For more than 30 years, the fundamental goal in molecular motility has been to resolve force-generating motor protein structural changes. Although low-resolution structural studies have provided evidence for force-generating myosin rotations upon muscle activation, these studies did not resolve structural states of myosin in contracting muscle. Using electron paramagnetic resonance, we observed two distinct orientations of a spin label attached specifically to a single site on the light chain domain of myosin in relaxed scallop muscle fibers. The two probe orientations, separated by a 36°؎ 5°axial rotation, did not change upon muscle activation, but the distribution between them changed substantially, indicating that a fraction (17% ؎ 2%) of myosin heads undergoes a large (at least 30°) axial rotation of the myosin light chain domain upon force generation and muscle contraction. The resulting model helps explain why this observation has remained so elusive and provides insight into the mechanisms by which motor protein structural transitions drive molecular motility.Muscle contraction results from the relative sliding between actin and myosin filaments, and most models suggest that filament sliding is driven by a large structural change of actin-bound myosin heads, usually depicted as an axial rotation on the order of 45°(1-3). However, resolving distinct myosin structural states in muscle has been difficult, because active muscle contains a heterogeneous population of myosin heads, each independently cycling through different structural states. Changes in x-ray diffraction (4), birefringence (5), electron microscopy (6), and fluorescence (7) upon muscle contraction are consistent with myosin head motions, but evidence for distinct myosin orientational states has not been provided by these techniques, suggesting that either distinct orientations do not exist or these techniques have insufficient orientational resolution to detect them.Electron paramagnetic resonance (EPR) has the orientational resolution needed to detect multiple orientations of nitroxide spin labels, because each spin label orientation corresponds to a unique splitting between the three narrow spectral lines. Therefore, by covalently attaching a spin label to a specific site on myosin in muscle and orienting the muscle fiber in the magnetic field, myosin orientations with respect to the fiber axis can be determined. Multiple orientations of myosin in muscle, as well as the degree of disorder about those orientations, can be determined by resolving the spectrum of an oriented fiber into a sum of spectra with different splittings (8).Previous EPR studies revealed two structural states of the spin-labeled catalytic domain, the globular region of myosin that regulates actin-myosin crossbridges through ATP binding and hydrolysis. These structural states are characterized by a single orientation in the strong-binding biochemical states (high actin affinity) and microsecond dynamic disorder in the weak-binding biochemical states (low actin aff...

We have used time-resolved phosphorescence anisotropy (TPA) to study the rotational dynamics of chicken gizzard regulatory light chain (RLC) bound to scallop adductor muscle myofibrils in key physiological states. Native RLC from scallop myofibrils was extracted and replaced completely with gizzard RLC labeled specifically at Cys 108 with erythrosin iodoacetamide (ErIA). The calcium sensitivity of the ATPase activity of the labeled myofibril preparation was quite similar to that of the native sample, indicating that the ErIA-labeled RLC is functionally bound to the myosin head. In rigor (in the absence of ATP, when all the myosin heads are rigidly bound to the thin filament), a slight decay was observed in the first few microseconds, followed by no change in the anisotropy. This indicates small-amplitude restricted motions of the RLC or the entire LC domain of myosin. Addition of calcium to rigor restricts these motions further. Relaxation with ATP (no Ca) causes a large decay in the anisotropy, indicating large-amplitude rotational motion with correlation times of 5-50 micros. Further addition of calcium, to induce contraction, resulted in a decrease in the rate and amplitude of anisotropy decay. In particular, there is clear evidence for a slow rotational motion with a correlation time of approximately 300 micros, which is not present either in rigor or relaxation. This indicates rotational motion that specifically correlates with force generation. The changes in the rotational dynamics of the light-chain domain in rigor, relaxation, and contraction support earlier work based on probes of the catalytic domain that muscle contraction is accompanied by a disorder-to-order transition of the myosin head. However, the motions of the LC domain are different from those of the catalytic domain, which indicates rotation of the two domains relative to each other.