In muscle each myosin head contains a regulatory light chain (LC2) that is wrapped around the head/rod junction, and an alkali light chain that is distal to LC2 (ref. 1). The role of these light chains in vertebrate skeletal muscle myosin has remained obscure. Here we prepare heavy chains that are free of both light chains in order to determine by a motility assay whether the light chains are necessary for movement. We find that removal of light chains from myosin reduces the velocity of actin filaments from 8.8 microns s-1 to 0.8 microns s-1 without significantly decreasing the ATPase activity. Reconstitution of myosin with LC2 or alkali light chain increases filament velocity to intermediate rates, and readdition of both classes of light chains fully restores the original sliding velocity. We conclude that even though the light chains are not essential for enzymatic activity, light-chain/heavy-chain interactions play an important part in the conversion of chemical energy into movement.

Several classes of the myosin superfamily are distinguished by their ''double-headed'' structure, where each head is a molecular motor capable of hydrolyzing ATP and interacting with actin to generate force and motion. The functional significance of this dimeric structure, however, has eluded investigators since its discovery in the late 1960s. Using an optical-trap transducer, we have measured the unitary displacement and force produced by double-headed and single-headed smooth-and skeletal-muscle myosins. Singleheaded myosin produces approximately half the displacement and force (Ϸ6 nm; 0.7 pN) of double-headed myosin (Ϸ10 nm; 1.4 pN) during a unitary interaction with actin. These data suggest that muscle myosins require both heads to generate maximal force and motion.Muscle shortening is driven by a cyclical interaction between the contractile proteins myosin and actin. During this cycle the dimeric molecular motor, myosin, transduces chemical energy into mechanical work. Although the contractile system has been investigated extensively, the details of this process and the functional significance of myosin's ''double-headed'' structure have remained unclear since the dimeric structure was discovered in the late 1960s (ref. 1; see refs. 2 and 3 for review). Based on the available biochemical and mechanical data (4-8), early models assumed that the two heads of myosin act independently (9, 10). Enzymatic assays clearly showed that the actin-activated ATPase activity per head was the same for proteolytically prepared myosin-head fragments (subfragment-1, S1) and for single-and double-headed heavy meromyosin (HMM; ref. 4). An even more compelling example of independent head action was observed when reconstituted actomyosin threads were used. In these experiments, ensembles of single-headed myosin generated half the force of native myosin (5). More recently, by using an optical trap, single S1 heads were shown to move actin and produce force, suggesting that a dimeric structure is not necessary for the production of work in vitro (11). Additional single-molecule experiments have shown that in synthetic thick filaments, single-and double-headed myosins produce similar unitary step displacements (12).However, evidence suggesting a functional difference between single-and double-headed myosin species came from measurements of the binding of S1 and HMM to actin in the absence of nucleotide (6, 7). These data showed a smaller association constant for HMM than would be expected if the heads bound independently, implying that the two heads are sterically constrained from simultaneously interacting with actin. More recent kinetic studies comparing S1 and HMM suggest that the two heads may be coordinated in their transition from the weakly bound to the strongly bound state (13).Because the functional significance of myosin's two-headed structure remains ambiguous, we have performed a singlemolecule mechanical comparison between single-and doubleheaded smooth-and skeletal-muscle myosins. Using an optical-trap assay ...

Myosin, a molecular motor that is responsible for muscle contraction, is composed of two heavy chains each with two light chains. The crystal structure of subfragment 1 indicates that both the regulatory light chains (RLCs) and the essential light chains (ELCs) stabilize an extended a-helical segment of the heavy chain. It has recently been shown in a motility assay that removal of either light chain markedly reduces actin frament sliding velocity without a significant loss in actin-activated ATPase activity. Here we demonstrate by single actin frament force measurements that RLC removal has little effect on isometric force, whereas ELC removal reduces isometric force by over 50%. These data are interpreted with a simple mechanical model where subfragment 1 behaves as a torque motor whose lever arm length is sensitive to light-chain removal. Although the effect of removing RLCs fits within the confines of this model, altered crossbridge kinetics, as reflected in a reduced unloaded duty cycle, probably contributes to the reduced velocity and force production of ELC-deficient myosins.

The myosin head (S1) consists of a wide, globular region that contains the actin- and nucleotide-binding sites and an alpha-helical, extended region that is stabilized by the presence of two classes of light chains. The essential light chain abuts the globular domain, whereas the regulatory light chain lies near the head-rod junction of myosin. Removal of the essential light chain by a mild denaturant exposes the underlying heavy chain to proteolysis by chymotrypsin. The cleaved fragment, or "motor domain" (MD), migrates as a single band on SDS-polyacrylamide gel electrophoresis, with a slightly greater mobility than S1 prepared by papain or chymotrypsin. Three-dimensional image analysis of actin filaments decorated with MD reveals a structure similar to S1, but shorter by an amount consistent with the absence of a light chain-binding domain. The actin-activated MgATPase activity of MD is similar to that of S1 in Vmax and Km. But the ability of MD to move actin filaments in a motility assay is considerably reduced relative to S1. We conclude that the globular, active site region of the myosin head is a stable, independently folded domain with intrinsic motor activity, but the coupling efficiency between ATP hydrolysis and movement declines markedly as the light chain binding region is truncated.