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THIN FILAMENT REGULATION
Michael A Geeves,
School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK
[email protected]
Key words: Actin, myosin, tropomyosin, troponin, calcium, cooperativity,
allosteric, coupled equilibria,
Synopsis:
The review summarizes the current state of knowledge of the calcium regulation of striated muscle
contraction via the thin filament proteins, tropomyosin and troponin. The description focuses on in
vitro studies of the thin filament and covers structural, biochemical and dynamic aspects of the thin
filament’s response to calcium binding. A reductionist approach has allowed many of the transitions
to be defined at the level of a single structural unit. Here an emphasis is placed on the cooperative
nature of the structural and biochemical transitions of the thin filament and the allosteric
relationship between calcium and myosin binding to the thin filament.
Glossary:
Thin filament.
The thinner of the two filaments found in muscle cells. It consists of a
polymer of several hundred actin monomers and contains the regulatory
proteins tropomyosin and troponin in a ratio of seven actins to one Tm and
one Tn (A7TmTn).
Motility
The velocity at which an actin filament moves over a surface coated with
myosin.
Cooperativity
A measure of the degree to which multiple ligands are required to induce a
structural and functional transition in a protein or protein complex.
Persistence length
A definition of the bending stiffness of a polymer chain. Average length over
which a thermally activated bend of 1 radian occurs
Inhibitory peptide
A short peptide of TnI that binds to actin, competes with myosin binding and
when bound to actin fixes the TmTn complex into the B or blocked position
on the actin surface.
Signal peptide
A peptide of TnI that binds to the N-terminus of TnC and enhances calcium
binding to TnC.
1
E-F hand
Abbreviations:
Tm
Tn
TnC
TnI
TnT
TnT1
TnT2
B-state
C-state
M-State
A calcium binding site of TnC. Named from the common peptide motif
(helix-loop helix) found in all calmodulin type calcium binding proteins.
tropomyosin
whole troponin
troponin C, the calcium binding component of Tn
troponin I, the inhibitory component of Tn
tropinin T the tropomyosin binding component of Tn
the N-terminal part of TnT
the C-terminal part of TnT
the blocked state of the thin filament
the closed or calcium induced state of the thin filament
the myosin induced state of the thin filament – also called the open state.
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1. INTRODUCTION
This review is focused on the regulation of muscle contraction via the thin filament in mammalian
striated muscle, which is the well studied muscle regulatory system. The regulation of contraction in
striated muscle is primarily via the ancillary thin filament proteins troponin and tropomyosin.
Tropomyosin (Tm) is a long coiled-coil protein which interacts with seven actin subunits in the actin
filament (1). Each tropomyosin is associated with one troponin protein complex (Tn) which is a
trimer of TnC (2), the calcium binding protein, TnI which interacts with actin and is known as the
inhibitory subunit (3) and TnT, the tropomyosin binding component (4), which keeps the whole
assembly together. The assembly of the TmTn complex in the filament is illustrated in Figure 1. Tm
polymerises head-to-tail along the actin filament with the TnI, TnC and the globular C-terminal part
of TnT, TnT2, associating with the middle of Tm and the extended N-terminal part of TnT (TnT1)
binding along Tm and reaching to the overlap between adjacent Tm molecules. The basic repeat
unit of the filament is therefore often referred to as A7TmTn, but since the seven actins of a single
long pitch strand of actin cannot exist in isolation, it is more accurately described in structural terms
as (A7TmTn)2 in which there are two parallel TmTn complexes on either side of the actin filament.
A low resolution cartoon of the thin filament structure. Spheres represent actin with the
coiled-coil of tropomyosin polymerised over the surface of seven actins. The extended N-terminal
part of TnT (TnT1) interacts with the overlap between adjacent Tm units and stabilises the Tm·Tm
interaction. TnT1 extends from the C terminus of Tm and with the C-terminal part of TnT (TnT2)
forms a compact structure with TnC and TnI at about 2/3 actins away from the Tm-Tm overlap.
From Heeley & Smillie 87(26).
