Sevrieva Ivanka R, Kampourakis Thomas, Irving Malcolm
Randall Centre for Cell & Molecular Biophysics, New Hunt's House, Guy's Campus, King's College London, London, UK.
Division of Cardiovascular Medicine, College of Medicine, University of Kentucky, Lexington, KY USA.
Biophys Rev. 2024 Oct 19;16(6):753-772. doi: 10.1007/s12551-024-01245-y. eCollection 2024 Dec.
Calcium binding to troponin triggers the contraction of skeletal and heart muscle through structural changes in the thin filaments that allow myosin motors from the thick filaments to bind to actin and drive filament sliding. Here, we review studies in which those changes were determined in demembranated fibres of skeletal and heart muscle using fluorescence for in situ structure (FISS), which determines domain orientations using polarised fluorescence from bifunctional rhodamine attached to cysteine pairs in the target domain. We describe the changes in the orientations of the N-terminal lobe of troponin C (TnC) and the troponin IT arm in skeletal and cardiac muscle cells associated with contraction and compare the orientations with those determined in isolated cardiac thin filaments by cryo-electron microscopy. We show that the orientations of the IT arm determined by the two approaches are essentially the same and that this region acts as an almost rigid scaffold for regulatory changes in the more mobile regions of troponin. However, the TnC orientations determined by the two methods are clearly distinct in both low- and high-calcium conditions. We discuss the implications of these results for the role of TnC in mediating the multiple signalling pathways acting through troponin in heart muscle cells and the general advantages and limitations of FISS and cryo-EM for determining protein domain orientations in cells and multiprotein complexes.
钙与肌钙蛋白结合，通过细肌丝的结构变化触发骨骼肌和心肌的收缩，这些变化使得粗肌丝上的肌球蛋白马达能够与肌动蛋白结合并驱动肌丝滑动。在此，我们回顾了一些研究，这些研究利用原位结构荧光法（FISS）在骨骼肌和心肌的去膜纤维中确定了这些变化，该方法利用附着在目标结构域半胱氨酸对上的双功能罗丹明的偏振荧光来确定结构域的方向。我们描述了骨骼肌和心肌细胞中与收缩相关的肌钙蛋白C（TnC）N端叶和肌钙蛋白I T臂方向的变化，并将这些方向与通过冷冻电子显微镜在分离的心脏细肌丝中确定的方向进行比较。我们表明，通过两种方法确定的T臂方向基本相同，并且该区域作为肌钙蛋白更易移动区域调节变化的几乎刚性的支架。然而，在低钙和高钙条件下，两种方法确定的TnC方向明显不同。我们讨论了这些结果对于TnC在介导心肌细胞中通过肌钙蛋白起作用的多种信号通路中的作用的意义，以及FISS和冷冻电镜在确定细胞和多蛋白复合物中蛋白质结构域方向方面的一般优点和局限性。
