Tropomyosin

Bailey discovered tropomyosin (TM) in 1946. Its solubility and structural properties show a similarity to those of myosin, hence its name. Tropomyosin is a rod-shaped molecule (about 400 A long and 20 A wide) with a molecular weight of 65,000-70,000. It consists of two a-helical chains arranged in a paralleled coiled-coil configuration. Tropomyosin molecules are bonded head to tail (Fig. TM1). In skeletal muscle, TM accounts to about 3% of the total muscle protein.

Fig. TM1. Head to tail joint in TM (lower part) and the interaction of TM with each of seven actin monomers (upper part) . (Smillie, 1996, with permission from Biochemistry of Smooth Muscle Contraction, 1996, Academic Press).

Tropomyosin isoforms: On SDS-PAGE, TM shows two bands, corresponding to the a (fast) and p (slow) chains. The molecular weight of the chains does not differ significantly, it is in the range of 33,000-35,000. Both chains have 284 amino acid residues, but they differ in 39 residues. The amino acid sequence shows a repeating pattern of nonpolar and polar residues, totaling of 7 residues.

The molecular length of a chain calculated from sequence, 284 x 1.49 A = 423 A, (number of residues multiplied by the effective residue translation in a coiled coil) is larger than the length of 400 A calculated from X-ray studies. This difference can be accounted for by assuming an overlap of 8-9 residues between the ends of TM molecules.

The ratio of the concentration of a and p subunit varies with the muscle type. Slow (red) skeletal muscle and fetal muscle contain a larger portion of the p subunit than fast (white) skeletal muscle, whereas rabbit and avian cardiac muscle contain only the a subunit.

Two dimensional gel electrophoresis revealed several isoforms of TM in different muscles. Furthermore, some of the TM isoforms can be phosphorylated. Changes in the distribution of TM isoforms were observed during development.

Structure of tropomyosin. The amino acid sequences of the a- and p- chains of rabbit skeletal muscle TM showed a repeating pattern of nonpolar and polar amino acids (reviewed by Smilie, 1996). This is characteristic for a coiled-coil structure in which two a-helices interact along their length by "knobes into holes" packing of nonpolar residues to form a hydrophobic core. Polar and ionic side chains are directed toward the exterior of the two-stranded ropelike arrangement, where they can interact with solvent and/or other protein molecules. Figure TM2 visualizes how the two helices in a coiled-coil interact.

Fig. TM2. Interaction between the two a-helices of TM coiled-coil. Each a-helix is shown with seven residues (a-g) in two turns. (A) End of view looking from N-terminus. The interface between the a-helices derives primarily from hydrophobic residues in core positions a and d. (B) The core interface viewed parallel to the coiled-coil axis shows how residues from one chain occupy the spaces between the corresponding residues from the second chain to give "knobs in holes" packing. (Reprinted with permission from Stewart, M., Structural basis for bending tropomyosin around actin in muscle thin filaments. Proc. Natl. Acad. Sci. USA, 98,8165-8166, 2001. Copyright (2001) National Academy of Sciences, USA.

X-ray diffraction studies of TM suffered from the 90% water content of the crystals yielding low resolution structures which identified only the general outline of the molecule, such as the coiled coil. Recently, the crystal structure of a 81 residue N-terminal fragment, comprising almost a third of TM, was determined at 2 A resolution.(Brown et al., 2001). This refined analysis revealed the two-stranded a-helical coiled structure with an axial sliding (about 1.2 A) of the two chains at clusters of alanine residues. The joining of these regions with neighboring segments, where the helices were in axial register, gave rise to specific bends in the molecular axis. This asymetric design allows the TM molecule to adopt multiple bent conformations. (there are seven alanine clusters) which may be involved in the regulatory role of TM in muscle contraction.

Binding properties: Tropomyosin has high affinity to actin, as evidenced by the difficulty in removing TM in course of actin purification. Each TM molecule binds 7 actin monomers in F-actin. When bound to actin, each TM is believed to be supercoiled with a radius of about 40 A and the molecular length is reduced to about 385 A. Since both actin and TM are abundant in charged residues, it is reasonable to assume that electrostatic interactions play a major role in the binding of TM to actin.

Electron microscopic image reconstruction and X-ray diffraction studies suggest that during muscle activation TM moves from its lateral position on the actin filament by a distance of 10-15 A toward the center of the groove in the actin double helix. It is postulated that in the resting muscle TM occupies the site of actin necessary for combination with the myosin head. The movement of TM, at the beginning of contraction, liberates the myosin-binding site, thus actomyosin can be formed and the muscle can contract. (This TM movement is discussed in the chapter of Regulation of Muscle Contraction).

The tropomyosin-binding component of troponin (TN-T) binds to TM; this will be described under Troponin. The encyclopaedic review of Perry (2001), about the distribution, properties, and function of vertebrate tropomyosin is a special learning experience.

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