INTRODUCTION
Since antiquity, motion has been looked upon as the index of life. The organ of motion
is muscle. Our present understanding of the mechanism of contraction is based on three
fundamental discoveries, all arising from studies on striated muscle. The modern era
began with the demonstration that contraction is the result of the interaction of
two proteins, actin and myosin with ATP, and that contraction can be reproduced in
vitro with purified proteins. The second fundamental advance was the sliding filament
theory, which established that shortening and power production are the result of interactions
between actin and myosin filaments, each containing several hundreds of molecules
and that this interaction proceeds by sliding without any change in filament lengths.
Third, the atomic structures arising from the crystallization of actin and myosin
now allow one to search for the changes in molecular structure that account for force
production.
Mostly I will discuss how biochemical studies from 1941 to 1972 contributed to our
understanding of contraction. I shall particularly focus on two aspects of the history:
the work of the Szeged school, since the papers in the Studies from the Institute
of Medical Chemistry University Szeged were and are not readily available;1 and the
history of the proteolytic fragments of myosin HMM and S1 that allowed studies of
in vitro contraction in solution. In a few cases more recent information will be quoted
for clarity.
The Cold Spring Harbor Meeting in 1972 was perhaps a watershed in muscle research
where the outlines of contraction and its regulation were enunciated. Indeed, the
general atmosphere at this time was most optimistic and it was thought that the full
solution to the problem was imminent. In fact, it took another 30 yr of intensive
study to begin to understand the conformational changes undergone by myosin during
the contractile cycle.2
The Beginning
A viscous protein was extracted from muscle with concentrated salt solution by Kühne
(1864), who called it “myosin” and considered it responsible for the rigor state of
muscle. Muralt and Edsall (1930) showed that the “myosin” in solution had a strong
flow birefringence with indications that the particles were uniform in size and shape.
Occasionally a preparation was obtained that lacked flow birefringence, which was
discarded. In 1935, Weber (1935) developed a new technique for the in vitro study
of contraction. He squirted “myosin” dissolved in high salt into water where it formed
threads that became strongly birefringent upon drying. Engelhardt and Lyubimova (1939)
reported in a careful study that myosin had ATPase activity. The importance of the
finding was underlined by the earlier findings of Lohmann (1934) that ATP was likely
to be the energy source for contraction. Nevertheless, the idea that myosin was an
ATPase was not universally accepted because enzymes were considered to be small globular
proteins, which myosin clearly was not; however, efforts by Polis and Meyerhof (1947)
to separate an ATPase from myosin failed. Engelhardt et al. (1941) also checked the
effect of ATP on the “myosin” fibers of H.H. Weber and found that the fibers became
more extensible.
Engelhardt and Lyubimova's experiments represented the opening salvo in the revolution
of muscle biochemistry. Albert Szent-Györgyi and colleagues then established that
the “myosin” used by previous investigators consisted of two proteins. These were
purified and shown to be necessary for the contraction elicited by ATP. This work
took place during the war years in complete scientific isolation, without access even
to Nature and Science. The results were published in three volumes of Studies of the
Institute of Medical Chemistry University of Szeged during the years 1941–1943.1 The
most important scientific instruments available were a simple Ostwald viscometer and
polarizing filters to detect double refraction of flow. Banga and Szent-Györgyi (1942)
observed that exposure of ground muscle to high salt concentrations for 20 min extracted
a protein of low viscosity (myosin A), whereas overnight exposure solubilized a protein
with high viscosity (myosin B). The viscosity of myosin B was reduced by adding ATP
while the viscosity of myosin A remained essentially unaffected. The effect of ATP
on Kühne's “myosin” was independently discovered by Needham et al. (1942). On account
of the war these two groups were never able to communicate. Needham et al. (1942)
found that ATP reduced the viscosity and flow birefringence of “myosin”. These changes
were reversed upon exhaustion of ATP. They proposed that ATP caused a reversible change
in the asymmetry of the “myosin” molecule possibly due to the shortening of the molecule,
or changes in the interaction between micellae formed by myosin molecules. They thought
it was likely that the change in birefringence resulted from enzyme-substrate combination.
Szent-Györgyi (1942a) discovered that the threads prepared from myosin B using H.H.
