Addition of Ca2+ and myosin heads to muscle thin filaments caused sequential 25-degree and 10-degree shifts in tropomyosin position, exposing myosin-binding sites and supporting steric-blocking models.
This study provides direct structural evidence that full activation of muscle thin filaments requires both Ca2+ and myosin head binding acting in sequence to reverse steric blocking.
The structural basis of thin filament-linked regulation of muscle contraction is not yet understood. Here we have used electron microscopy and three-dimensional image reconstruction to observe the effects of Ca2+ and myosin head binding on thin filament structure, especially on the position of tropomyosin. Thin filaments isolated in EGTA were treated with Ca2+ or myosin heads (S-1) and negatively stained. Tropomyosin strands were directly visualized in electron micrographs, and distinct EGTA, Ca2+ and S-1-dependent positions were apparent in reconstructions. By fitting reconstructions to the atomic model of F-actin, clusters of amino acids on actin lying beneath tropomyosin were defined under each set of conditions. In the presence of Ca2+, tropomyosin moved 25 degrees away from its low Ca2+ position, exposing most, but not all, of the previously blocked myosin-binding sites. Saturation of filaments with myosin heads produced a further 10 degrees shift in tropomyosin position, thereby exposing the entire myosin-binding site. Our results thus suggest that full switching-on of thin filaments by reversal of steric-blocking requires both Ca2+ and the binding of myosin heads, acting in sequence. By using filaments which were partially decorated with heads, tropomyosin movement was shown to be cooperative, and the size of the actin-tropomyosin cooperative unit was estimated directly. Our results provide direct structural support for previous models of thin filament activation based on kinetics of actin-myosin interaction.
Vibert et al. (1997) studied Muscle contraction (basic science). Ca2+ and myosin heads (S-1) vs. EGTA (low Ca2+) was evaluated on Tropomyosin position shift. Addition of Ca2+ and myosin heads to muscle thin filaments caused sequential 25-degree and 10-degree shifts in tropomyosin position, exposing myosin-binding sites and supporting steric-blocking models.
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