Biophysical Society Thematic Meeting | Canterbury 2023

Towards a More Perfect Union: Multi-Scale Models of Muscle and Their Experimental Validation

Monday Speaker Abstracts

COMPARISON OF THE CARDIAC TROPONIN CONFORMATION DETERMINED BY POLARIZED FLUORESCENCE AND CRYO-EM Ivanka R Sevrieva 1 ; Andrea C Knowles 1 ; Thomas Kampourakis 1 ; Yin-Biao Sun 1 ; Malcolm Irving 1 ; 1 King's College London, Randall Centre for Cell and Molecular Biophysics, London, United Kingdom Recent advances in cryo-electron microscopy (cryo-EM) led to 7Å-resolution structural models of the cardiac troponin core domain in isolated thin filaments at different calcium concentrations (Yamada et al. Nat Comm 2020; Risi et al. PNAS 2021) and a better understanding of the changes in thin filament structure associated with calcium binding. However, these structural models may not fully reproduce native thin filament structure in the intact lattice of thin and thick filaments in a muscle cell. Polarized fluorescence from sets of bifunctional probes attached to specific helices in a target protein provides a complementary approach that can be applied in native intracellular conditions, and we previously used that approach to determine the in situ orientations of the N- and C-terminal lobes of TnC during relaxation and active contraction of cardiac muscle cells (Sevrieva et al. JMCC 2014). The C-lobe of cTnC is part of a well-defined domain of troponin called the ‘IT arm’, and our results showed that the long axis of the IT arm is at about 65° to the filament axis independently of calcium concentration [Ca 2+ ]. The orientation of the IT arm in the cryo-EM models at high [Ca 2+ ] is almost identical to that determined in situ by polarised fluorescence, but that at low [Ca 2+ ] is slightly more parallel to the filament axis. However, in contrast with the cryo-EM models, the in situ method shows that the N-lobe of cTnC does not take up a single conformation on the thin filament at either low or high [Ca 2+ ], suggesting that it is in dynamic equilibrium between multiple conformations. In this respect, the cardiac thin filament is distinct from that of skeletal muscle, in which the N-lobe of sTnC does take up a well-defined conformation at both low and high [Ca 2+ ].

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