Weber's method shortened on addition of boiled muscle juice (Fig. 1), but when fibers
of myosin A were tested these remained unchanged. The shortening was apparently due
to exclusion of water. The active material in the boiled extract was identified as
ATP. In his autobiography, Szent-Györgyi (1963) describes that “to see them (the threads)
contract for the first time, was perhaps the most thrilling moment of my life.”
Figure 1.
Contraction of actomyosin threads (“myosin B”) on addition of ATP. Shown are the same
thread A before and B after addition of boiled muscle juice (a source of ATP) (Szent-Györgyi,
1942a).
Straub joined Szent-Györgyi about this time and it became clear that the difference
between myosin B and A was due to the presence of another protein that they called
“actin”, which, when combined with myosin, was responsible for the high viscosity
and for contractility. Myosin A was purified as paracrystals by Szent-Györgyi (1943a)
and retained the name myosin. In a very elegant series of experiments actin was purified
by Straub (1942). Myosin B was renamed actomyosin (Szent-Györgyi, 1942b). Straub (1943)
showed that the newly discovered protein existed in two forms: globular actin (G-actin)
that was stable in the absence of salt, and in the presence of ions it polymerized
to form fibrous actin (F-actin). The steady-state ATPase activity of actomyosin, but
not of myosin alone, was activated by magnesium (Banga, 1942). The effect of ATP on
viscosity and birefringence was imitated only by one other trinucleotide ITP (Needham
et al., 1941; Dainty et al., 1944). Contraction was not elicited by ADP (Szent-Györgyi,
1943b). The formation of a rapidly sedimenting coarse precipitate of suspended actomyosin
formed by adding ATP and called “superprecipitation” was also taken as a measure of
contraction. Furthermore, Szent-Györgyi demonstrated that ATP had a dual function
that depended on ionic strength. At low ionic strength ATP induced contraction, at
high ionic strength it dissociated actin from myosin (Guba, 1943). It was realized
that the rigor state was due to the formation of actomyosin in the absence of ATP.
In fact, rigor mortis was the result of the depletion of ATP (Erdös, 1943). It was
further shown that the steady-state ATPase activity was increased during the contraction
of actomyosin or of minced and washed muscle (Biró and Szent-Györgyi, 1949). The claim
that the in vitro contraction of actomyosin threads and superprecipitation (mostly
due to dehydration) were equivalent to contraction of living muscle was not universally
accepted. Astbury (1947), in his Croonian lecture, proposed that the cross-β pattern
(a structure produced by stretching and releasing hair) represented contracted myosin.
Both he and Meyerhof believed that superprecipitation was an artifact. (In spite of
their scientific differences Astbury and Szent-Györgyi remained close friends and
Astbury spent the summer of 1953 in Szent-Györgyi's cottage in Woods Hole.) However,
the development and the behavior of the glycerol extracted psoas muscle preparation
by Szent-Györgyi (1949) brought conclusive evidence that the interaction of ATP with
actomyosin was the basic contractile event. The glycerol extracted psoas muscle preparation
consists of a chemically skinned muscle fiber bundle that is permeable to ions. On
addition of Mg2+ ATP the preparation develops a tension that is comparable to the
tension development of living muscle. Moreover, the preparation behaves somewhat like
actomyosin. The glycerinated psoas muscle preparation, with some modification, is
still used today for structural studies.
The demonstration that contraction can be reproduced in vitro by two proteins, actin
and myosin, opened up the modern phase of muscle biochemistry. It made possible the
interpretation of structural features of striated muscle that formed the basis of
the sliding filament theory. It simplified the study of contraction, allowed one to
focus on the way the ATP energy is used and facilitated the beginning of the discussion
that relates structural changes with biochemical events.
The Myosin Molecule
About 400 myosin molecules assemble to form a filament, which interacts with actin
filaments containing about the same number of actin monomers (Hanson and Lowy, 1963;
Huxley, 1963). Similar filaments form readily in vitro by self-assembly except that
they display variable filament lengths (Huxley, 1963).
