Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

A band H zone

I band Z line

High viscosity in an aqueous solution is a sign of long-range molecular interactions, defined as interactions that extend through and connect many molecules. Concentrated sugar solutions (molasses, for example) are viscous because of the extensive hydrogen-bonding networks established by the multiple hydroxyl

groups of sugar molecules. Solutions of DNA are highly viscous because isolated DNA fibers are extremely long (often in the millimeter size range) and highly hydrated. As Szent-Györgyi discovered, the extensive aggregates formed by myosin and actin also produce highly viscous solutions.

(a)

ATP

ATP

ATP binding causes dissociation of myosin from actin

4

2

Low-energy conformation of the myosin head

Power stroke

ADP and Pi

are released ADP +

The optical trapping experiment involves the attachment of myosin molecules to silica beads that are immobilized on a microscope coverslip (see figure). Actin filaments are then prepared such that a polystyrene bead is attached to each end of the filament. These beads can be “caught” and held in place in solution by a pair of “optical traps”—two high-intensity infrared laser beams, one focused on the polystyrene bead at one end of the actin filament and the other focused on the bead at the other end of the actin filament. The force acting on each bead in such a trap is proportional to the position of the bead in the “trap,” so that displacement and forces acting on the bead (and thus on the actin filament) can both be measured. When the “trapped” actin filament is brought close to the silica bead, one or a few myosin molecules may interact with sites on the actin, and ATPinduced interactions of individual myosin molecules with the trapped actin filament can be measured and quantitated. Such optical trapping experiments have shown that a single cycle or turnover of a single myosin molecule along an actin filament involves an average movement of 4 to 11 nm (40–110 Å) and generates an average force of 1.7 to 4 10 12 newton (1.7–4 piconewtons (pN)).

The magnitudes of the movements observed in the optical trapping experiments are consistent with the movements predicted by the cryoelectron microscopy imaging data. Can the movements and forces detected in a single contraction cycle by optical trapping also be related to the energy available from hydrolysis of a single ATP molecule? The energy required for a contraction cycle is defined by the “work” accomplished by contraction, and work (w) is defined as force (F) times distance (d):

w F d

For a movement of 4 nm against a force of 1.7 pN, we have

w (1.7 pN) (4 nm) 0.68 10 20 J

For a movement of 11 nm against a force of 4 pN, the energy requirement is larger:

w (4 pN) (11 nm) 4.4 10 20 J

If the cellular free energy of hydrolysis of ATP is taken as 50 kJ/mol, the free energy available from the hydrolysis of a single ATP molecule is

G ( 50 kJ/mol)/6.02 1023 molecules/mol) 8.3 10 20 J

Thus, the free energy of hydrolysis of a single ATP molecule is sufficient to drive the observed movements against the forces that have been measured.

Movements of single myosin molecules along an actin filament can be measured by means of an optical trap consisting of laser beams focused on polystyrene beads attached to the ends of actin molecules. (Adapted from Finer et al., 1994. Nature 368:113 – 119. See also Block, 1995. Nature 378:132–133.)

by Calcium Channels and Pumps

The trigger for all muscle contraction is an increase in Ca2 concentration in the vicinity of the muscle fibers of skeletal muscle or the myocytes of cardiac and smooth muscle. In all these cases, this increase in Ca2 is due to the flow of Ca2 through calcium channels (Figure 17.24). A muscle contraction ends when the Ca2 concentration is reduced by specific calcium pumps (such as the SR Ca2 - ATPase, Chapter 10). The sarcoplasmic reticulum, t-tubule, and sarcolemmal membranes all contain Ca2 channels. As we shall see, the Ca2 channels of the SR function together with the t-tubules in a remarkable coupled process.

Ca2 release in skeletal and heart muscle has been characterized through the use of specific antagonist molecules that block Ca2 channel activity. The dihydropyridine (DHP) receptors of t-tubules, for example, are blocked by dihydropyridine derivatives, such as nifedipine (Figure 17.25). The purified DHP receptor of heart muscle can be incorporated into liposomes, whereupon it shows calcium channel activity. The channel displays voltage-dependent gating and is selective for divalent cations over monovalent cations. Thus, the heart muscle DHP receptor is a voltage-dependent Ca2 channel. Other evidence suggests that the skeletal muscle DHP receptor is a voltage-sensing protein; it presumably undergoes voltage-dependent conformation changes.

The DHP receptor from t-tubules consists of five different polypeptides, designated 1 (150 to 173 kD), 2 (120 to 150 kD), (50 to 65 kD), (30 to 35 kD), and (22 to 27 kD). The 2- and -subunits are linked by a disulfide bond. The 1, 2- , , and stoichiometry is 1 1 1 1. The 2-subunit is glycosylated, but 1 is not. 1 is homologous with the -subunit of the voltagesensitive sodium channel (Chapter 34). The sequence of 1 contains four internal sequence repeats, each containing six transmembrane helices, one of which is positively charged and is believed to be a voltage sensor (Figure 17.26). The loop between helices 5 and 6 contributes to the pore. These six segments share many similarities with the corresponding segments of the sodium channel. The1-subunit of the DHP receptor in heart muscle is implicated in channel formation and voltage-dependent gating.

