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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >The SAH domain extends the functional length of the myosin lever
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The SAH domain extends the functional length of the myosin lever

机译：SAH域延长了肌球蛋白杠杆的功能长度

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摘要

Stable, single alpha-helix (SAH) domains are widely distributed in the proteome, including in myosins, but their functions are unknown. To test whether SAH domains can act as levers, we replaced four of the six calmodulin-binding iQ motifs in the levers of mouse myosin 5a (Myo5) with the putative SAH domain of Dictyostelium myosin MyoM of similar length. The SAH domain was inserted between the IQ motifs and the coiled coil in a Myo5 HMM construct in which the levers were truncated from six to two IQ motifs (Myo5-2IQ). Electron microscopy of this chimera (Myo5-2IQ-SAH) showed the SAH domain was straight and 17 nm long as predicted, restoring the truncated lever to the length of wild-type (Myo5-6IQ). The powerstroke (of 21.5 nm) measured in the optical trap was slightly less than that for Myo5-6IQ but much greater than for Myo5-2IQ. Myo5-2IQ-SAH moved processively along actin at physiological ATP concentrations with similar stride and run lengths to Myo5-6IQ in in-vitro single molecule assays. In comparison, Myo5-2IQ is not processive under these conditions. Solution biochemical experiments indicated that the rear head did not mechanically gate the rate of ADP release from the lead head, unlike Myo5-6IQ. These data show that the SAH domain can form part of a functional lever in myosins, although its mechanical stiffness might be lower. More generally, we conclude that SAH domains can act as stiff structural extensions in aqueous solution and this structural role may be important in other proteins.
机译：稳定的单个α-螺旋（SAH）域在蛋白质组中广泛分布，包括在肌球蛋白中，但其功能尚不清楚。为了测试SAH结构域是否可以充当杠杆，我们用相似长度的推测的Dictyostelium肌球蛋白MyoM的SAH结构域替换了小鼠肌球蛋白5a（Myo5）杠杆中六个钙调蛋白结合iQ基序中的四个。 SAH域插入在Myo5 HMM构建体的IQ模体和螺旋线圈​​之间，其中杠杆从6个被截断为2个IQ模体（Myo5-2IQ）。此嵌合体（Myo5-2IQ-SAH）的电子显微镜观察显示，SAH域是直的，如预期的那样长17 nm，将截短的杆恢复为野生型（Myo5-6IQ）的长度。在光阱中测得的功率冲程（为21.5 nm）略小于Myo5-6IQ，但远大于Myo5-2IQ。在体外单分子测定中，Myo5-2IQ-SAH在生理ATP浓度下沿肌动蛋白进行性移动，步幅和游动长度与Myo5-6IQ相似。相比之下，Myo5-2IQ在这些条件下无法进行。溶液生化实验表明，与Myo5-6IQ不同，后部头部没有机械地控制ADP从引线头部释放的速度。这些数据表明，尽管SAH域的机械刚度可能较低，但它可以构成肌球蛋白中功能性杠杆的一部分。更一般地说，我们得出的结论是SAH域可以在水溶液中充当刚性结构延伸，并且这种结构作用在其他蛋白质中可能很重要。

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    Thomas G; Baboolal; Takeshi Sakamoto; Eva Forgacs; Howard D. White; Scott M. Jackson; Yasuharu Takagi; Rachel E. Farrow; Justin E. Molloy; Peter J. Knight; James R. Sellers; Michelle Peckham;

  • 作者单位

    Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom;

    Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, Bethesda, MD 20892 Department of Physics and Astronomy, Wayne State University, 66G West Hancock Avenue, Suite 287, Detroit, Ml 48201;

    Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA 23507;

    Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA 23507;

    Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom;

    Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, Bethesda, MD 20892;

    Division of Physical Biochemistry, Medical Research Council, National Institute for Medicai Research, The Ridgeway, London, NW7 1AA, United Kingdom;

    Division of Physical Biochemistry, Medical Research Council, National Institute for Medicai Research, The Ridgeway, London, NW7 1AA, United Kingdom;

    Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom;

    Laboratory of Molecular Physiology, National Heart, Lung and Blood Institute, Bethesda, MD 20892;

    Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    ATPase; electron microscopy; optical trap; single alpha helix;

    机译：ATP酶电子显微镜;光学陷阱单α螺旋;

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