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Domain dynamics of the chicken gizzard smooth muscle myosin catalytic domain (heavy chain Cys-717) and regulatory domain (regulatory light chain Cys-108) were determined in the absence of nucleotides using saturation-transfer electron paramagnetic resonance. In unphosphorylated synthetic filaments, the effective rotational correlation times, tau(r), were 24 +/- 6 micros and 441 +/- 79 micros for the catalytic and regulatory domains, respectively. The corresponding amplitudes of motion were 42 +/- 4 degrees and 24 +/- 9 degrees as determined from steady-state phosphorescence anisotropy. These results suggest that the two domains have independent mobility due to a hinge between the two domains. Although a similar hinge was observed for skeletal myosin (Adhikari and Fajer (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 9643-9647. Brown et al. (2001) Biochemistry 40, 8283-8291), the latter displayed higher regulatory domain mobility, tau(r)= 40 +/- 3 micros, suggesting a smooth muscle specific mechanism of constraining regulatory domain dynamics. In the myosin monomers the correlation times for both domains were the same (approximately 4 micros) for both smooth and skeletal myosin, suggesting that the motional difference between the two isoforms in the filaments was not due to intrinsic variation of hinge stiffness. Heavy chain/regulatory light chain chimeras of smooth and skeletal myosin pinpointed the origin of the restriction to the heavy chain and established correlation between the regulatory domain dynamics with the ability of myosin to switch off but not to switch on the ATPase and the actin sliding velocity. Phosphorylation of smooth muscle myosin filaments caused a small increase in the amplitude of motion of the regulatory domain (from 24 +/- 4 degrees to 36 +/- 7 degrees ) but did not significantly affect the rotational correlation time of the regulatory domain (441 to 408 micros) or the catalytic domain (24 to 17 micros). These data are not consistent with a stable interaction between the two catalytic domains in unphosphorylated smooth muscle myosin filaments in the absence of nucleotides.

EPR of spin labeled TnC at Cys98 was used to explore the possible structural coupling between TnC in the thin filament and myosin trapped in the intermediate states of ATPase cycle. Weakly attached myosin heads (trapped by low ionic strength, low temperature and ATP) did not induce structural changes in TnC as compared to relaxed muscle, as spin labeled TnC displayed the same narrow orientational distribution (Li, H.-C., and Fajer, P. G. (1994) Biochemistry 33, 14324). Ca 2+ -binding alone resulted in disordering of the labeled domain of TnC. Additional conformational changes of TnC occurred upon the attachment of strongly bound, prepower stroke myosin heads (trapped by AlF4 - ). These changes were not present in ghost fibers which myosin had been removed, excluding direct effects of AlF4 - on the orientation of TnC in muscle fibers. The postpower stroke heads (rigor‚ADP/Ca 2+ and rigor/Ca 2+ ) induced further changes in the orientational distribution of labeled domain of TnC irrespective of the degree of cooperativity in thin filaments. We thus conclude that troponin C in thin filaments detects structural changes in myosin during force generation, implying that there is a structural coupling between actomyosin and TnC. Muscle contracts when the thick and thin filaments slide past each other to relieve the strain generated by the interaction of the globular head of myosin with actin. In vertebrate skeletal muscle, the thin filament proteins sactin, tropomyosin (Tm), 1 and troponin (Tn)sregulate contraction.

The relative movement of the catalytic and regulatory domains of the myosin head (S1) is likely to be the force generating conformational change in the energy transduction of muscle (Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261 ,5 8-65). To test this model we have measured, using frequency-modulated FRET, three distances between the catalytic domain and regulatory domains and within the regulatory domain of myosin. The donor/acceptor pairs included MHC cys707 and ELC cys177; ELC cys177 and RLC cys154; and ELC cys177 and gizzard RLC cys108. The IAEDANS (donor) or acceptor (DABMI or IAF) labeled light chains (ELC and RLC) were exchanged into monomeric myosin and the distances were measured in the putative prepower stroke states (in the presence of MgATP or ADP/AlF4 - ) and the postpower stroke states (ADP and the absence of nucleotides). For each of the three distances, the donor/acceptor pairs were reversed to minimize uncertainty in the distance measured, arising from probe orientational factors. The distances obtained from FRET were in close agreement with the distances in the crystal structure. Importantly, none of the measured distances varied by more than 2 A, putting a strong constraint on the extent of conformational changes within S1. The maximum axial movement of the distal part of myosin head was modeled using FRET distance changes within the myosin head reported here and previously. These models revealed an upper bound of 85 A for a swing of the regulatory domain with respect to the catalytic domain during the power stroke. Additionally, an upper bound of 22 A could be contributed to the power stroke by a reorientation of RLC with respect to the ELC during the power stroke.

We have used electron paramagnetic resonance to describe the orientational changes of troponin C (TnC) accompanying muscle activation by Ca2+. Rabbit skeletal TnC was labeled with maleimide spin label (MSL) at Cys-98 and reconstituted into an oriented skinned muscle fiber. About 70% of endogenous troponin C was replaced with labeled TnC, with a concomitant recovery of 80-90% of muscle tension. The nanosecond domain mobility present in solution, as determined from the EPR spectra of randomized samples, is fully inhibited in the reconstituted fibers. The orientational analysis revealed a bimodal orientational distribution of TnC in the absence Ca2+ and attached myosin heads. One of the components is well-ordered with its probe axis inclined at 22 degrees to the fiber axis, while the other is more disordered and inclined at 58 degrees. Ca2+ and/or cross-bridge binding significantly disordered the labeled domain and increased the average probe axis angle by 20-30 degrees away from the fiber axis. The order for the magnitude of angular tilt was Ca2+myosin cross-bridgesCa2+ and cross-bridges. Thus, TnC exists in many different orientational conformations depending on which ligand is bound. We believe that these conformations reflect different activation mechanisms by Ca2+ and cross-bridge binding.