Myosin therefore has multiple functions: filament formation, ATPase activity, and
reversible combination with actin. The use of proteolytic enzymes revealed which regions
of the myosin molecule were responsible for each of these different functions. This
approach was initiated independently by Gergely (1950) and by Perry (1951), who wanted
to see if the ATPase activity might be separated from myosin. Both of these authors
observed that the ATPase activity was solubilized by a short tryptic digestion. However,
they concluded that the soluble fraction did not combine with actin since the rise
of viscosity on addition of actin was relatively small (Gergely, 1953). Further investigations
by Mihályi and Szent-Györgyi (1953a) confirmed that the ATPase was solubilized, but
ultracentrifuge evidence indicated that the soluble fraction also bound to actin.
Tryptic digestion of myosin resulted in the formation of two well-defined components
that sedimented differently at high ionic strength (0.6 M KCl). The two new peaks
formed in an all or none fashion, at intermediate digestion times only the two new
peaks and that of intact myosin were observed. In the presence of actin the faster
peak sedimented very rapidly, indicating that it bound to actin (Mihályi and Szent-Györgyi,
1953a). The splitting of the native myosin into two main components during the first
rapid phase of tryptic digestion did not decrease ATPase activity. The viscosity of
the digestion products was considerably reduced; nevertheless the amount of actin
needed to saturate the increase in viscosity was not altered by the rapid phase of
tryptic digestion. The two principle components were separated using differential
centrifugation in the presence of actin, which was removed by a second centrifugation
in the presence of ATP. Both ATPase activity and the ability to combine with actin
was retained by the rapidly sedimenting component (Mihályi and Szent-Györgyi, 1953b).
Further investigations used a simple method for the separation of the two components
of tryptic digestion (Szent-Györgyi, 1953). The slow component was named light meromyosin
(LMM), and the faster sedimenting component heavy meromyosin (HMM). The separation
was based on differences of solubility at low concentrations of monovalent cations
and also in fractionation by ammonium sulfate. LMM had a very similar solubility to
intact myosin in both of these reagents; it precipitated below 0.2 M KCl and at 30%
ammonium sulfate saturation. HMM remained fully soluble at low ionic strengths while
LMM formed paracrystals.
The region connecting LMM with HMM was also available to some other proteolytic enzymes,
such as chymotrypsin, producing fragments very similar to the meromyosins (Gergely
et al., 1955). Mihályi and Harrington (1959) reported that trypsin rapidly attack
a 64-residues long region between LMM and HMM, suggesting the possible absence of
the coiled coil structure at the LMM-HMM junction. The paracrystals of LMM showed
a 420 ± 25 Å periodicity even without staining or shadowing (Philpott and Szent-Györgyi,
1954). This periodicity agreed with the fundamental fiber period obtained by Huxley
(1953a), indicating a structural role for LMM in filament formation and interactions
between myosin molecules. LMM was further purified by ethanol precipitation (Szent-Györgyi
et al., 1960), probably removing peptide material attached to myosin or formed during
digestion (LMM fraction 1). It showed the characteristic periodic structure in electron
microscopy. Fibers prepared from purified LMM fraction 1 showed 10 orders of 428 A
repeat with strong meridional reflections at 143 and 70 Å. Optical rotatory dispersion
indicated that LMM is a fully coiled α-helix (Cohen and Szent-Györgyi, 1957). Sedimentation
data suggested that LMM and HMM were linearly attached in myosin (Lauffer and Szent-Györgyi,
1955)
The division of roles between LMM and HMM then became clear. LMM is responsible for
filament formation, whereas HMM contains the sites responsible for ATPase activity
and also the sites for interacting with actin. The working out of how the meromyosins
form a myosin molecule was made difficult by three factors: the low estimates of the
molecular weights of the meromyosins—uncertainties of the molecular weight of myosin
ranging from 400,000 to 1,000,000; uncertainty in the number of parallel peptide chains;
and uncertainties in the estimation of lengths both of myosins and meromyosins. Clarification
came from two directions: electron microscopy and analysis of the products of prolonged
tryptic digestion.
Mueller and Perry (1962) showed that exposure of HMM to trypsin for an extended time
converted it to a single major component sedimenting more slowly, called subfragment
1 (S1), but also yielded a more heterogeneous component known as subfragment 2 (S2).
