THE TROPOMYOSIN DOMAIN IS FLEXIBLE AND DISORDERED IN RECONSTITUTED THIN-FILAMENTS

We have used EPR spectroscopy to study the rotational motion and orien tation of tropomyosin labeled with maleimide spin-label, in skeletal m uscle fibers. Fibers depleted of intrinsic myosin, troponin, and tropo myosin were reconstituted with labeled tropomyosin. The 3-7 ns mobilit y of the labeled domains was only slightly (2-fold) inhibited by recon stitution into fibers. No motional changes were observed on addition o f 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 angul ar 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 tropomyos in. Overall, the observed changes are very small, implying a loose ass ociation of the probed domain of tropomyosin with the thin filament.