Contractile Proteins



Skeletal muscle contains 70 -100 mg of myosin per gram of fresh muscle weight; this corresponds to 40-50% of the total muscle proteins. Myosin is a globulin, soluble at high salt concentration, e.g. 0.6 M KCl, and insoluble at low salt concentration, e.g. 0.03 M KCl. Thus, myosin can be extracted from the muscle with 0.6 M KCl solution and purified by dilution of the extract with 20 volumes of distilled water. However, under these conditions, some actin remains bound to myosin and special procedures are required to prepare myosin free of actin.

Size and shape of the myosin molecule: Myosin is a large asymmetric molecule, it has a long tail and two globular heads (Fig. M1). The tail is about 1500 Å long and 20 Å wide. Each head is about 165 Å long, 65 Å wide and 40 Å deep at its thickest part. The molecular weight of myosin is about 500,000. In strong denaturing solutions, such as 5 M guanidine-HCl or 8 M urea, myosin dissociates into six polypeptide chains: two heavy chains (molecular weight of each heavy chain about 200,000) and four light chains (two with a molecular weight of 20,000, one with 15,000 and another with 25,000). The two heavy chains are wound around each other to form a double helical structure. At one end both chains are folded into separate globular structures to form the two heads. In the muscle, the long tail portion forms the backbone of the thick filament and the heads protrude as crossbridges toward the thin filament. Each head contains two light chains.

Fig. M1. Scheme of the myosin molecule

Proteolytic fragments of myosin: When myosin is exposed to the proteolytic enzyme trypsin, fragmentation occurs in the middle of the tail yielding heavy meromyosin (HMM, molecular weight about 350,000) and light meromyosin (LMM, molecular weight about 150,000). HMM can be further split by proteolytic enzymes, such as papain, into subfragment 1 (S1, molecular weight about 110,000) and subfragment 2 (S2). HMM and S1 bind actin, hydrolyze ATP and are water-soluble. LMM has no sites for actin or ATP binding, but inherits the solubility of myosin in 0.6 M KCl and the self-assembling property of myosin in 0.03 M KCl. S2 is water-soluble. The regions of enzymatic fragmentation may serve as hinges.

The heavy chain of S1 can still be further split by proteolytic enzymes into three fragments: 25 kDa (N terminal), 50 kDa (central) and 20 kDa (C terminal) fragments. These fragments played a role in the understanding of the three-dimensional structure of subfragment 1.

Myosin and its proteolytic fragments can be visualized by electron microscopy as shown in Figure M2.

Fig. M2. Electron micrographs of myosin and its proteolytic fragments. (From Lowey et al., 1969).

Myosin light chains: These were discovered by analytical ultracentrifugation; succinylated, or K2CO3 treated myosin exhibited a small slowly sedimenting component behind the main myosin peak. Subsequently, one-dimensional sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) of purified skeletal muscle myosin showed three light chain bands in addition to the heavy chain bands (Fig. M3). The light chain bands were called LC1, LC2, and LC3 with decreasing molecular weight. LC1 and LC3 can be isolated after alkali treatment of myosin, therefore, they are also called Alkali 1 and Alkali 2 light chains; they are also referred to as essential light chains. Alkali 1 and Alkali 2 are identical over their 142 amino acid residues at the C terminal. These isozymes arise from a single gene by alternative splicing. LC2 can be phosphorylated and is also referred to as regulatory light chain. Each myosin head contains 1 essential and 1 regulatory light chain.

 Fig. M3. SDS-PAGE of skeletal muscle myosin (the heavy chain of myosin on the top of the second column is not indicated).

Review: An encyclopedic review about myosin, containing 2354 references, has been written by Sellers and Goodson (1995).



Lowey, S., Slayter, H.S., Weeds, A.G., and Baker, H. (1969). Substructure of the myosin molecule I. Subfragments of myosin by enzymic degradation. J. Mol. Biol., 42, 1-20.

Sellers, J.R. and Goodson, H.V. (1995). Motor proteins 2: myosin. Protein Profile, 2, 1323-1423.

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