Kominz et al. (1965), using papain, were able to obtain a rather homogeneous S1 component,
which very likely was responsible for the thickening seen at the ends of the 1,600
Å long myosin molecules (Huxley, 1963). The length of LMM was estimated to be ∼900
Å from gap-overlap structures of LMM paracrystals (Huxley, 1963). The important electron
microscope studies of Slayter and Lowey (1967) established that a myosin molecule
ended in two globules (heads). This proved that myosin was made up of two parallel
peptide chains, in contrast to previous studies proposing a three-chain structure
(Woods et al., 1963). HMM is therefore a two-headed molecule connected to LMM via
S2; LMM plus S2 forms the rod portion of the molecule, which for most of its length
is a coiled-coil α-helix (Fig. 2). Lowey et al. (1969) showed that, at low ionic strengths,
papain or chymotrypsin splits myosin into S1 and rod. The S1 combined with actin and
was a fully active ATPase. In addition, a homogeneous helical S2 fragment was obtained
(Lowey et al., 1967). Later a longer S2 was isolated (Sutoh et al., 1978), which was
flexible. A portion of it had a low melting point. It appears therefore that the LMM
is responsible for the core of the filaments and the flexible S2 allows the myosin
heads to reach out to the actin filaments. Thus, HMM consists of two cross-bridges
connected by a short rod. Moreover S1, containing the active site and the site interacting
with actin, is essentially an isolated cross-bridge and is a very suitable preparation
for the study of most aspects of the cross-bridge cycle (Huxley, 1963).
Figure 2.
The myosin molecule (adapted from Alberts et al., 2002).
The Light Chains
The presence of small subunits of myosin was first observed by Tsao (1953), who found
that prolonged urea treatment produced low molecular weight components in addition
to the larger subunits. Various denaturing agents including alkali treatment (Kominz
et al., 1959) also dissociated small molecular weight components from purified myosin
preparations. To decide if these were true subunits, their stoichiometry, their effects
on ATPase activity, and their location within the myosin molecule had to be demonstrated.
Two classes of light chains have been reported. One class could be removed with LiCl
with the concomitant loss of ATPase activity (Stracher 1969; Dreizen and Gershman,
1970). Partial recovery of ATPase was reported by recombination of the isolated light
and heavy chains after removal of LiCl. This class of light chains was named the “essential
light chains”. Another class of light chains could be reversibly removed by 5,5′-dithiobis-(2-nitrobenzoic
acid) (DTNB) treatment without significant loss of ATPase activity (Weeds, 1969; Gazith
et al., 1970). This class of light chains was named the “regulatory light chains”
because they are directly involved in regulation of ATPase activity and contraction
in molluscan muscles (see Szent-Györgyi, 1975). There are two moles of regulatory
and two moles of essential light chains in a mole of myosin (Lowey and Risby, 1971;
Weeds and Lowey, 1971). In myosin from fast rabbit muscle there are two types of essential
light chains, A1 and A2. A1 has an extra 41 amino acid extension possibly indicating
the presence of two populations of myosin in adult rabbit (Weeds and Lowey, 1971).
The light chains bind to S1 (Trotta et al., 1968; Weeds and Lowey, 1971). S1 obtained
by digestion with chymotrypsin at low ionic strengths contains only essential light
chains (Weeds and Lowey, 1971), However, significant amounts of the regulatory light
chains are preserved in S1 that is prepared by digestion with papain at low ionic
strengths. Sequences of both light chains indicate the presence of 4 EF hands; but
only one of these contain the ligands necessary for divalent cation binding (Collins,
1991). The light chains play a crucial role in regulating contraction in molluscan
muscles and phosphorylation of the light chains is necessary for smooth muscle activity.
Actin
Actin was discovered by Straub (1942). Together with myosin and ATP it constitutes
the contractile system. In the absence of salt, actin molecules are stable as monomers
(G-actin); in the presence of salt, especially divalent cations, actin polymerizes.