Figure. 1:
The basic mechanism of the regulation of contraction and the structural changes involved has been
known for more than 40 years when the Steric Blocking model was first proposed (5-7). A modern
interpretation of this model is shown in Figure 2a & b. In the absence of calcium, TnI interacts with
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a specific actin subunit and fixes Tm on the surface of the actin filament in a position that sterically
blocks the myosin binding site on actin. Calcium binding to TnC induces it to bind TnI dissociating
TnI from its site on actin. TnI dissociation from actin releases Tm which moves to its preferred
position on actin, allowing myosin to bind to actin and allowing muscle contraction. TnT prevents
complete dissociation of TnI and TnC from the complex such that when the calcium concentration
drops, TnI readily rebinds to actin, i.e. when the calcium concentration falls, calcium dissociates from
TnC, and TnC detaches from TnI allowing TnI to rebind to actin. This much of the mechanism has
broad agreement and has been reviewed (8-9). However, debates remain on key issues, including
determining (a) the extent to which Tm movement is cooperative along the actin filament, (b) the
presence, and possible regulatory role, of a third structural a state of Tm on the actin filament
(Figure 2C), (c) if actin is a passive partner in the regulation (as implied by the structures in Figure 2)
or is some of the cooperativity communicated through structural changes in actin, and (d) strongly
bound myosin heads in the absence of ATP can activate the filament (10-11); what role do these
strongly attached cross-bridges have in the regulation of muscle contraction when ATP is present?
Figure. 2: Cartoon of the relationship between calcium binding to Tn, the thin filament structure
and myosin binding to actin. A. The relaxed or blocked state of the thin filament. A single strand of
A7TmTn is depicted. Actin as dark circles (dark blue) , Tm as a black line along 7 actins. TnI is a light
(blue) oval which in the absence of calcium binds tightly to one actin site and fixes Tm over the
surface of the myosin binding site on actin (circle segment red) i.e. myosin binding is sterically
blocked. TnC is shown in as a semi circle (brown) and in the absence of calcium has a weak
interaction with TnI. TnT (thin black line) holds TnC and TnI in close proximity to Tm. B. The calcium
induced structure of the thin filament; the C-state. When calcium binds to TnC this enhances the
interaction of TnC with TnI causing TnI to dissociate from actin. The complex remains bound to Tm
through TnT. When TnI dissociates from actin Tm reverts to its preferred binding site on actin and
exposes the sites on actin for myosin (triangle) to bind. C. The myosin induced structure; the Mstate. Full strong binding of myosin to actin requires Tm to move further away from the myosin
binding site into the M-state. Note each of these transitions is drawn as a change in the position of
the components and their interaction with their partners. Each of these transitions is driven by
changes in protein conformations and it is these changes in conformation that in many cases need to
defined.
While the basic structure of the thin filament has been known for more than 40 years, we still have
no high resolution structure of the filament nor of the detailed structural changes of the filament
that are involved in the regulation of muscle contraction. The essential problem is that the
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functional unit is a part of a filament; there is no crystal structure of the actin filament with or
without the TmTn components attached. What we do have is a series of low resolution structure of
the filament and high resolution structures of the different components. We also have functional
studies of the regulatory mechanism in vitro and in the muscle fibre. Putting all of this together
remains a challenge. One problem remaining is that while the reductionist approach has been very
successful at understanding the components and their contributions to regulation, we still lack a
clear understanding of regulation at the muscle fibre level. There remains a debate about the extent
to which regulation is a sum of the properties of the components or if there are “emergent
properties” that can only be studied at the whole filament or whole muscle level (see (12-14). In
essence, does an understanding of the behaviour of a single (A7TmTn)2 unit provide a complete
description of the system or can this only be discerned at a higher level? Some studies suggest that
a whole thin filament is for example either off or on (12).
This review is limited to in vitro studies of the regulatory mechanism and reference will be made to
some of the studies in muscle fibre. However, the underlying philosophy is that we need to
understand how the properties of individual components can account for calcium regulation before
invoking emergent properties of the system as an explanation. The reader is referred to the major
review by Gordon et al (2000) for an authoritative overview of the regulation at the muscle fibre
level which despite being 10 years old remains a comprehensive review of the system. For a review
of the cardiac muscle regulation system see (15).
This review will examine the structural studies of the thin filament before going on to look at other
in vitro data that reveal details of the mechanism of contraction regulation.
