Abstract
In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P2〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads.
Highlights
Contraction of striated muscle is initiated by calciuminduced changes in the structure of the actin-containing thin filament
Temperature dependence of the order parameters of regulatory light chain (RLC) probes in relaxed muscle fibers Mutants of chicken skeletal RLC, with native cysteines replaced by alanine and new cysteine pairs introduced in its C-lobe at positions 95–103 (E helix), 131–138 (G helix), 151–158 (H helix), or 122–134, were labeled by cross-linking cysteine pairs with bifunctional sulforhodamine (BSR) (Fig. 1; Fig. S1; Movie S1)
Each BSR-RLC was exchanged into permeabilized fibers from rabbit psoas muscle using a mild exchange protocol to better preserve the native structure and function of the thick filaments. This protocol led to replacement of ~26% of the native RLC by BSR-RLC, which was uniformly distributed along the A-band of the sarcomere and across the fiber width (Fig. S3)
Summary
Contraction of striated muscle is initiated by calciuminduced changes in the structure of the actin-containing thin filament. Calcium ions released in the cytoplasm following electrical stimulation bind to troponin, triggering the movement of tropomyosin around the filament and uncovering the actin binding sites for the motor or head domains of myosin emerging from the overlapping thick filament [1,2]. Subsequent electron microscopy (EM) studies of isolated thick filaments from invertebrate skeletal muscle identified an asymmetric arrangement of the two heads of each myosin molecule folded back against the myosin tails in the filament backbone [7,8] This conformation, called the J motif or interacting heads motif (IHM), inhibits the ATPase activity of intrinsically regulated myosin filaments (i.e., in muscles lacking the troponin/tropomyosin system) and is associated with the relaxed or OFF state of smooth muscle myosin [9]. The interaction between this potential regulatory structural switch in the thick filament and the well-known regulatory switch in the thin filaments remains obscure
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