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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 34.2Myosins Move Along Actin Filaments

Myosins, kinesins, and dyneins move by cycling between states with different affinities for the long, polymeric macromolecules that serve as their tracks. For myosin, the molecular track is a polymeric form of actin, a 42-kd protein that is one of the most abundant proteins in eukaryotic cells, typically accounting for as much as 10% of the total protein. Actin polymers are continually being assembled and disassembled in cells in a highly dynamic manner, accompanied by the hydrolysis of ATP. On the microscopic scale, actin filaments participate in the dynamic reshaping of the cytoskeleton and the cell itself and in other motility mechanisms that do not include myosin. In muscle, myosin and actin together are the key components responsible for muscle contraction.

34.2.1. Muscle Is a Complex of Myosin and Actin

Vertebrate muscle that is under voluntary control has a banded (striated) appearance when examined under a light microscope. It consists of multinucleated cells that are bounded by an electrically excitable plasma membrane. A muscle cell contains many parallel myofibrils, each about 1 μm in diameter. The functional unit, called a sarcomere, typically repeats every 2.3 μm (23,000 Å) along the fibril axis in relaxed muscle (Figure 34.13). A dark A band and a light I band alternate regularly. The central region of the A band, termed the H zone, is less dense that the rest of the band. The I band is bisected by a very dense, narrow Z line.

Figure 34.13. Sarcomere.

Figure 34.13

Sarcomere. (A) Electron micrograph of a longitudinal section of a skeletal muscle myofibril, showing a single sarcomere. (B) Schematic representations of cross sections correspond to the regions in the micrograph. [Courtesy of Dr. Hugh Huxley.]

The underlying molecular plan of a sarcomere is revealed by cross sections of a myofibril. These cross sections show the presence of two kinds of interacting protein filaments. The thick filaments have diameters of about 15 nm (150 Å) and consist primarily of myosin. The thin filaments have diameters of approximately 9 nm (90 Å) and consist of actin as well as tropomyosin and the troponin complex. Muscle contraction is achieved through the sliding of the thin filaments along the length of the thick filaments, driven by the hydrolysis of ATP (Figure 34.14). Tropomyosin and the troponin complex regulate this sliding in response to nerve impulses. Under resting conditions, tropomyosin blocks the intimate interaction between mysosin and actin. A nerve impulse leads to an increase in calcium ion concentration within the muscle cell. A component of the troponin complex senses the increase in calcium and, in response, relieves the inhibition of myosin - actin interactions by tropomyosin.

Figure 34.14. Sliding-Filament Model.

Figure 34.14

Sliding-Filament Model. Muscle contraction depends on the motion of thin filaments (blue) relative to thick filaments (red). [After H. E. Huxley. The mechanism of muscular contraction. Copyright © 1965 by Scientific American, Inc. All rights reserved.] (more...)

Image caduceus.jpg Although myosin was discovered through its role in muscle, other types of myosin play crucial roles in a number of biological contexts. Some defects in hearing in both mice and human beings have been linked to mutations in particular myosin homologs that are present in cells of the ear. For example, Usher syndrome in human beings and the shaker mutation in mice have been linked to myosin VIIa, expressed in hair cells (Section 32.4.1). The mutation of this myosin results in the formation of splayed stereocilia that do not function well. Myosin VIIa differs from muscle myosin in that its tail region possesses a number of amino acid sequences that correspond to domains known to mediate specific protein - protein interactions.

34.2.2. Actin Is a Polar, Self-Assembling, Dynamic Polymer

The structure of the actin monomer was determined to atomic resolution by x-ray crystallography and has been used to interpret the structure of actin filaments, already somewhat understood through electron microscopy studies at lower resolution. Each actin monomer comprises four domains (Figure 34.15). These domains come together to surround a bound nucleotide, either ATP or ADP. The ATP form can be converted into the ADP form by hydrolysis.

Figure 34.15. Actin Structure.

Figure 34.15

Actin Structure. Image mouse.jpg (Left) Five actin monomers of an actin filament are shown explicitly, one in blue. (Right) The domains in the four-domain structure of an actin monomer are identified by different shades of blue.

Actin monomers (often called G-actin for globular) come together to form actin filaments (often called F-actin; see Figure 34.15). F-actin has a helical structure; each monomer is related to the preceding one by a translation of 27.5 Å and a rotation of 166 degrees around the helical axis. Because the rotation is nearly 180 degrees, F-actin resembles a two-stranded cable. Note that each actin monomer is oriented in the same direction along the F-actin filament, and so the structure is polar, with discernibly different ends. One end is called the barbed (plus) end, and the other is called the pointed (minus) end. The names “barbed” and “pointed” refer to the appearance of an actin filament when myosin S1 fragments are bound to it.

