Actin & Myosin Account for 75% of the Protein Mass in Muscle
Actin and myosin contribute 20% and 55% of the protein mass in muscle, respectively. The monomeric form of actin, G-actin (43 kDa; G, globular) polymerizes in the presence of Mg2+ to form an insoluble double helical filament called F-actin (Figure 1). The F-actin fiber, also known as the thin filament, is 6- to 7-nm thick and has a structure that repeats every 35.5 nm.
Fig1. Schematic representation of the thin filament, showing the spatial configuration of its three major protein components: actin, troponin, and tropomyosin. The upper panel shows individual molecules of G-actin. The middle panel shows actin monomers assembled into F-actin. Individual molecules of tropomyosin (two strands wound around one another) and of troponin (made up of its three subunits) are also shown. The lower panel shows the assembled thin filament, consisting of F-actin, tropomyosin, and the three subunits of troponin (TpC, TpI, and TpT).
Myosin-I, which exists in cells as a monomer, links cytoskeletal microfilaments to the plasma membrane. The pre dominant form of myosin in contractile tissues is myosin II (≈ 460 kDa), hereafter referred to simply as myosin, an asymmetric hexamer. The hexamer consists of one pair of heavy (H) chains (≈200 kDa), and two pairs of light (L) chains (≈20 kDa), referred to as essential and regulatory. The two heavy chains are intertwined, forming an extended helical tail, each capped by a globular head domain to which the light chains associate (Figure 2). Myosin possesses low, but detectable levels of ATPase activity in vitro: the catalysis of the hydrolysis of ATP by water to form ADP and Pi . Low levels of ATPase activity are a common feature of enzymes that employ ATP as a substrate. When skeletal muscle myosin is complexed to actin to form actomyosin complex, its ATPase activity greatly increases.
Fig2. Diagram of a myosin molecule showing the two intertwined α-helices (fibrous portion), the globular region or head (G), the light chains (L), and the effects of proteolytic cleavage by trypsin and papain. The globular head region, which contains an actin-binding site and an L chain-binding site, attaches to the remainder of the myosin molecule.
The Structural & Functional Organization of Myosin Was Mapped by Limited Proteolysis
Limited proteolysis with trypsin yielded two myosin frag ments called light and heavy meromyosin (HMM, ≈ 340 kDa). Light meromyosin (LMM) consists of the aggregated α-helical fibers from the tail of myosin (see Figure 2). It does not hydrolyze ATP or bind to F-actin. By contrast, HMM is a soluble protein that possesses both fibrous and globular regions (see Figure 2). HMM exhibits ATPase activity and binds to F-actin. Limited proteolysis of HMM with the protease papain cleaves it into two subfragments: S-1 globular region (≈115 kDa) and S-2 fibrous region. Only the S-1 fragment, exhibits ATPase activity and binds both actin and myosin light chains (see Figure 2).
Tropomyosin & the Troponins Are Key Components of the Thin Filaments in Striated Muscle
In striated muscle, there are two other proteins that are minor in terms of their mass but important in terms of their function. Tropomyosin, present in all muscle and muscle-like structures, is a fibrous molecule that consists of two chains, α and β. The chains attach to F-actin in the groove between its filaments (see Figure 1). The troponin complex is unique to striated muscle and consists of three polypeptides. Troponin T (TpT) binds to tropomyosin as well as to the other two troponin components. Troponin I (TpI) inhibits the F-actin myosin interaction and also binds to the other components of troponin. Troponin C(TpC) is a calcium-binding polypeptide that is structurally and functionally analogous to calmodulin, an important calcium-binding protein widely distributed in nature. Up to four calcium ions can bind per molecule of troponin C or calmodulin.
