Coordination of the two heads of myosin during muscle contraction

Abstract

We have used luminescence resonance energy transfer between regulatory light chains (RLC) to detect structural changes within the dimeric myosin molecule in contracting muscle fibers. Fully functional scallop muscle fibers were prepared such that each myosin molecule contained a terbium-labeled (luminescent donor) RLC on one head and a rhodamine-labeled (acceptor) RLC on the other. Time-resolved luminescence energy transfer between the two heads increased upon the transition from relaxation (ATP) to contraction (ATP plus Ca) and increased further in rigor (no ATP). Combined with experiments on mutant RLCs labeled specifically at other sites, these results support a model in which the forcegenerating weak-to-strong transition causes one myosin LC domain to tilt through a 30°angle toward the other, thus acting as a coordinated lever arm.

Site-Directed Spectroscopic Probes of Actomyosin Structural Dynamics

“…LRET, using Tb 3+ chelates bound to single-Cys RLC mutants, was used to measure distances between LCDs on the two heads of myosin in scallop muscle fibers (42) and in skeletal muscle HMM (15). In the muscle fiber study, the head-head separation was greatest in relaxation, least in rigor, and intermediate in contraction.…”

“…In the muscle fiber study, the head-head separation was greatest in relaxation, least in rigor, and intermediate in contraction. Using four different labeling sites on RLC, it was found that the relative tilt of the two LCDs decreases by 30° in the force-generating weak-to-strong transition (42). The HMM study concluded that the two heads of myosin bind to actin in rigor in a strained configuration that is distinct from that of single-headed binding (15).…”

“…In spectroscopy, there have also been significant technological advances, such as time-resolved luminescence resonance energy transfer (LRET) (42, 83, 84), single-molecule fluorescence (92), total internal reflection fluorescence (TIRF) detection (26), time-domain electron paramagnetic resonance (EPR) (36), multifrequency EPR (50), and electrostatic potential measured by EPR (71b). These breakthroughs have allowed spectroscopy to test previously untestable molecular models of muscle structure and function.…”

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).…”

Actomyosin-ADP States, Interhead Cooperativity, and the Force-Velocity Relation of Skeletal Muscle

“…One of these states (denoted AM*ADP) is separated from the actual ADP-release step by a strain-dependent isomerization (5,6,16,21). Since a similar state has been implicated as being critical for the processivity of nonmuscular myosins (e.g., myosin V (22)), it is of interest to consider the possibility that processivity may also occur with myosin II (6,13,(23)(24)(25)(26), and that the AM*ADP state may play a role in this connection.…”