How are actin filaments formed? Like many biological structures, actin filaments self-assemble; that is, under appropriate conditions, actin monomers will come together to form well-structured, polar filaments. The aggregation of the first two or three monomers to form a filament is somewhat unfavorable. Once such a filament nucleus exists, the addition of subunits is more favorable. Let us consider the polymerization reaction in more detail. We designate an actin filament with n subunits An. This filament can bind an additional actin monomer, A, to form An+1.

Image ch34e1.jpg
Image ch34fu2.jpg

The dissociation constant for this reaction, Kd, defines the monomer concentrations at which the polymerization reaction will take place, because the concentration of polymers of length n + 1 will be essentially equal to that for polymers of length n. Thus,

Image ch34e2.jpg

In other words, the polymerization reaction will proceed until the monomer concentration is reduced to the value of Kd. If the monomer concentration is below the value of Kd, the polymerization reaction will not proceed at all; indeed, existing filaments will depolymerize until the monomer concentration reaches the value of Kd. Because of these phenomena, Kd is referred to as the critical concentration for the polymer. Recall that actin contains a nucleotide-binding site that can contain either ATP or ADP. The critical concentration for the actin - ATP complex is approximately 20-fold lower than that for the actin - ADP complex; actin - ATP polymerizes more readily than does actin - ADP.

Actin filaments inside cells are highly dynamic structures that are continually gaining and losing monomers. The concentration of free actin monomers is controlled by several mechanisms. For example, actinsequestering proteins such as β-thymosin bind to actin monomers and inhibit polymerization. Furthermore, the concentration and properties of actin filaments are closely regulated by proteins that sever an actin filament into two or that cap one of the ends of a filament. Regulated actin polymerization is central to the changes in cell shape associated with cell motility in amebas as well as in human cells such as macrophages.

Image tree.jpg A well-defined actin cytoskeleton is unique to eukaryotes; prokaryotes lack such structures. How did filamentous actin evolve? Comparison of the three-dimensional structure of G-actin with other proteins revealed remarkable similarity to several other proteins, including sugar kinases such as hexokinase (Figure 34.16; see also Section 16.1.1). Notably, the nucleotide-binding site in actin corresponds to the ATP-binding site in hexokinase. Thus, actin evolved from an enzyme that utilized ATP as a substrate.

Figure 34.16. Actin and Hexokinase.

Figure 34.16

Actin and Hexokinase. Image mouse.jpg A comparison of actin (blue) and hexokinase from yeast (red) reveals structural similarities indicative of homology. Both proteins have a deep cleft in which nucleotides bind.

More recently, a closer prokaryotic homolog of actin was characterized. This protein, called MreB, plays an important role in determining cell shape in rod-shaped, filamentous, and helical bacteria. The internal structures formed by MreB are suggestive of the actin cytoskeleton of eukaryotic cells, although they are far less extensive. Even though this protein is only approximately 15% identical in sequence with actin, MreB folds into a very similar three-dimensional structure. It also polymerizes into structures that are similar to F-actin in a number of ways, including the alignment of the component monomers.

34.2.3. Motions of Single Motor Proteins Can Be Directly Observed

Muscle contraction is complex, requiring the action of many different myosin molecules. Studies of single myosin molecules moving relative to actin filaments have been sources of deep insight into the mechanisms underlying muscle contraction and other complex processes.

A powerful tool for these studies, called an optical trap, relies on highly focused laser beams (Figure 34.17). Small beads can be caught in these traps and held in place in solution.

Figure 34.17. Watching a Single Motor Protein in Action.

Figure 34.17

Watching a Single Motor Protein in Action. (A) An actin filament (blue) is placed above a heavy meromyosin (HMM) fragment (yellow) that projects from a bead on a glass slide. A bead attached to each end of the actin filament is held in an optical trap (more...)

The position of the beads can be monitored with nanometer precision. James Spudich and coworkers designed an experimental arrangement consisting of an actin filament that had a bead attached to each end. Each bead could be caught in an optical trap (one at each end of the filament) and the actin filament pulled taut over a microscope slide containing other beads that had been coated with fragments of myosin such as the heavy meromyosin fragment (see Figure 34.17). On the addition of ATP, transient displacements of the actin filament were observed along its long axis. The size of the displacement steps was fairly uniform with an average size of 11 nm.

