Actin

Straub, a young biochemist in Szent-Györgyi's laboratory, discovered actin in 1942. Previously Szent-Györgyi has shown that brief extraction of minced rabbit muscle with an alkaline 0.6 M KCl solution in the cold room yields a myosin with low viscosity (myosin A), whereas when the muscle mince was left in the 0.6 M KCl for a day a very viscous myosin solution was extracted (myosin B). Straub thought that the difference between myosin B and A is caused by the extraction of a new protein that makes the one-day extract viscous. Accordingly, he extracted myosin A from the muscle, then left the residue in the cold room for a day. The muscle residue was washed with distilled water to remove the KCl and remaining cytoplasmic proteins, and finally the residue was dried with acetone. The protein extracted from the acetone-dried residue formed a very viscous complex with myosin A, similarly to myosin B, "it activated myosin" and hence it was named actin. In skeletal muscle, actin comprises about 15% of the total protein.

The two forms of actin: After an improved procedure for actin preparation, Straub has found that water extraction of the acetone-dried muscle residue yielded an actin solution with low viscosity, globular or G-actin, that upon addition of salts (at physiological concentrations) polymerized to a highly viscous actin gel, fibrous or F-actin. Straub followed the polymerization of actin by viscometry, shown in Fig. A1.

Fig. A1. Polymerization of actin in the presence of various ions. Curve 1) 110 mM NaCl, 3 mM KCl, 3 mM CaCl2, and 10 mM MgSO4; Curve 2) same as 1 but without Mg2+; Curve 3) same as 1 but without Ca2+; Curve 4) same as 1 but without K+. Temperature 24o C (From Feuer et al., 1948).

Ionic strength, temperature, and pH affect polymerization. Optimal conditions are: 0.1 M salts concentration, 37^o C, and pH 6.5-7.5.

In 1949 Straub and Feuer reported that G-actin contains bound ATP and during polymerization of actin the ATP is hydrolyzed to bound ADP and P_i. Straub postulated that the transformation of G-actin-ATP to F-actin-ADP plays a key role in muscle contraction, however, this could not be demonstrated in skeletal muscle of live animals (Martonosi et al., 1960). Actin polymerization with concomitant ATP hydrolysis may take place in non-muscle cells and may provide the mechanochemistry for motility (see Cell Motility chapter).

Electron micrograph of fibrous actin filaments reveals that the structure consists of twin strings of actin globules wound around each other in a double helix. The subunit repeat is about 55 Å and the helical repeat is about 370 Å.

Fig. A2. Electron micrograph of actin filament (From Huxley, H.E., 1972).

Janmey et al. (1999) reviewed the characteristic properties of actin filaments.

Actin-myosin binding: F-actin combines with myosin to form actomyosin. In 0.6 M KCl actomyosin forms a viscous solution; upon addition of ATP, actomyosin dissociates into its components actin and myosin, with accompanying reduction of the viscosity. At physiological ionic strength actomyosin is insoluble, the same way as in the muscle.

F-actin also combines with the proteolytic fragments of myosin, HMM or S1. The complex formed actoheavymeromyosin or actosubfragment 1 remains soluble at low ionic strength. When HMM or S1 is added to muscle thin filament it attaches to the actin component of the filament, forming a specific "arrow head" structure (Fig. A3). This suggests a structural polarity for the thin filament.

Fig. A3. Electron micrograph of thin filament decorated with HMM. (From. Huxley, H.E., 1972).

 

Based on this observation, H. E. Huxley postulated that the structural polarity of thin and thick filaments allows the sliding force to move the thin filaments toward the center of the sarcomere (Fig. A4).

Fig. A4. Diagram for the structural polarity of thin and thick filaments (From Huxley, H.E., 1972).

 

Three-dimensional structure of actin: Kabsch and collaborators (1990) were the first to crystallize G-actin and determined its structure (Fig. A5).

Fig. A5. Scheme for the structure of actin. (From Holmes and Kabsch, 1991).

Folding of the actin molecule is represented by ribbon tracing of the a-carbon atoms. N and C correspond to the amino- and carboxyl-terminals, respectively. The letters followed by numbers represent amino acids in the polypeptide chain. A hypothetical vertical line divides the actin molecule into two domains "large", left side, and "small", right side. ATP and Ca²⁺ are located between the two domains. These two domains can be subdivided further into two subdomains each, the small domain being composed of subdomains 1 and 2, and the large domain of subdomains 3 and 4. (Subdomain 2 has significantly less mass than the other three subdomains and this is the reason of dividing actin into small and large domains). The four subdomains are held together and stabilized mainly by salt bridges and hydrogen bonds to the phosphate groups of the bound ATP and to its associated Ca²⁺ localized in the center of the molecule. Because of the less mass in subdomain 2, the actin molecule is distinctly polar in the direction from subdomains 1 and 3, called the "barbed end", toward subdomains 2 and 4, called the "pointed end". This polarity defines the orientation of the actin molecule in the myosin HMM decoration pattern of the thin filament, shown in Fig. A3.

The intersubunit contacts in the F-actin filament: In helical structures, such as the F-actin filament, two types of intersubunit contacts are possible in principle: those along and those between the long-pitch helical strands. In the atomic model of the F-actin filament, 24 amino acid residues per subunit are involved in contacts along the long-pitch helical strands. By contrast, only 15 residues per subunit mediate the weaker contacts between the two strands.

Localization of actin in the structure of muscle: Under the microscope, myosin extracted myofibrils exhibit the thin filaments, attached to the Z line. When 0.6 M KI solution, that dissolves F-actin, is added to such a myofibrillar ghost the structure disappears, indicating that the thin filaments are composed of actin. In the structure of muscle, the I band contains thin filaments whereas the A band.contains both thick and thin filaments.

Structure of the thin filament: Fig. A6 shows the structure: actin molecules form two strings wound around each other, in the grove is the tropomyosin strand and at regular intervals troponin molecules are attached to tropomyosin.

Fig. A6. Model for the structure of the thin filament (From Huxley, H.E., 1972).

References

Feuer, G, Molnár, F., Pettkó, E. and Straub, F.B. (1948). Studies on the composition and polymerization of actin. Hungarica Acta Physiologica, 1, 150-163.

Holmes, K.C. and Kabsch, W. (1991). Muscle proteins: actin. Current Opinion in Structural Biology, 1, 270-280.

Huxley, H.E. (1972). Molecular basis for contraction in cross-striated muscles. In The structure and function of muscle, ( G.H. Bourne, Ed.) second edition, vol. I, Part 1, pp. 301-387.

Janmey, P.A., Tang, J.X., and Schmidt, C.F. (1999). Actin filaments. In Supramolecular Assemblies, (V. Bloomfield, Ed.), Biophysical Society Homepage.

Kabsch, W., Mannherz, E.G., Suck, D., Pai, E.F., and Holmes, K.C. (1990). Atomic structure of the actin:DNase I complex. Nature, 347, 37-44.

Martonosi, A., Gouvea, M.A., and Gergely, J. (1960). Studies on actin III. G-F transformation of actin and muscular contraction (experiments in vivo). J. Biol. Chem. 235, 1707-1710.

Straub, F.B. and Feuer, G. (1949). Adenosinetriphosphate the functional group of actin. Biochim. Biophys. Acta. 4, 455-470.

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