2. THE PLAYERS TROPOMYOSIN AND TROPONIN
2.1 Troponin C
Troponin C is a classical small calcium binding protein (18 kDa) of the calmodulin family with 4calcium E-F hands (Figure 3); two at the C-terminus with a high calcium affinity (~0.1 M) that can
also bind magnesium and two at the N terminus with low calcium affinity (1 M)(16). In cardiac TnC
only one of the N-terminal sites can bind calcium. The crystal structure of TnC in both the calcium
bound and calcium free states has been solved (2, 17 PBD 2TN4). The C-terminal sites are known as
structural sites as calcium binding does not regulate muscle contraction. In the cell, the C-terminal
calcium binding sites will always be occupied by either calcium or magnesium but predominantly
magnesium in a relaxed muscle when the calcium concentration is low (< 0.1 µM). Under what
conditions magnesium will exchange for calcium in the cell is not known.
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2.2 Troponin I
Troponin I is a globular protein which on its own
can bind to actin in a 1:1 stoichiometry and inhibit
myosin binding (reviewed by (3). This inhibition is
consistent with TnI and myosin competing for the
same site on actin with little or no coopertivity in
the interaction. A similar inhibition can be induced
by a fragment of TnI, the inhibitory peptide
(residue 96-115 rabbit TnI). When Tm is present,
TnI can inhibit myosin binding to actin at a lower
stoichiometry of one Tn to seven actin subunits
(18-19). This suggests that TnI binds to Tm and
together they occupy a single position on the actin
surface such that the myosin binding is blocked by
Tm. The alternative is that TnI induces a structural
change in actin which can be communicated to
adjacent actins only in the presence of Tm (20).
TnI can form a complex with TnC in the presence of
calcium, and TnC will limit the ability of TnI to
inhibit myosin binding to actin. This property is
associated with a second region of TnI known as
the switch peptide (residues 115-131) that has
shown to be able to bind to the N-terminal region
of TnC in the presence of calcium. The interaction
of the C-terminal domain of TnC with the essential
regions of TnI (and TnT) is shown in crystal
structures (21 PDB 1YTZ & PDB 1YV0, 22, 23 PDB
1A2X) and by NMR (24). The major interaction
sites between TnI and the rest of the thin filament
proteins are illustrated in Figure 4.
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Figure. 3: Structure of troponin C. The
image shows the main chain structure of
chickenfast muscle TnC with the
Nterminal regulatory domain shown at the
top and the C-terminal structural domain
at the bottom. The bound calcium ions
are indicated by blue spheres. The
structure was produce with MOLMOL
(Koradi et al.(1996) J Mol Graphics 14:
51-55]) with the coordinates PDB 1TCF .
With thanks to M. J. Bloemink & D.
Pearson.
Figure. 4: Cartoon of the calcium switch of the Tn complex in the thin filament based
on crystal structures of TnC with TnI and TnT2. This shows the role of the switch
(residues 116-131) and inhibitory (residues 104-115) peptides of TnI (blue) in transmitting
the calcium binding to TnC(in red) to the tropomyosin (pale blue strand)) position on actin
filament (grey strand). Upper panel shows the calcium bound state. In the absence of TnT1
(absent from the crystal structure there is litte contact between Tm or actin and Tn). Calcium
(black dots) binding to the N-terminal domain causes the regulatory domain to open allowing
the TnI signal peptide to bind in the cleft (the TnC & switch peptide contact surface is shown
in green). This straightens the TnC helix and prevents the inhibitory peptide from binding to
its site on actin. Tm sits in its preferred site on actin. The lower panel shows the calciumfree state with the inhibitory TnI peptide binding to actin and locating Tm in the blocked
position on actin. Note the collapse of the central helix of TnC and the closed regulatory
domain of TnC. Note also that the diagram shows the role of the inhibitory peptide (residues
104-115) but not the rest of the C-terminus of TnI (140-148) which also contributes to the
actin binding site. This is based on the crystal structure of skeletal muscle troponin core
domain(21). The crystal structure of the cardiac troponin core domain(21) does not show
the central helix straightening as depicted here. This region may therefore be quite mobile.
Figure courtesy of M. Vinogradova and R. Fletterick . Based on PDB 1YTZ & PDB 1YV0
2.3 Troponin T
Troponin T is a structural component which binds TnI, TnC and Tm (4). TnT can be cleaved into (or
expressed as) two fragments, the N-terminal TnT1 and the C-terminal TnT2. (25). TnT2 binds to both
TnI & TnC and with Tm forms a unit capable of regulating actin-myosin interaction. The TnIC-T2
complex has most of the calcium sensitivity of the parent whole Tn (26-28). Figure 4 shows the
TnICT2 complex as resolved in crystal structures.(21-22 PDB 1J1D & PDB 1J1E) TnT1 binds to Tm and
strengthens the Tm-Tm contacts through its binding in the Tm-Tm overlap region. TnT1 thereby
enhances both Tm binding to actin and cooperativity between Tm strands along the actin filament
(28-29). The fact that calcium regulation appears to work reasonably well in the absence of TnT
leads to a simple model of TnT as a structural component holding the TnC & TnI together with Tm.
