Structural changes in troponin during activation of skeletal and heart muscle determined in situ by polarised fluorescence.

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_N) 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_N 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_N 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.