有变化（Miki et al. (1998) J. Biochem. 123, 1104-1111）。这与最近的电子冷冻显微镜分析一致，该分析显示原肌凝蛋白的G端有三分之一向肌动蛋白外域明显移动，而原肌凝蛋白的n端只有一半移动了一点（Narita et al. (2001) J. Mol. Biol. 308, 241-261）。为了检测原肌凝蛋白c端相对于肌动蛋白的位置变化，我们在c端区域的237、245、247或252位置产生了具有独特半胱氨酸残基的突变原肌凝蛋白分子。能量供体探针附着在原肌球蛋白的这些位置上，受体探针附着在肌动蛋白的Cys-374或Gln-41上。这些探针标记的突变原肌球蛋白分子保留了与肌钙蛋白和Ca2+一起调节act - s1 atp酶活性的能力。原肌凝蛋白和肌动蛋白这些点之间的荧光共振能量转移显示出很高的转移效率，这应该对附着在肌动蛋白和原肌凝蛋白上的探针之间距离的变化非常敏感。然而，Ca2+离子去除后，原肌球蛋白的转移效率并没有明显的变化，这表明在Ca2+浓度的变化下，原肌球蛋白的c端相对于肌动蛋白在重组细丝上的移动并不明显。
Fluorescence resonance energy transfer between points on tropomyosin (positions 87 and 190) and actin (Gln-41, Lys-61, Cys-374, and the ATP-binding site) showed no positional change of tropomyosin relative to actin on the thin filament in response to changes in Ca2+ concentration (Miki et al. (1998) J. Biochem. 123, 1104-1111). This is consistent with recent electron cryo-microscopy analysis, which showed that the G terminal one-third of tropomyosin shifted significantly towards the outer domain of actin, while the N-terminal half of tropomyosin shifted only a little (Narita et al. (2001) J. Mol. Biol. 308, 241-261). In order to detect any significant positional change of the C-terminal region of tropomyosin relative to actin, we generated mutant tropomyosin molecules with a unique cysteine residue at position 237, 245, 247, or 252 in the C-terminal region. The energy donor probe was attached to these positions on tropomyosin and the acceptor probe was attached to Cys-374 or Gln-41 of actin. These probe-labeled mutant tropomyosin molecules retain the ability to regulate the acto-S1 ATPase activity in conjunction with troponin and Ca2+. Fluorescence resonance energy transfer between these points of tropomyosin and actin showed a high transfer efficiency, which should be very sensitive to changes in distance between probes attached to actin and tropomyosin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that the C-terminal region of tropomyosin did not shift significantly relative to actin on the reconstituted thin filament in response to the change of Ca2+ concentration.