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However, the remarkable number of mutations in TnT linked with human cardiomyopathies suggest
that the TnT can have a significant role in modulating the mechanism of regulation(30).
2.4 Tropomyosin
Tropomyosin is a linear parallel dimer comprised of a pair of alpha helical chains forming a single
relatively rigid coil-coil (1, 31 PDB 2TMA, 32 PDB 1C1G). There are two isoforms in striated muscle
 & β and in skeletal muscle the dimer can be either αα or αβ. The ββ structure is not stable at
physiological temperatures and is rarely found. The αβ complex can vary from < 10% to 50% of the
Tm in adult mammalian muscle. The properties of the αα vs αβ have not been well defined not least
because unlike smooth muscle Tm the skeletal muscle αβ isoform is difficult to isolate or to
assemble from α and β monomers. Tropomyosin can polymerise head-to-tail in solution to make
longer filaments but these tend to be unstable in solution and the filaments dissociate at high ionic
strengths (33-34). There are low resolution x-ray crystals of the polymerised Tm and both crystals
and NMR structures of tropomyosin fragments(35 PDB 1MV4). Of significance is that the recent Xray crystal structures of the Tm overlap region between the N and C terminal regions confirm the
morphology shown in earlier NMR structures (36 PDB 3MTU & PDB 3MDU, 37 PDB 2G9J, 38).
These show that the N-terminal coiled-coil of Tm remains intact at the N-terminus and this inserts
between the two C terminal chains which splay over the last 10-15 residues to form a 4--helix
bundle. The end-to-end interacting region of skeletal muscle Tm is relatively weak (11 amino acid
overlap) compared to smooth muscle and non-muscle Tms (15-16 amino acid overlap) and in
skeletal muscle Tm the overlap is stabilized by interaction with the N-terminal region of TnT. We
still need to know how the TnT N-terminal region interacts with the Tm-Tm overlap region of skeletal
muscle Tm. An interesting finding of the crystal structures of the overlap in smooth muscle Tm is
that the angle between the two sets of helices varied for the different constructs that were
crystallized. This may show that the region (at least in the absence of actin) has some flexibility and
could allow movement between adjacent Tms along the actin filament without breaking the contact.
Whether this is a component of the cooperativity between Tms along actin remains to be defined
but the structures do potentially provide a strategy to test this via mutagenesis in the overlap region.
Electron micrographs of Tm molecules and polymerised filaments together with molecular dynamic
simulations provide additional information on the structure of the molecule (39-42). These
experiments suggest that the Tm has a natural curvature which matches closely the structure of
actin. Thus, Tm requires little structural change to bind to the actin filament and conversely actin
provides a surface to allow Tm polymerisation and stabilises that most favourable form of Tm (4243). The molecular dynamics modelling also shows that the thermal motions of the Tm are evenly
distributed along its length – providing little evidence of kinks or specific bends in the structure. This
contrasts with other evidence suggesting specific bends or flexible joints in the coiled-coil, produced
by alanine clusters (44) or the presence of charge residues in the a and d positions of the heptad
repeat (45); places in a stable coiled-coil structure normally expected to be small hydrophobic
residues (46-47).
3. STRUCTURAL CHANGES WITHIN THE REGULATED ACTIN FILAMENTS.
Tropomyosin polymerises along the actin filament and interacts with 7 actins along one side of the
helical actin filament. Tropomyosin has 7-quasi repeat sequences in its structure providing potential
interacting sites with each actin subunit(46). Both electron micrograph images and low angle x-ray
scattering of muscle fibres suggests that Tm does not make direct specific contacts with the actin
surface but lies at a radius of 39-40 Å, significantly above the actin surface(48). This means that
there is little direct contact between Tm and actin and therefore Tm is bound via an electrostatic
potential well. This interpretation is compatible with the very low affinity of single Tm molecules
with actin filaments ( > 20 M (14, 49) and the view that Tm essentially polymerises on the surface
of actin. In such a model the Tm is expected to have significant thermal motion away from its most
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stable average position. The degree of movement will be constrained by the shape of the potential
well and the flexibility in the Tm structure, i.e. its persistence length on the surface of actin. While
the measured apparent persistence length of Tm in the absence of actin has been estimated as 140
nm (42) with an even longer dynamic persistence length (450 nm) how this is altered when it is
bound to actin is more difficult to define.
