• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Structural kinetics of myosin by transient time-resolved FRET
【24h】

Structural kinetics of myosin by transient time-resolved FRET

机译：瞬态时间分辨FRET对肌球蛋白的结构动力学

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

For many proteins, especially for molecular motors and other enzymes, the functional mechanisms remain unsolved due to a gap between static structural data and kinetics. We have filled this gap by detecting structure and kinetics simultaneously. This structural kinetics experiment is made possible by a new technique, (TR)~2FRET (transient time-resolved FRET), which resolves protein structural states on the submillisecond timescale during the transient phase of a biochemical reaction. (TR)~2FRET is accomplished with a fluorescence instrument that uses a pulsed laser and direct waveform recording to acquire an accurate subnanosecond time-resolved fluorescence decay every 0.1 ms after stopped flow. To apply this method to myosin, we labeled the force-generating region site specifically with two probes, mixed rapidly with ATP to initiate the recovery stroke, and measured the interprobe distance by (TR)~2FRET with high resolution in both space and time. We found that the relay helix bends during the recovery stroke, most of which occurs before ATP is hydrolyzed, and two structural states (relay helix straight and bent) are resolved in each nucleotide-bound biochemical state. Thus the structural transition of the force-generating region of myosin is only loosely coupled to the ATPase reaction, with conformational selection driving the motor mechanism.
机译：对于许多蛋白质，特别是对于分子马达和其他酶而言，由于静态结构数据和动力学之间存在差距，因此功能机制仍未解决。通过同时检测结构和动力学，我们填补了这一空白。通过新技术（TR）〜2FRET（瞬态时间分辨FRET）使这种结构动力学实验成为可能，该技术可在生化反应瞬态阶段的亚毫秒级尺度上解析蛋白质结构状态。 （TR）〜2FRET使用荧光仪器完成，该仪器使用脉冲激光并直接记录波形，以在停止流动后每隔0.1 ms获取一次精确的亚纳秒时间分辨的荧光衰减。为了将该方法应用于肌球蛋白，我们用两个探针专门标记了力产生区域的位点，与ATP迅速混合以启动恢复冲程，并通过（TR）〜2FRET在空间和时间上以高分辨率测量了探针间距离。我们发现中继螺旋在恢复冲程中弯曲，其中大多数发生在ATP水解之前，并且两个结构状态（继电器螺旋直和弯曲）在每个核苷酸结合的生化状态中被解析。因此，肌球蛋白的力产生区的结构转变仅松散地与ATPase反应偶联，而构象选择驱动马达机制。

著录项

  • 来源
  • 作者

    Yuri E. Nesmelov; Roman V. Agafonov; Igor V. Negrashov; Sarah E. Blakely; Margaret A. Titus; David D. Thomas;

  • 作者单位

    Department of Physics and Optical Science, University of North Carolina,Charlotte, NC 28223;

    Department of Biochemistry, Molecular Biology and Biophysics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN 55455;

    Department of Biochemistry, Molecular Biology and Biophysics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN 55455;

    Department of Biochemistry, Molecular Biology and Biophysics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN 55455;

    Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN 55455;

    Department of Biochemistry, Molecular Biology and Biophysics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN 55455;

  • 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

    disorder-to-order transition; myosin ii; dictyostelium;

    机译：无序转变;肌球蛋白Ⅱ;双歧杆菌;

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号