Subsequent work by Taylor and others revealed that the above reaction is much more complex than shown in the equation. Part b of Fig. M6 shows a recent version of the ATPase cycle and Part a of Fig. M6 shows the coupling of the ATPase cycle to the crossbridge cycle.

Fig. M6. Crossbridge cycle (Part a) correlated with the ATPase cycle (Part b). (From Perry, 1996).

ATPase activity of myosin and speed of muscle shortening: The ATPase activity of myosin was determined in 25 different muscles with a 250-fold variation in the speed of shortening. A correlation was found between the ATPase activity of myosin and the speed of shortening (Fig. M7). This suggests that the myosin ATPase determines the speed of muscle shortening (Barany, 1967).

Cross-innervation studies provided further evidence for a physiological role of the myosin ATPase - muscle shortening relationship (Barany and Close, 1971). In rat, the fast extensor digitorum longus muscle was cross innervated with the nerve of the slow soleus muscle and vice versa. This transformed the fast extensor to a slow muscle and the slow soleus to a fast muscle. The changes in the myosin ATPase activity closely followed the changes in the speed of shortening of the cross-innervated muscles (Fig. M8). The high actin-activated ATPase activity of myosin from the extensor muscle was reduced to the low ATPase activity of myosin from the soleus. At the same time the myosin ATPase activity of the cross-innervated soleus was elevated to the level of the normal extensor. A new type of myosin is synthesized in the cross-innervated muscle, which carries the genetic information necessary to determine the speed of muscle contraction.

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Fig. M8. The relationship between speed of muscle shortening and actin-activated ATPase activity of myosin in cross-innervated muscles. The symbols represent normal or cross-innervated extensor digitorum longus and soleus muscles.

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Myosin heavy chains: The myosin heavy chain (MHC) of different skeletal muscles exhibits diversity. MHC from slow muscle has a slightly lower molecular weight than MHC from the fast muscle, as assessed by SDS-PAGE. The mHc isoform expressed in a single muscle fiber is correlated with the contraction speed of the fiber (Reiser et al., 1985).

During development of the muscle, its contractile properties and myosin isozyme composition are changing. As the muscle speed is increasing more of the fast type MHC is incorporated into the fibers. Furthermore, changes in MHC composition were demonstrated upon increased use of muscle, resulting in hypertrophy, or upon denervation causing atrophy.

Three-dimensional structure of subfragment 1: Rayment and collaborators (1993) crystallized myosin subfragment 1 and determined its three-dimensional structure by x-ray diffraction at 2.8-A resolution. The structure of myosin S1 is illustrated in Fig. M9. The picture shows the head of S1 consisting of a seven-stranded beta-sheet and a C-terminal tail containing the regulatory light chain (magenta) and the essential light chain (yellow). The proteolytic fragments of S1 are color coded as follows: 25K (N-terminal), green; 50K, red; and 20K (C-terminal) blue. The 50K fragment spans two domains: the 50K upper domain and the 50K lower domain or actin-binding domain (grey). Part of the 50-kDa and 20-kDa fragments form the actin-binding site, whereas part of the 50-kDa and 25-kDa fragments of S1 form the ATP-binding site. The ATP-binding site is about 4 nm from the actin-binding site. The ATP-binding site has the sequence GLY-GLU-SER-GLY-ALA-GLY-LYS-THR, which is similar to the sequences found in the active sites of other ATPases. The ATP-binding site was identified as a pocket. There is a cleft in the upper part of the head that extends from under the ATP-binding site to the actin interface. Both portions, above and below the cleft are involved in the actin binding. When ATP binds to S1, the pocket most likely closes and the cleft widens disturbing the S1-actin binding, that is ATP dissociates S1 from actin. When ATP is hydrolyzed by S1 to ADP and Pi, actin recombines with S1. The accompanying structural changes are the narrowing of the cleft and opening of the ATP-binding pocket. These subtle changes are called conformational changes that play a key role in the mechanism of muscle contraction.

The Converter Domain (Houdusse and Cohen, 1996) is seen on the bottom of Fig. M9. This is a small compact domain that functions as socket for the C-terminal tail of S1, which is its regulatory domain.

The regulatory and essential light chains wrap around the 20-kDa fragment of S1, which forms an 85A-long a helix that spans much of the S1. Rayment suggested that the conformational changes in the nucleotide-binding pocket could be transmitted to the 85A-long a helix. This structure may play the role of a lever arm, which magnifies small conformational changes to larger movements. The lever arm hypothesis is presented in the Actin-Myosin Interaction chapter.

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Fig. M9. Ribbon representation of the structure of S1 (From Geeves and


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Structure-function relatonship in myosin: Two prominent trypsin sensitive surface loops form the border of the 25-, 50-, and 20-kDa subdomains of S1. The first loop, loop 1, spanning the 25/50-kDa junction is situated near the nucleotide binding site and is involved in determining the rate of ADP release. The 50/20-kDa junction, loop 2, is involved in the interaction between actin and myosin. Nine residues were deleted from loop 2 without affecting the nucleotide binding properties of myosin (Knetsch et al., 1999). However, the deletion affected actin binding and the communication between the actin and nucleotide binding sites. Thus, these studies demonstrated different pathways of communication between the actin and nucleotide binding sites. Further studies by a different group (Joel et al., 2001) showed that elimination of two highly conserved lysines at the C-terminal end of loop 2 specifically blocked the ability of HMM to undergo a week to strong binding transition with actin in the presence of ATP. Removal of these lysines had no effect on strong binding in the absence of nucleotide, on the rate of ATP binding or release, or on the basal ATPase activity. The data suggested that the interaction of the two conserved lysines with acidic residues in subdomain 1 of actin either triggers a structural change or stabilizes a conformation that is necessary for actin-activated Pi release and completion of the ATPase cycle.

Of the functional sites of S1 there is a helix containing the reactive sulfhydryls, Cys707 (SH1) and Cys697 (SH2). The SH1-SH2 helix can undergo structural changes in the presence of ATP and its derivatives. For instance SH1 and SH2 can be cross-linked in the presence of nucleotides with reagents of spans ranging from 5 to 15 A, although the distance between the sulfur atoms in the native helix is as much as 19 A. Therefore, in the presence of nucleotides the helix must undergo some conformational changes in order for SH1 and SH2 to come close to each other. Using the cross-linking approach, it was shown that nucleotides induce a flexibility of the SH1 and SH2 helix, in other words nucleotides shift the equilibria among conformational states of the helix (Nitao and Reisler, 1998).

Myosin filament: At low ionic strength, e.g. 0.03 M KCl, myosin precipitates and forms filaments. Electron micrographs reveal the specific structure of the filaments, that is their central shaft and side projections (Fig. M10).

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