Abstract
The sarcomere, the minimal mechanical unit of muscle, is composed of myosins, which self-assemble into thick filaments that interact with actin-based thin filaments in a highly-structured lattice. This complex imposes a geometric restriction on myosin in force generation. However, how single myosins generate force within the restriction remains elusive and conventional synthetic filaments do not recapitulate the symmetric bipolar filaments in sarcomeres. Here we engineered thick filaments using DNA origami that incorporate human muscle myosin to directly visualize the motion of the heads during force generation in a restricted space. We found that when the head diffuses, it weakly interacts with actin filaments and then strongly binds preferentially to the forward region as a Brownian ratchet. Upon strong binding, the two-step lever-arm swing dominantly halts at the first step and occasionally reverses direction. Our results illustrate the usefulness of our DNA origami-based assay system to dissect the mechanistic details of motor proteins.
Highlights
The sarcomere, the minimal mechanical unit of muscle, is composed of myosins, which self-assemble into thick filaments that interact with actin-based thin filaments in a highlystructured lattice
One reason is that muscle myosin is nonprocessive when isolated and works as a group with other muscle myosins in the highly-structured sarcomere, the minimal mechanical unit of muscle, which is composed of rigorously arranged myosin II-based thick filaments (Fig. 1a) and actinbased thin filaments
When the thick filament formed a rigor complex with an actin filament, the myosin heads strongly bound with actin at ~36 nm intervals (Fig. 1f), resulting in an anisotropy of linkers that was fixed (39 ± 9° SD) (Fig. 1e) and slightly dependent on position (Fig. 1g)
Summary
The sarcomere, the minimal mechanical unit of muscle, is composed of myosins, which self-assemble into thick filaments that interact with actin-based thin filaments in a highlystructured lattice This complex imposes a geometric restriction on myosin in force generation. One reason is that muscle myosin is nonprocessive when isolated and works as a group with other muscle myosins in the highly-structured sarcomere, the minimal mechanical unit of muscle, which is composed of rigorously arranged myosin II-based thick filaments (Fig. 1a) and actinbased thin filaments This design and the non-processivity make it difficult to directly observe the internal dynamics of the rapid and minute displacements of myosin II at the single molecular level. To observe the motion of the heads in thick filaments in vitro, synthesis of the thick filaments is necessary; synthetic filaments composed of purified myosin II self-assembled in a conventional manner[26] do not mimic the symmetric bipolar filaments observed in sarcomeres and instead assemble randomly[27]
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