The results of these studies, performed in the presence of varying concentrations of ATP, are interpreted as showing that individual myosin heads bind the actin filament and undergo a conformational change (the power stroke) that pulls the actin filament, leading to the displacement of the beads. After a period of time, the myosin head releases the actin, which then snaps back into place.

34.2.4. Phosphate Release Triggers the Myosin Power Stroke

How does ATP hydrolysis drive the power stroke? A key observation is that the addition of ATP to a complex of myosin and actin results in the dissociation of the complex. Thus, ATP binding and hydrolysis cannot be directly responsible for the power stroke. We can combine this fact with the structural observations described earlier to construct a mechanism for the motion of myosin along actin (Figure 34.18). Let us begin with myosin-ADP bound to actin. The release of ADP and the binding of ATP to actin result in the dissociation of myosin from actin. As we saw earlier, the binding of ATP with its γ-phosphate group to the myosin head leads to a significant conformational change, amplified by the lever arm. This conformational change moves the myosin head along the actin filament by approximately 110 Å. The ATP in the myosin is then hydrolyzed to ADP and Pi, which remain bound to myosin. The myosin head can then bind to the surface of actin, resulting in the dissociation of Pi from the myosin. Phosphate release, in turn, leads to a conformational change that increases the affinity of the myosin head for actin and allows the lever arm to move back to its initial position. The conformational change associated with phosphate release corresponds to the power stroke. After the release of Pi, the myosin remains tightly bound to the actin and the cycle can begin again.

Figure 34.18. Myosin Motion Along Actin.

Figure 34.18

Myosin Motion Along Actin. A myosin head (yellow) in the ADP form is bound to an actin filament (blue). The exchange of ADP for ATP results in (1) the release of myosin from actin and (2) substantial reorientation of the lever arm of myosin. Hydrolysis (more...)

How does this cycle apply to muscle contraction? Myosin molecules self-assemble into thick bipolar structures with the myosin heads protruding at both ends of a bare region in the center (Figure 34.19). Approximately 500 head domains line the surface of each thick filament. These domains are paired in myosin dimers, but the two heads within each dimer act independently. Actin filaments associate with each head-rich region, with the barbed ends of actin toward the Z-line. In the presence of normal levels of ATP, most of the myosin heads are detached from actin. Each head can independently hydrolyze ATP, bind to actin, release Pi, and undergo its power stroke. Because few other heads are attached, the actin filament is relatively free to slide. Each head cycles approximately five times per second with a movement of 110 Å per cycle. However, because hundreds of heads are interacting with the same actin filament, the overall rate of movement of myosin relative to the actin filament may reach 80,000 Å per second, allowing a sacromere to contract from its fully relaxed to its fully contracted form rapidly. Having many myosin heads briefly and independently attaching and moving an actin filament allows for much greater speed than could be achieved by a single motor protein.

Figure 34.19. Thick Filament.

Figure 34.19

Thick Filament. (A) An electron micrograph of a reconstituted thick filament reveals the presence of myosin head domains at each end and a relatively narrow central region. (B) A schematic view shows how myosin molecules come together to form the thick (more...)

34.2.5. The Length of the Lever Arm Determines Motor Velocity

A key feature of myosin motors is the role of the lever arm as an amplifier. The lever arm amplifies small structural changes at the nucleotide-binding site to achieve the 110-Å movement along the actin filament that takes place in each ATP hydrolysis cycle. A strong prediction of the mechanism proposed for the movement of myosin along actin is that the length traveled per cycle should depend on the length of this lever arm. Thus, the length of the lever arm should influence the overall rate at which actin moves relative to a collection of myosin heads.

This prediction was tested with the use of mutated forms of myosin with lever arms of different lengths. The lever arm in muscle myosin includes binding sites for two light chains (Section 34.1.2). Thus investigators shortened the lever arm by deleting the sequences that correspond to one or both of these binding sites. They then examined the rates at which actin filaments were transported along collections of these mutated myosins (Figure 34.20). As predicted, the rate decreased as the lever arm was shortened. A mutated form of myosin with an unusually long lever arm was generated by inserting 23 amino acids corresponding to the binding site for an additional regulatory light chain. Remarkably, this form was found to support actin movement that was faster than the wild-type protein. These results strongly support the proposed role of the lever arm in contributing to myosin motor activity.

Figure 34.20. Myosin Lever Arm Length.

Figure 34.20

Myosin Lever Arm Length. Examination of the rates of actin movement supported by a set of myosin mutants with different numbers of light-chain binding sites revealed a linear relation; the greater the number of light-chain binding sites (and, hence, the (more...)

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22418

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