A large and distinct rotation of the myosin light chain domain occurs upon muscle contraction

Abstract

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...

Invertebrate muscles: Thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

“…Relatively early work showed that mollusc myosin has the typical two headed structure of other myosins (Elliott et al, 1976), that in the rest state ADP binds to both heads (Marston and Lehman, 1974;Shibata-Sekiya, 1982), that ATP binding alters tryptophan fluorescence (Kondo et al, 1979;, that a tryptophan present in the skeletal ATP binding site is replaced in scallop by an arginine (Kondo et al, 1979;Kerwin and Yount, 1992), and identified portions of the heavy chain and myosin head required for ATPase activity (Szentkiralyi, 1987) and actin binding (Castellani et al, 1987). Electron paramagnetic resonance showed that force generation (but not ATP hydrolysis) is associated with light chain rotation (Baker et al, 1998;Roopnarine et al, 1998;Cooke, 1998;BrustMascher et al, 1999;LaConte et al, 2003), and measurement of luminescence resonance energy transfer between the regulatory light chains showed that the light chains of both heads rotate together to act as a coordinated lever arm (Lidke and Thomas, 2002).…”

Tryptophan 512 Is Sensitive to Conformational Changes in the Rigid Relay Loop of Smooth Muscle Myosin during the MgATPase Cycle

“…16). Electron paramagnetic resonance studies, utilizing a probe on the regulatory light chain have shown a 30°rotation of this region (17), suggesting that the lever arm region rotates relative to the catalytic domain during muscle contraction. In addition, fluorescence studies utilizing a fluorescent probe at the regulatory light chain demonstrated rotation of the lever arm in contracting muscle fibers (15).…”

Conformationally Trapping the Actin-binding Cleft of Myosin with a Bifunctional Spin Label

“…Force generation by myosin is associated with the transition from a state of weak actin binding, characterized by dynamic disorder, to an ordered, strong actin-binding state (1). The weak-to-strong transition is associated with large-scale rotation of the light-chain domain (LCD) 4 relative to the catalytic domain (CD) (1)(2)(3)(4)(5). The rotation of the LCD, acting as a lever arm, displaces the associated actin filament, generating movement.…”