The electron micrograph images of actin.Tm and actin.TmTn filaments provide evidence of where
the average position of Tm is on actin but less about its thermal motion. Since we still have no high
resolution structure of the actin filament or Tm it remains problematic to model the precise
interactions between Tm and actin or to estimate the energy of the interaction sites. Fluorescence
and spin probes of Tm should be able to provide some information of Tm dynamic movement on the
surface of actin but the time scale of the probe dynamics is often not ideal and the probe movement
itself may be more extensive than local Tm dynamics. (50) Bi-functional probes coupled to stable helices, as used to orientate TnC within the thin filament(51) may provide another route to address
this question. To date environmental probes and FRET probes have provided contradictory evidence
of the type of Tm movements on the actin surface. This is partly because the Tm provides a rather
complex system for such studies. Tm is a dimer and in most studies the homodimer is used which
provides at least 2 equivalent locations for any probe. Using the FRET approach, with a second
probe site on actin, means that multiple distances are possible between both the Tm backbone sites
and the different actin subunits within a few nm.(52-55)
Helical averaging and single particle EM approaches to the thin filament structure have revealed
more details about the thin filament structure and how it changes in response to calcium or myosin
binding. These studies have revealed three distinct positions of Tm on the actin surface, see Figure
5. The position of Tm in these images was later shown to be compatible with low angle scattering
data from muscle fibres stretched to non overlap (48). Tm alone or TmTn in the presence of calcium
is found in what has been called the calcium induced or C-position. This is therefore assumed to be
the default position in which Tm preferentially sits as defined by the charge on the actin surface.
When Tn is present in the absence of calcium the Tm is moved over the surface where it blocks a
significant part of the myosin binding site on actin and is referred to as the blocked or B-position.
The charged N-terminal region of actin is still available and this is a region where both the inhibitory
peptide of TnI and the lower 50kD domain and Loop 2 of myosin are thought to compete for binding
to actin. When myosin binds to the filament (in the presence or absence of calcium) the Tm is
displaced further away from the B-position and is known as the M-position (myosin induced). In
summary, the Tm is observed in 3 distinct positions. In the presence of myosin (independent of Tn
and calcium) the Tm is displaced to the M position, away from the myosin binding sites on actin.
When myosin is removed Tm reverts to its preferred position, the C-position (a 10° movement from
the M-position). When Tn I is present and there is no calcium such that TnI binds strongly to actin
Tm is moved a further 15° to the B-position. Here Tm blocks a significant part of the myosin binding
site. Note that the images place Tm in the B-site at a place directly between the upper and lower
50kD domains of myosin and so it could block the binding of either domain to actin.
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Figure. 5: Three positions of Tm on the surface of actin compatible with EM images of
the thin filament and low angle X-ray scattering from muscle fibres. The image shows
actin subunits and tropomyosin as ribbon diagrams based on crystal structures of the
monomers docked in the EM and X-ray data. Individual actin subunits are shown in white,
sky blue and dark blue. Tm is shown in the three distinct positions, Red: B or blocked
position covering a significant part of the myosin binding site. Yellow: C or calcium induced
position in which most of the myosin binding site is exposed. Green the position observed
when myosins is bound to actin ( the M-or myosin induced position). From Poole et al.
2006(48).
In a related study, EM imaging has suggested the location of a TnI peptide in the filament in a
position for the Tn to interact not just with the Tm to which it is held by TnT but it could reach round
the actin filament for TnI to bind the actin site on the opposite strand. In doing so it may help to
position Tm on the other strand. If correct this means that the structural unit comprises 14 actins
and 2 molecules of TmTn(56). This has implications for the measurements of the cooperativity within
the filament and how this is modelled.
