Fig. TM1. Head to tail joint in TM (lower part) and the interaction of TM with each of seven actin monomers (upper part). (From Smillie, 1996).
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 Å long and 20 Å wide) with a molecular weight of 65,000-70,000. It consists of two alpha 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.
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Fig. TM1. Head to tail joint in TM (lower part) and the interaction of TM with each of seven actin monomers (upper part). (From Smillie, 1996). |
Tropomyosin isoforms: On SDS-PAGE, TM shows two bands, corresponding to the a (fast) and b (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 Å = 423 Å, (number of residues multiplied by the effective residue translation in a coiled coil) is larger than the length of 400 Å 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 b subunit varies with the muscle type. Slow (red) skeletal muscle and fetal muscle contain a larger portion of the b 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.
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 Å and the molecular length is reduced to about 385 Å.
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 Å 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.
Another important protein interaction is the binding of troponin to TM. One mole of troponin is bound per mole of TM.
Ebashi discovered troponin (TN) in 1963. It is a globular molecule, which consists of three subunits (Fig. TN1), each possesses a specific function.
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Fig. TN1. SDS-PAGE of troponin. |
Troponin C is the Ca2+ receptor in the thin filament. It has been crystallized and its three-dimensional structure determined (Fig. TN2).
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Fig. TN2. Ribbon diagram of TN-C. (From Garrett and Grisham, 1995). The structure revealed that TN-C belongs to the Ca2+-binding protein family. Its Ca2+-binding domains comprise a short a-helix, a Ca2+-binding loop and a second short a-helix. The domain folds around the Ca2+ ion in a manner of a clenched right hand, with the extended forefinger and thumb representing the helices and the middle finger forming the loop structure. This domain is termed an E-F hand. Two E -F hand domains form a stable pair in many Ca2+-binding proteins. In TN-C, the two E-F hand domains are connected by a long a-helix resulting in four Ca2+-binding sites per molecule. Two of the four sites have high affinity to Ca2+ and are located in the carboxyl-terminal domain, whereas the other two sites have low affinity to Ca2+ and are located in the amino-terminal domain. Only the low affinity Ca2+-binding sites of TN-C are involved in the regulation of muscle contraction. |
Troponin I inhibits the Mg2+-activated ATPase of actomyosin (identical with the actin and Mg2+-activated myosin ATPase). TN-I is a basic protein that readily complexes with the acidic TN-C; importantly Ca2+ strengthens the complex formation. Thus, upon muscle stimulation TN-C binds Ca2+ and then complexes with TN-I; this provides a simple mechanism to relieve the inhibition of the actomyosin Mg2+-ATPase by TN-I. Several isoforms of TN-I are known in the molecular mass range of 21-23 kDa.
Troponin T is an asymmetric molecule that interacts with TM. Various studies located the binding of TN-T at the C terminal end of TM; the a-helix of TN-T and the coiled coil of TM participate in the binding. The TM - TN-T interaction serves to fix the position of the entire TN complex within the thin filament, so that subtle changes in the conformation of these proteins may regulate contraction. In this respect, it is important that TN-T also binds TN-C, thus the Ca2+-induced conformational changes in TN-C are transmitted through TN-T to TM. Recently, a highly conserved protein domain was described in the amino acid sequence of TN-T (Stefancsik et al., 1998), that is characterized by a heptad repeat motif with a potential for a -helical coiled coil formation. A similar, potentially coiled coil forming domain is also conserved in all known TN-I sequences, suggesting that these protein domains play a role in TN-T - TN-I interaction. This is another example of how specific interactions of the TN subunits build the entire TN molecule.
TroponinT, similarly to TN-C and TN-I, has several isoforms in the molecular mass range of 30-35 kDa.
The book of Perry (1996) is a good source for learning about the structure and properties of the troponin-tropomyosin system.
References
Bailey, K. (1946). Tropomyosin a new asymmetric protein component of muscle. Nature, 157, 368.
Ebashi, S. (1963). Third component participating in the superprecipitation of actomyosin. Nature, 200, 1010.
Garrett, R.H. and Grisham, C.M. (1995). Molecular Aspects of Cell Biology. Saunders College Publishing.
Perry, S.V. (1996). Molecular mechanisms in striated muscle. Cambridge University Press.
Smillie, L.B. (1996). Tropomyosin. In Biochemistry of smooth muscle contraction (M. Bárány, Ed.), pp. 63-90. Academic Press.
Stefancsik, R, Jha, P.K., and Sarkar, S. (1998). Identification and mutagenesis of a highly conserved domain in troponin T responsible for troponin I binding: Potential role for coiled coil interaction. Proc. Natl. Acad. Sci. USA , 95, 957-962.
Vassylyev, D.G., Takeda, S., Vakatsuki, S. Maeda, K., and Maeda, Y. (1998). Crystal structure of troponin C in complex with troponin I fragments at 2.3-Å resolution. Proc. Natl. Acad. Sci. USA, 95, 4847-4852.