The Ca2 -release channel from the terminal cisternae of sarcoplasmic reticulum has been identified by virtue of its high affinity for ryanodine, a toxic alkaloid (Figure 17.25). The purified receptor consists of oligomers, containing four or more subunits of a single large polypeptide (565 kD). Electron microscopy reveals that the purified ryanodine receptor (Figure 17.27) is in fact the foot structure observed in native muscle tissue. Image reconstructions reveal that the receptor is a square structure with fourfold symmetry, containing a central pore with four radially extending canals (Figure 17.28). These

Lumen of SR

Calcium

Ca2+ release channel

SR membrane

Channel opening may be

voltage-sensitive or Ca2+-sensitive

ADP

Ca2+

Ca2+

Ca2+

● Ca2 is the trigger signal for muscle contraction. Release of Ca2 through voltageor Ca2 -sensitive channels activates contraction. Ca2 pumps induce relaxation by reducing the concentration of Ca2 available to the muscle fibers.

Nifedipine

HO

Ryanodine

● (a) Electron micrograph images of foot structures of terminal cisternae. (b, c) Foot structures appear as trapezoids and diamonds on the surface of the membrane. The central canal (CC), radial canals (RC), and peripheral vestibules (PV) are indicated. (d) The relationship between the foot structures, t- tubule, terminal cisternae, and muscle fiber.

(Photo courtesy of Sidney Fleischer, Vanderbilt University)

17.3 ● Skeletal Muscle Myosin and Muscle Contraction

FIGURE 17.28 ● Image reconstructions of the junctional channel complex of a foot structure. (Photo courtesy of Sidney Fleischer, Vanderbilt University)

radial canals each extend to openings in the periphery of the structure and are therefore contiguous with the myoplasm.

So how do the foot structures effect the release of Ca2 from the terminal cisternae of the SR? The feet that join the t-tubules and the terminal cisternae of the SR are approximately 16 nm thick. The feet apparently function by first sensing either a voltage-dependent conformation change (skeletal muscle) or the transport of Ca2 across the voltage-sensitive Ca2 channel (heart muscle) of the t-tubule and then facilitating the release of large amounts of Ca2 through the foot structure from the SR. The reconstructed image (Figure 17.28) for the foot structure suggests a possible pathway for Ca2 transport from the lumen of the SR to the myoplasm via the ryanodine receptor. A Ca2 - or voltage-dependent conformation change may serve to gate open the cen-

tral canal of the foot structure. On entering the central canal, calcium ions TnI Actin move outward through the radial canals to the outer vestibule regions and into

the myoplasm adjacent to the triad junctions, where binding to the muscle fibers induces contraction.

Regulation of Contraction by Ca2

557

Myosin head

Early in this chapter, the importance of Ca2 ion as the triggering signal for muscle contraction was described. Ca2 is the intermediary signal that allows striated muscle to respond to motor nerve impulses (Figure 17.24). The importance of Ca2 as a contraction signal was understood in the 1940s, but it remained for Setsuro Ebashi, a pioneer of muscle research, to show in the early 1960s that the Ca2 signal is correctly interpreted by muscle only when tropomyosin and the troponins are present. Specifically, actomyosin prepared from pure preparations of actin and myosin (thus containing no tropomyosin and troponins) was observed to contract when ATP was added, even in the absence of Ca2 . However, actomyosin prepared directly from whole muscle would contract in the presence of ATP only when Ca2 was added. Clearly the muscle extracts contained a factor that conferred normal Ca2 sensitivity to actomyosin. The factor turned out to be the tropomyosin–troponin complex.

Actin thin filaments consist of actin, tropomyosin, and the troponins in a 7 1 1 ratio (Figure 17.15). Each tropomyosin molecule spans seven actin molecules, lying along the thin filament groove, between pairs of actin monomers. As shown in a cross-section view in Figure 17.29, in the absence of Ca2 , troponin I is thought to interact directly with actin to prevent the interaction of actin with myosin S1 heads. Troponin I and troponin T interact with tropomyosin to keep tropomyosin away from the groove between adjacent actin

TnC

TnT

MyosinTropomyosin binding

site

Ca2+

Ca2+

FIGURE 17.29 ● A drawing of the thick and thin filaments of skeletal muscle in cross-sec- tion showing the changes that are postulated to occur when Ca2 binds to troponin C.

558 Chapter 17 ● Molecular Motors

FIGURE 17.30 ● (a) A ribbon diagram and

(b) a molecular graphic showing two slightly different views of the structure of troponin C. Note the long -helical domain connecting the N-terminal and C-terminal lobes of the molecule.

monomers. However, the binding of Ca2 ions to troponin C appears to increase the binding of troponin C to troponin I, simultaneously decreasing the interaction of troponin I with actin. As a result, tropomyosin slides deeper into the actin-thin filament groove, exposing myosin-binding sites on actin, and initiating the muscle contraction cycle (Figure 17.23). Because the troponin complexes can interact only with every seventh actin in the thin filament, the conformational changes that expose myosin-binding sites on actin may well be cooperative. Binding of an S1 head to an actin may displace tropomyosin and the troponin complex from myosin-binding sites on adjacent actin subunits.

The Interaction of Ca2 with Troponin C

There are four Ca2 -binding sites on troponin C—two high-affinity sites on the carboxy-terminal end of the molecule, labeled III and IV in Figure 17.30, and two low-affinity sites on the amino-terminal end, labeled I and II. Ca2 binding to sites III and IV is sufficiently strong (KD 0.1 M) that these sites are presumed to be filled under resting conditions. Sites I and II, however, where the KD is approximately 10 M, are empty in resting muscle. The rise of Ca2 levels when contraction is signaled leads to the filling of sites I and II, causing a conformation change in the amino-terminal domain of TnC. This conformational change apparently facilitates a more intimate binding of TnI to TnC that involves the C helix, and also possibly the E helix of TnC. The increased interaction between TnI and TnC results in a decreased interaction between TnI and actin.