The images of the Tm on thin filament are striking and it is tempting to think that this is what would
be seen if we could look at single thin filaments. However, it has to be remembered that these are
fixed and/or averaged structures and that under physiological conditions protein structures are
dynamic on the sec time scale. The images report the average position of Tm. What the images do
not tell us is how the Tm moves from one position to the other, how much time the Tm spends in
positions other than the average shown. These relatively low resolution images also do not tell us
whether the actin structure changes in response to Tm binding or to the position of Tm on the actin
surface. These questions lead onto the major question of how cooperative are the structural
changes? Do single TmTn molecules move between the three positions or are movements
coordinated along the thin filament? The more rigid the Tm is thought to be and the stronger the
end to end interactions between adjacent Tms, the harder it is to imagine a single Tm being able to
move without the neighbours being perturbed. At the other extreme if the whole filament were to
change structure simultaneously, as suggested in some interpretations, the thermodynamics of the
switch are hard to imagine. What is the energy barrier between the positions that allows a m Tm
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strand to move as a single unit over the surface of actin yet have a well defined average position that
can block myosin binding? The question arises how many calciums binding to the TnCs along the
actin filament are required before the blocked state is lost? It is expected that occupancy of a single
TnC will locally weaken the TnI interaction with actin. How many TnI must dissociate before the Tm
strand moves taking the remaining TnI’s with it? Similarly how many myosins are required to bind
to actin before the whole filament adopts the M-state. On a simple steric blocking model, a myosin
cannot bind locally until the Tm has moved into the M-position. This implies that no myosin binding
occurs until the whole filament moves to the M-position. These are a few of the many questions that
arise when the cooperativity within the filament is considered. This cooperativity is the subject of
section 5.
4. BIOCHEMICAL STUDIES OF THE THIN FILAMENT REGULATION
The biochemical evidence for different states of the thin filament in solution has been reviewed
several times in recent years (8, 57-58) and only a short summary will be presented here. When
Tm and Tn, are present the removal of calcium results in inhibition of the ATPase activity of myosin,
inhibition of strong actin binding to myosin, and inhibition of actin motility over a surface coated
with myosin. Most commentators agree that there are more than 2 simple states (off and on) of the
thin filament, but there remain questions about the exact properties of each of the proposed
additional biochemical states; whether these are sufficient to explain all of the experimental data
and how they relate to the three structural states of Tm defined by EM and X-ray scattering. The
three state model as proposed by McKillop & Geeves (59) is shown in an extended version of Figure
2 in Figure 6 . Superficially, the three positions of Tm correspond to the three structural states
described in section 3. The biochemical model was defined from the ability of TmTn to control the
equilibrium and kinetics of binding of myosin to actin. Other models have been proposed based on
different types of experimental approach. Most, however, use the data on the equilibrium binding of
myosin or myosin nucleotide complexes to actin as the starting point. The basic observation is that
equilibrium binding of myosin to pure actin filaments is not cooperative but gives a simple
hyperbolic binding isotherm. When either Tm alone or TmTn are on the filament the binding is
cooperative. A caveat of these statement is that the binding of myosin S1 to actin is very tight ( ~1050 nM ) at standard 0.15 M ionic strength, so the binding isotherms need to be defined at very low
actin concentrations for precision. Early work used actin at micromolar concentrations which limited
the precision of the measurements. Later work was able to use phalloidin stabilised actin filaments
to attain lower actin concentrations and hence better precision. The sigmoid curves produced by
such titrations require at minimum, two states of the filament; an initial form that binds myosin
weakly and then a cooperative transition to a form that binds myosin tightly (see fig 2). Analysis of
the isotherms predicts a poised equilibrium between these states, and the data indicate 80% of the
low affinity form and 20% of the high affinity form in the presence of calcium. The absence of
calcium reduces the high affinity on state to much less than 20%. To have so few on-states in the
presence of calcium was surprising and indicated that full activation of the filament required binding
of myosin heads as well as the presence of calcium. It had long been known that the ATPase was
activated by myosin heads at low calcium (see below (11, 60).
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Figure. 6: The McKillop & Geeves cooperative 3-state model of myosin binding to
regulated actin filament. The figure uses the same symbols as in Fig 2. Note KB and KT
are properties of the thin filament and are calcium sensitive. K1 and K2 describe the two step
binding of myosin to actin and are properties of the myosin. K1 and K2 are independent of
calcium but are sensitive to the nucleotide bound to myosin. The 3-states were originally
referred to as the Blocked, Closed and Open states, the corresponding positions of Tm
observed in electron micrographs were called Blocked, Calcium-induced and the Myosininduced positions.
An analysis of the rate of myosin binding to actin filaments revealed a different class of off-states.
The experiment originally done by Trybus & Taylor (61) was extended by McKillop & Geeves (59).