The high asymmetry of the polymerized actin (F-actin) is indicated by its high viscosity,
thyxotropy and strong double refraction (Straub, 1942). The preparation of actin necessitated
overcoming a number of problems. Actin in muscle is present as F-actin that usually
is extracted together with myosin after ATP is completely hydrolyzed. The strategy
therefore was to remove myosin from fresh muscle still containing ATP. The remainder
of the myosin was denatured by acetone. Actin then was depolymerized and extracted
in a mildly alkaline salt-free solution. Use of salt or acidity below pH 6.0 had to
be avoided to prevent repolymerization (Straub, 1943). Straub and Feuer (1950) found
that ATP was a functional group of G-actin, its removal from G-actin by dialysis resulted
in loss of polymerizability. Asakura (1961), using sonication, demonstrated that polymerization
is associated with ATP hydrolysis. However, hydrolysis of nucleotides is not essential
for F-actin formation. ADP alone can support a slow polymerization (Hayashi and Rosenbluth,
1960). Nonhydrolizable ATP analogs are also effective (Cooke and Murdoch, 1973). Polymerization
begins very slowly, because nucleation is slow, but introduction of nuclei in the
form of small amounts of sonicated fragments leads to an explosive reaction (Higashi
and Oosawa, 1965). It was proposed that F-actin formation is a condensation process
and that a nucleus consists of about four monomers (Asakura et al., 1963).
Actin polymerizes in the presence of salts to form a long-pitch two-stranded helix
with a periodicity of ∼360 Å (Hanson and Lowy, 1963). In negatively stained actin
filaments ∼13 actin subunits can be visualized within the two strands that form the
helical repeat of 360 Å.
The Sliding Filament
Cross-striated muscle is organized in sarcomeres, repeating units ∼2–3-μm long (Fig.
3). Huxley, in his Ph.D. thesis in 1952 (see Huxley, 1953a), observed that the basic
meridional periodicities of muscle remain constant at various muscle lengths. Equatorial
reflections indicated the presence of two filamentous structures. Moreover, electron
microscopy revealed the presence of two types of filaments: 1.6-μm long thick filaments
located in the A-band and 1-μm long thin filaments stretching from the Z-band to the
H-zone (Huxley, 1953b, 1957a). Hanson and Huxley (1953) observed that high concentration
of salts in the presence of ATP removed the A-band protein, which was therefore identified
as myosin. Hasselbach (1953) also observed independently the removal of myosin from
the A-band with pyrophosphate solution. Myosin added to the ghost fibers bound to
the thin filaments demonstrating that these contained actin. Light microscopic observations
distinguished a zone of high refractive index, the A (anisotropic) band from the I
(isotropic) band.
Figure 3.
Cross-striated muscle is organized in sarcomeres that extend from one Z-line to the
next. The distance between Z-lines is 2–3 μm. The thin filaments contain actin and
the thick filaments contain myosin. The thick filaments have bipolar symmetry with
a central bare zone in which there are no cross-bridges. The actin fiber symmetry
reverses in the Z-line. The area not penetrated by the thin filaments is variable,
and is known as the H-zone.
The sliding filament theory was based on the observations of constancy of the length
of the A-band and the shortening of the I band during a contraction. As pointed out
by A.F. Huxley, this observation was made by applying interference microscopy to the
most differentiated motile system available, namely intact frog muscle fibers (Huxley
and Niedergerke, 1954). A very similar observation was made on glycerol-extracted
myofibrils using phase contract microscopy (Huxley and Hanson, 1954). These authors
were also able to associate actin with the thin filaments and myosin with the thick
filaments. The sliding filament hypothesis was proposed to explain these observations
(Fig. 4). The association of thin filaments with actin and thick filaments with myosin
was later verified by electron microscopy (Huxley, 1957a; Hanson and Lowy, 1963).
Previous theories took it for granted that contraction was the result of a length
change in long polymer-like molecules. Until the epoch-making papers in 1954 the idea
that movement might result from a process other than the shortening of molecular structures
had just not been considered. However, the studies cited above clearly showed that
the filaments did not change their lengths during contraction and thus heralded a
new era.
Figure 4.
2-μm long myosin containing thick filaments with cross-bridges and 1-μm long actin
containing thin filaments are shown. As the sarcomere shortens, the myosin cross-bridges
react with actin and propel the thin filaments toward the center of the sarcomere.
Both filament types remain at constant lengths during contraction. The sliding of
the filaments explains the constancy of the A-band and the changes of the I-band and
the H-zone.
