The tropomyosin domain is flexible and disordered in reconstituted thin filaments.

We have used EPR spectroscopy to study the rotational motion and orientation of tropomyosin labeled with maleimide spin-label, in skeletal muscle fibers. Fibers depleted of intrinsic myosin, troponin, and tropomyosin were reconstituted with labeled tropomyosin. The 3-7 ns mobility of the labeled domains was only slightly (2-fold) inhibited by reconstitution into fibers. No motional changes were observed on addition of troponin, irrespective of the presence of Ca2+; however, the binding of extrinsic myosin heads increased the rate of domain motion to that observed in solution. Orientational studies demonstrate a broad angular distribution of the labeled domain of tropomyosin, with respect to the fiber axis. Troponin reduces the orientational disorder, while the binding of Ca2+ to troponin partially reverses this ordering effect. Myosin S1 has no effect on the orientational distribution of tropomyosin. Overall, the observed changes are very small, implying a loose association of the probed domain of tropomyosin with the thin filament.