This showed that the rate of myosin binding to pure actin, actin·Tm, and actin·TmTn were all very
similar provided a small number of myosin heads were allowed to bind to actin ( ~10%) to avoid the
complication of myosin induced cooperativity. However, when the reaction was repeated in the
absence of calcium the rate was slowed to about 30%. This is compatible with 70% of the actin sites
being unavailable for myosin to bind and therefore in a blocked conformation. To resolve the
different estimates of on and off states McKillop & Geeves proposed that the kinetic data only
resolved the blocked state, the rate of binding to the M & C states were similar, while the
equilibrium binding data could not distinguish between two classes of weak binding (weak and very
weak), both being difficult to detect. This lead to the three state model with the proportion of
B/C/M state being ~0.7/0.5/<0.05 in the absence of calcium and ~<0.05/ 0.8/0.2 in the presence of
calcium.
Since the model was first proposed we know much more about the binding of myosin to actin, and
the binding sites of Tm, Tn and myosin on actin. Geeves & Holmes (62)discussed the potential
pathway of docking of myosin on actin via initial electrostatic interactions (myosin loop 2 and actin
N-terminal acidic residues), followed by stereospecific hydrophobic interaction possibly via the lower
50 kDa domain of myosin. The final step would be closure of the 50 kDa domain cleft and formation
of the full interaction site. Cleft closure would initiate the changes in myosin structure that result in
product release and force generation. This outline remains speculation until a high resolution
structure is available, but the model is consistent with the known facts. As outlined in section 3
above, Tm in the B position does not block the ionic sites on the N-terminus of actin and would allow
the long rang ionic interactions between actin and myosin. At normal ionic strength these
interactions are very weak (Kd > 100 µM) but can be strengthened at lower ionic strength (Kd ~10
µM). Thus at very low salt concentration a complex between actin and myosin can still form and is
still regulated by calcium. Such complexes have been defined for M.ATP and M·ADP·Pi at very low
salt (63-64). However the blocked position of Tm would interfere with the formation of a stereospecific interaction between actin and myosin. In the C position the closing of the myosin cleft and
12
formation of the full binding site requires a further movement of Tm. The recent 6.6 Å resolution
structure of F-actin suggests that we may soon have much better picture of the actin - myosin, and
actin - TmTn interfaces that has been possible to date(65 PDB 3MFP).
Other regulation models have been proposed based on differing types of experimental approach.
For example the model of Tobacman (20)compares the effects of tropomyosin on myosin affinity for
actin and visa-versa. Since both myosin and tropomyosin cooperatively enhance each other’s
affinity for actin, the model assumes that this is an effect transmitted via the actin structure and
both Tm and myosin bind preferentially to an altered conformation of actin. In the absence of
additional evidence for a structural change in actin, other models tend to treat actin as a passive
partner in the process with the change in conformation being primarily a change in the Tm position
on the actin surface as suggested by the structural analysis. The observation that yeast
tropomyosins appear to induce cooperative binding of myosin to actin without an enhancement of
either myosin or tropomyosin binding suggests that such effects, while very strong in mammalian
tropomyosins are not fundamental to the mechanism of regulation (66-67). Undoubtedly, the
binding of myosin to actin will induce local structural changes in actin. The extent of these changes
and the degree to which such changes can be communicated to adjacent actins – directly through
actin-actin contacts (68)partners are required to assess how changes in actin structure may
contribute to regulation and the cooperativity along the filament.
Chalovich has developed a model based on the earlier 2-state models of Hill ((69-70). This model
assumed two states of A7TmTn and allowed for long range cooperativity between adjacent structural
units. The model was originally conceived to deal with marked cooperativity in the actin filament
particularly for myosin ADP complexes. This was later shown to be due to small contamination of
ATP in the ADP, and the real cooperativity was much smaller (71). However, the cooperativity
remains greater than can be accounted for by a single A7TmTn unit, and the model requires more
than two simple states(72). Chalovich also focuses on the ATPase cycle and argues that the myosin
ATPase is regulated by controlling the rate of Pi release not just the access of myosin to types of
binding site on actin. The difference between controlling an isomerisation that allows Pi release and
controlling Pi release itself ultimately becomes a semantic argument until the structural changes are
defined. These long running arguments have not been resolved in recent years and current methods
are unlikely to resolve the differences between the models. The reader is referred to the older
literature for some of the arguments (72-74). However one challenge that remains for all models is
to define and account for the calcium dependence of each of the model parameters.
