Abstract
Rabbit skeletal myosin forms stable filaments under physiological conditions, and only a small amount stays as a monomer in equilibrium with filaments. The myosin monomers were observed in two conformational states, as extended and folded forms upon electron microscopy and gel filtration high performance liquid chromatography. The fraction of monomers in the folded conformation increased with a decrease in the concentration of NaCl below 0.2 M, and the conformational state was affected neither by the presence of ATP nor by the phosphorylation of regulatory light chain. In most of the folded monomers, the tail bent back toward the heads at one region, 45 nm apart from the head-tail junction, and the remaining tail portion containing the C-terminal tip appeared to interact with the head-tail junction. Only a small percentage of the folded monomers was in a more compact conformation close to the 10 S conformation of vertebrate smooth muscle and non-muscle myosins. The folded monomers, however, may not trap the products of ATP hydrolysis as assessed by single turnover experiments. The percentage of monomers in the 10 S-like conformation was increased by the exchange of a regulatory light chain with the smooth muscle light chain, indicating the participation of head-tail junction, including the regulatory light chain in the formation of folded conformation. The folded conformation may be common to various myosin IIs, suggestive of common roles for the folded monomers.
Highlights
Myosin II consists of two globular heads connected to one long ␣-helical coiled coil tail
Under physiological ionic conditions in the presence of ATP, vertebrate smooth muscle and non-muscle myosins are folded into the 10 S folded conformation in which the tail bends at two regions [1,2,3] and are unfolded to the 6 S extended conformation by the phosphorylation of regulatory light chain (RLC) [2,3,4,5,6]
Proteins—Skeletal muscle myosin was prepared from rabbit back muscle as described by Perry [12], and smooth muscle myosin was from porcine aorta as described previously [13, 14]
Summary
Proteins—Skeletal muscle myosin was prepared from rabbit back muscle as described by Perry [12], and smooth muscle myosin was from porcine aorta as described previously [13, 14]. Rabbit skeletal myosin in which RLC was exchanged to aorta RLC was prepared by incubating myosin at 37 °C with an 8-fold molar excess of aorta RLC as described previously [19]. Electron Microscopy—Myosin (0.2 mg/ml) in 0.12– 0.5 M NaCl, 1 mM MgCl2, 0.1 mM EGTA, and 10 mM imidazole (pH 7.0) was ultracentrifuged at 150,000 ϫ g for 10 min at 4 °C in the presence or absence of 1 mM ATP. Gel Filtration HPLC—Myosin (0.2 mg/ml) in a solution containing 0.08 – 0.4 M NaCl, 1 mM MgCl2, 0.1 mM EGTA, and 10 mM imidazole (pH 7.0) in the presence or absence of 0.1 mM ATP was passed through a 0.5-m membrane filter (Millipore) to remove filaments. The amount of phosphate bound to myosin was estimated by subtracting the amount of phosphate in the filtrate from that in the reaction mixture
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