During a contraction actin filaments move toward the center from both halves of the
sarcomere. This necessitates a change in direction (orientation) of the actin filaments
every half sarcomere. The directionality is built into the way actin and myosin assembled
into filaments. Thick filaments are bipolar structures. Their assembly begins with
the tail-to-tail association of the LMM fractions so that the heads come out pointing
in opposite directions. Then filaments grow by addition of myosin molecules onto these
bipolar nuclei. The overall result is a smooth central region 0.2-μm long that is
free of myosin heads, while the molecules in the two halves of the filaments face
in opposite direction (Huxley, 1963). The polarity of the actin filaments was established
from the asymmetric structures seen by electron microscopy when complexed with S1
or HMM (“decorated actin”). The filaments showed a pointed and a barbed end. The barbed
ends attached to both sides of the Z-line so that the actin molecules faced toward
the center of the sarcomere (Huxley, 1963). This symmetric structure of the filaments
attached to the Z-line assures that actin containing filaments slide toward the center
of the sarcomere.
Contraction is driven by cross-bridge activity. Cross-bridges were clearly visualized
by Huxley (1957a) by electron microscopy of ultra-thin sections. Shortly afterwards,
Huxley (1958) proposed a mechanical cross-bridge cycle that is similar to present-day
models. A.F. Huxley (1957b) investigated the idea that the entropy of cross-bridge
attachment may be used to drive the cross-bridge cycle, which is still a relevant
idea. However, the first direct evidence for a change in cross-bridge shape that might
provide the basis for movement was obtained by Reedy et al. (1965), who discovered
that the angle of the myosin cross-bridge in insect muscles depended on the state
of the muscle. Electron microscopy combined with X-ray diffraction showed that at
rest the cross-bridges extended at right angle from the thick filament (90°), whereas
in rigor (no ATP present) the cross-bridges protruded at an acute angle (45°). Therefore,
when Huxley (1969) put forward a swinging cross-bridge model, proposing that the myosin
head attached to actin changes its angle during the contraction cycle, the idea was
widely supported. Nevertheless, in point of fact it took many years to produce direct
evidence in support of the swinging cross-bridge model.
The Cross-bridge Cycle
Filament sliding is generated by interactions of the cross-bridges with actin. This
interaction can be studied with the soluble fragments of myosin, S1, and HMM. The
use of transient kinetics to explore the steps of the cross-bridge cycle was introduced
by Tonomura and colleagues, who showed that there is an initial rapid liberation of
phosphate by myosin (Kanazawa and Tonomura, 1965). The kinetic analysis with S1 indicated
the existence of several ATP states and several ADP states (Bagshaw and Trentham,
1974). Kinetic analysis also demonstrated that the bound ATP was in equilibrium with
the bound ADP and inorganic phosphate. An equilibrium constant of ∼7 indicated the
reversibility between the states of the bound ATP and bound ADP and Pi. Therefore,
hydrolysis of the ATP does not dissipate its energy while the nucleotide is bound.
The cross-bridge cycle was proposed by Lymn and Taylor (1971) based on two fundamental
findings: they provided evidence that hydrolysis of ATP occurs in the detached state
when myosin is not bound to actin; they also showed that the addition of ATP to myosin
results in a burst of ATP hydrolysis that was nearly stoichiometric with the myosin
heads. The burst occurred when the active site was unoccupied, so this finding indicated
that the dissociation of ADP was the limiting reaction of the cycle. They proposed
a four-state model (Fig. 5). In the detached state the myosin undergoes a structural
change from the state reached at the end of contraction to the state formed after
ATP hydrolysis. When attached to actin the conformation of the cross-bridge is altered,
which propels actin forward by a step. Force production is associated with products
release whereupon the cross-bridge assumes the rigor configuration. The release of
ADP enables the rebinding of ATP. The affinity of myosin to actin is greatly reduced
by ATP binding and myosin detaches from actin. This is a simplified model omitting
a number of intermediates, nevertheless it describes the essential steps of the cycle
and continues to be used. The ATP and the product-bound states are weak-binding states;
the transition to the strong-binding state and concomitant release of products is
required for force production. In striated muscle there is a strong coupling between
ATP binding to myosin and actin binding to myosin. This is reflected in a large mutual
reduction in affinities.
Figure 5.
The cross-bridge cycle. Note that ATP hydrolysis takes place in the detached state.
