Investigation on the functional significance of myosin subfragment-2

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We investigated the functional significance of myosin subfragment-2 (S-2) connecting the head and rod domains of the molecule. The head contains actin-binding site and ATP-hydrolysis pocket and is considered to be pivotal in mechanochemical function of the molecule. Myosin molecules bind together at the rod domains to form a thick filament in muscle cells. While several studies suggested significant role of the S-2 region, located in the head-rod junction, in force generation and transduction, the functional role of the S-2 region is not clearly elucidated yet. We used chicken smooth muscle myosin as a model and expressed heavy meromyosin (HMM) with baculoviral vector in cultured insect cells. We purified wild-type and two mutants that had deletions of 20(Δ20) and 49 (Δ49) amino-acid residues in the S-2 region. The Δ49 mutant had longer deletion than Δ20, but conserved the seven amino-acid residues periodicity (heptad repeat) in this region. We analyzed the molecular motor function usi … More ng the in vitro motility assay and laser trap. We measured the sliding velocity of fluorescently labeled actin filaments (VEL) over myosin layer fixed on a coverslip. We also measured the velocity in the presence of α-actinin which imposed mechanical load upon the sliding filaments. The α-actinin concentration (ACT), at which the load was balanced with the generated force of interacting myosin molecules and the sliding movement stopped, was measured as an index of the average force of myosin molecules. In laser trap experiments, the unitary displacement (Duni),the unitary force (Funi) and the duration of a single event of the actin-myosin interaction (Ton) were determined. Both VEL and ACT were decreased in the mutants (Δ20 and Δ 49) than in the wild-type. Especially, the decreases compared to the wild-type were greater in Δ20 than Δ49 albeit the shorter deletion length (VEL,-38% vs. -23%;ACT,-67% vs. -38%,respectively). Neither Duni nor Funi was different between the mutants and the wild-type or among the mutants. Notably, Ton was 26% longer in Δ20 as compared to the wild-type. In conclusion, the S-2 region may play significant roles in motor function of the molecule, but does not affect step size or unitary force of a single molecule. It is also suggested that the 7 amino-acid residue periodicity may be important for the motor function. Less