Although widely accepted, the steric-blocking model of vertebrate skeletal muscle regulation has not been confirmed. Previous attempts to directly visualize tropomyosin in relaxed skeletal muscle and demonstrate that it interferes with the crossbridge-thin filament contractile cycle were unsuccessful. In the work reported here, tropomyosin was resolved in electron micrographs of native thin filaments isolated from relaxed vertebrate striated muscle. Three-dimensional helical reconstructions of these filaments showed continuous narrow strands of density, representing tropomyosin, which followed the outer domains of successive actin monomers. The results obtained from fitting the atomic model of filamentous actin to these reconstructions illustrate, and are consistent with, the mechanism of steric-blocking, since tropomyosin was found to be positioned on the actin surface of thin filaments over clusters of identifiable amino acids required for myosin crossbridge docking.f2
Caldesmon inhibits actomyosin ATPase and filament sliding in vitro, and therefore may play a role in modulating smooth and non-muscle motile activities.A bacterially expressed caldesmon fragment, 606C, which consists of the C-terminal 150 amino acids of the intact molecule, possesses the same inhibitory properties as full-length caldesmon and was used in our structural studies to examine caldesmon function.Three-dimensional image reconstruction was carried out from electron micrographs of negatively stained, reconstituted thin filaments consisting of actin and smooth muscle tropomyosin both with and without added 606C.Helically arranged actin monomers and tropomyosin strands were observed in both cases.In the absence of 606C, tropomyosin adopted a position on the inner edge of the outer domain of actin monomers, with an apparent connection to sub-domain 1 of actin.In 606C-containing filaments that inhibited acto-HMM ATPase activity, tropomyosin was found in a different position, in association with the inner domain of actin, away from the majority of strong myosin binding sites.The effect of caldesmon on tropomyosin position therefore differs from that of troponin on skeletal muscle filaments, implying that caldesmon and troponin act by different structural mechanisms.
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