In the actin-bound state contraction is associated with the dissociation of the hydrolysis
products; recovery of the resting state structure follows dissociation of myosin from
actin by ATP (see Taylor, 2001).
A mechanistic relationship between possible cross-bridge movements and the mechanical
properties of muscle first was proposed by Huxley (1957a). However, it turned out
that an understanding of the structural changes that the myosin cross-bridge undergoes
during a cycle necessitated the determination of the structures of actin and the myosin
cross-bridge at atomic resolution. This took another 30 yr! In the meantime, several
important techniques were developed and yielded new understanding at the molecular
level. Application of electron spin resonance and fluorescence energy transfer for
the study of muscle proteins were introduced by M.F. Morales (Dos Remedios et al.,
1972). New techniques were developed to deal with the physiology of single molecules—the
mechanical equivalent of patch-clamps. The first was a direct demonstration of the
in vitro sliding of actin filaments over lawn of myosin molecules attached to a cover-slip
(Kron and Spudich, 1986). Later came measurements of the step size and tension induced
by single myosin molecules acting on an actin filament attached to a very thin glass
needle (Kishino and Yanagida, 1988). This method was refined by holding the actin
filament between beads in a laser trap (Finer et al., 1994). Other very important
developments were the expression of myosin (De Lozanne and Spudich, 1987), the production
of mutants in Dictyostelium discoideum (Patterson and Spudich, 1995), and the expression
of smooth muscle myosin in insect cells using baculo virus as a vector (Trybus, 1994).
Tropomyosin
Tropomyosin was discovered and isolated by Bailey (1946)(1948). The molecule has a
very high α-helix content. (Cohen and Szent-Györgyi, 1957). The presence of nonpolar
side chains in every three or four residues in the amino acid sequences indicate that
it is a two-stranded coiled coil (Hodges and Smiley, 1972). Tropomyosin is located
in the thin filaments, lying on a flat surface formed by the two strands of actin.
In studies using electron microscopy and small angle X-ray diffraction (Cohen and
Longley, 1966) magnesium salts of tropomyosin form paracrystals that have a repeat
period of 396 Å, indicating an elongated structure with an end-to-end overlap. Tropomyosin
can be removed from actin at low temperatures (Drabikowski and Gergely, 1962). It
also combines with troponin, the complex responsible for thin filament regulation
by blocking the actin sites necessary for binding myosin in a calcium-dependent manner.
Regulation of Contraction
By the time of the Cold Spring Harbor Symposium in 1972, the basic principles of regulation
were understood. A low Ca2+ concentration in the sarcoplasm activated the muscle;
removal of Ca2+ resulted in relaxation. Marsh (1951), working in Bailey's laboratory,
followed contraction by measuring the ATP-induced loss in centrifuged volume of homogenized
muscle fibrils. He observed that soluble muscle extract prevented the volume change,
i.e., prevented contraction. Boiling or acid treatment of the extract inactivated
the factor, which was also nondialyzable, indicating that it was a protein. The effect
of the factor was inhibited by 2 mM Ca2+. Bendall (1952) tested the muscle extract
on glycerinated fibers and found that it relaxed the fibers. Initially the “relaxing
factor”, or Marsh-Bendall factor, was considered to be an ATP-regenerating enzyme
such as creatine kinase or myokinase. However, it was not really soluble and could
be collected by high-speed centrifugation. Moreover, Kumagai et al. (1955) found it
to be identical with the Kielley-Meyerhof granular ATPase. The factor was later identified
by Hasselbach and Makinose (1961)(1963) and by Ebashi and Lipmann (1962) as fragmented
sarcoplasmic reticulum that acted as Ca2+-pump. The triggered release of sequestered
Ca2+ to ∼10 μM caused muscle to contract.
