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Biophys J, August 1999, p. 985-992, Vol. 77, No. 2

Tropomyosin Positions in Regulated Thin Filaments Revealed by Cryoelectron Microscopy

C. Xu,* R. Craig,# L. Tobacman,§ R. Horowitz,# and W. Lehman*

 *Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118 USA;  #Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655 USA; and  §Departments of Internal Medicine and Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242 USA

Past attempts to detect tropomyosin in electron micrograph images of frozen-hydrated troponin-regulated thin filaments under relaxing conditions have not been successful. This raised the possibility that tropomyosin may be disordered on filaments in the off-state, a possibility at odds with the steric blocking model of muscle regulation. By using cryoelectron microscopy and helical image reconstruction we have now resolved the location of tropomyosin in both relaxing and activating conditions. In the off-state, tropomyosin adopts a position on the outer domain of actin with a binding site virtually identical to that determined previously by negative staining, although at a radius of 3.8 nm, slightly higher than found in stained filaments. Molecular fitting to the atomic model of F-actin shows that tropomyosin is localized over sites on actin subdomain 1 required for myosin binding. Restricting access to these sites would inhibit the myosin-cross-bridge cycle, and hence contraction. Under high Ca2+ activating conditions, tropomyosin moved azimuthally, away from its blocking position to the same site on the inner domain of actin previously determined by negative staining, also at 3.8 nm radius. These results provide strong support for operation of the steric mechanism of muscle regulation under near-native solution conditions and also validate the use of negative staining in investigations of muscle thin filament structure.

Biophys J, August 1999, p. 985-992, Vol. 77, No. 2
© 1999 by the Biophysical Society   0006-3495/99/08/985/08  $2.00


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Copyright © 1999 by the Biophysical Society.