Actin-linked Regulation
Perry and Grey (1956) reported that EDTA inhibited only a crude actomyosin preparation
but not a synthetic preparation, a first indication of the involvement of a protein
modulating the activity of actomyosin. Weber and Winicur (1961) observed that the
Ca2+ sensitivity of different actomyosin preparations varied and that the variation
was due to differences in the way the actin was prepared. Ebashi and colleagues discovered
that for the relaxing effect tropomyosin was required. However, only “native” tropomyosin
was effective. This was due to an additional protein, named troponin (Ebashi, 1963;
Ebashi and Ebashi, 1964). In the thin filaments tropomyosin lies on the flat surface
formed between the two strands of actin. Tropomyosin's length somewhat exceeds the
pitch of the long actin helix so that the tropomyosin molecules overlap when binding
to actin. The presence of tropomyosin and the overlap between the tropomyosin molecules
confers cooperativity to the regulatory system (Bremel and Weber, 1972). Troponin
is arranged periodically, each tropomyosin binds one troponin molecule (Ohtsuki et
al., 1967). Greaser and Gergely (1971) showed that troponin consists of three different
subunits. TroponinC (TnC) binds Ca2+ and is related to calmodulin; troponinI (TnI)
is an inhibitory subunit that binds to TnC and to actin and troponinT (TnT) in a Ca2+-dependent
manner, and TnT binds to tropomyosin. It is thought that in the absence of Ca2+ the
affinity between TnC and TnI is strong so that tropomyosin is held over the myosin-binding
site of actin. In the presence of Ca2+ the binding between TnI and TnC weakens, tropomyosin
is allowed to roll azimuthally around actin to open up the binding-site for myosin.
Combination with S1 evidently leads to its further movement. This steric hindrance
model of regulation was based on observations of the low angle X-ray diffraction patterns
from muscle fibers. The second-order reflection of the actin period changes dramatically
on activation (Haselgrove, 1973; Huxley, 1973; Parry and Squire, 1973). This result
was later supported by electron microscopy (Vibert et al., 1997).
Myosin-linked Regulation
Molluscan myosins are regulated molecules. In contrast to skeletal muscle myosins,
these myosins have a specific high affinity Ca2+ binding-site. Binding of Ca2+ to
these sites is necessary for activity (Kendrick-Jones et al., 1970). A detailed study
of scallop myosin has shown that the light chains are regulatory subunits. Depletion
of the regulatory light chains by EDTA results in the loss of regulation and of the
ability to bind Ca2+. Rebinding of the regulatory light chains restores regulation.
Only two-headed molecules, myosin and HMM, are regulated. While retaining the ability
to bind Ca2+, S1 is fully active in its absence, although it can still be regulated
by the troponin-tropomyosin system (Szent-Györgyi et al., 1973). Ca2+ binding requires
that both regulatory and essential light chains are complexed with the heavy chain.
Isolated light chains are unable to bind Ca2+. Later studies have shown why this is
so. Szentkiralyi (1984) isolated the light chain-binding region, called the regulatory
domain, consisting of both light chains complexed with the associated heavy chain
fragment. The atomic structure of the regulatory domain located the bound Ca2+ on
domain 1 of the essential light chain. The Ca2+-binding loop is stabilized by the
regulatory light chain, mainly by interactions between Gly23 of the essential light
chain and Gly117 of the regulatory light chain (Xie et al., 1994). The mutation of
Gly117 to alanine abolished Ca2+ binding. Furthermore, conversion of cysteine into
glycine of the inactive skeletal regulatory light chain resulted in a “gain in function”
mutation (Jancsó and Szent-Györgyi, 1994).
Epilogue
The years of research between 1941 and 1972 were exciting. The results were often
quite surprising. However, peu a peu, a solid foundation for our understanding of
muscle function at the molecular level was established. The realization that movement
and force production require interaction between two proteins, the discovery of actin,
the sliding filament mechanism, the way contraction is controlled, and an understanding
of the manner in which the energy of ATP may be used all opened up new vistas. The
field also served as an example of how the combination of structural and biochemical
approaches can lead to a detailed understanding of a cellular function. The success
of the analysis was beyond most expectations. The road was not easy and errors abounded.
Nevertheless, a solid base for the next 30 yr of research has been established which,
no doubt, will often lead to results no one foresaw. The importance of a detailed
knowledge of motility in cell biology is manifest. Moreover, discoveries remain surprising
and repeatedly find unexpected applications in numerous cellular functions. In spite
of the expectations of 1972, the work is still far from complete. One may quote Albert
Szent-Györgyi:
“A discovery is said to be an accident meeting a prepared mind.”
We should stay prepared.