A prediction for each model is that some model parameters are independent of calcium while others
show calcium dependence in a way that correlates with the occupancy of TnC with calcium. This has
been tested for high and low calcium but has not yet been tested over the range calcium
concentrations for any of the models.
5. COOPERATIVITY
The McKillop & Geeves 3-state model (Figure 6) defines KB and KT as equilibrium constants between
the three positions of Tm. The values of these constants are a property of the thin filament, are
affected by the binding of calcium to TnC, but are independent of myosin binding to the thin
filament. In contrast K1 and K2 define the equilibrium constants of the two-step binding of myosin to
actin; initial weak binding followed by isomerisation to the strongly bound rigor form. The values of
these two constants are properties of myosin and are independent of the state of the thin filament.
Note that myosin cannot bind to the Blocked-state and that step 2 the transition from the weakly
bound to the strongly bound rigor-like state is only possible for the M or myosin induced state of the
thin filament.
13
The original version of the model assumed that the cooperative unit was the structural A7TmTn unit,
but the data required additional longer range cooperativity. This was dealt with pragmatically by
introducing a term n, the apparent cooperative unit size, which is the apparent number of myosin
binding sites activated by each tightly bound myosin. This is equivalent in many respects to the
classic Hill coefficient which is used to define the degree of cooperativity in ligand binding assays. A
later version of the model attempted to give physical meaning to the term by modelling Tm as a
continuous semi-rigid cable on the surface of actin and relating the myosin induced movement to
the persistence length of the confined Tm strand (75-76). The alternative is the model of TL Hill
(69), developed more recently by Chalovich (72, 77), which allows for cooperativity between
adjacent A7Tm units. Both approaches can deal with the longer range cooperativity required by
equilibrium binding data but any such method losses sensitivity for cooperativity that extends
beyond two A7Tm units. I.e. the differences between a cooperativity affecting 14 actins vs 21 actins
is difficult to define experimentally.
Here I will focus on the correspondence between the biochemical data and the structural data and
how this helps us to understand the complexities of the calcium regulation system. The major
argument that I will set out here is that the cooperativity within the actin filament depends upon the
feature of the filament being observed. The degree of cooperativity in the filament is not a single
parameter. It is possiblet to describe a specific behaviour or property of the filament as having a
defined degree of cooperativity. A complete description of course will bring all perspectives
together but for the individual investigator each experiment can reveal a different facet of the
cooperative interaction.
5.1 Calcium binding to TnC
Calcium binding to TnC has been well documented using a range of different methods, including
NMR(78 PDB 1TNQ, 79-80) and fluorescence reporters in the TnC structure (81-83). These studies
show that calcium binding to isolated skeletal muscle TnC is cooperative and that calcium binding to
the N-terminal sites are required for activation. Significantly, calcium binding to the N-terminal sites
is influenced by TnI as an allosteric effector. When TnI is present, the affinity of calcium for TnC is
enhanced almost 10 fold from ~ 1 M to 0.1 M (84). If actin is present, it competes with TnC for
TnI and so the apparent affinity of TnC falls back to 1 M. If myosin is also present then myosin
competes with TnI for binding to actin. Thus TnI cannot bind to actin and will therefore interact with
TnC resulting in tighter apparent calcium binding to TnC when myosin is bound to actin.
This
mechanism is a classic cooperative calcium binding system that requires two forms of TnC, a low
(TnC) and high (TnC’) calcium affinity form as show in Figure 7 (68, 85). The first calcium to bind TnC
switches the equilibrium between the two forms of TnC from the predominant low affinity
conformation (TnC, KTnC = 0.1) to a balance position (KTnC = 1). The second calcium causes a further
shift so that the high affinity form predominates (TnC’ KTnC = 10). If TnI’s bind preferentially to the
TnC’ form and TnI cannot bind to both actin and TnC’ at the same time, then the structural model
gives a complete description of the TnC calcium binding properties in a regulatory system. A
cartoon of the Ca switching system based on the structural studies (shown in Figure 4) gives a
depiction of the system compatible with all of the calcium binding data. Calcium binding causes an
opening of the N-terminal domain of TnC which exposes the TnI binding site, allowing TnI to bind.
The binding of the switch peptide to this TnC site requires the dissociation of the inhibitory peptide
of TnI from actin. Thus the system is a set of poised equilibria. In the absence of calcium the
inhibitory peptide binds to actin. But when calcium is bound to TnC the preferred state is one in
which the switch peptide is bound to TnC, and the inhibitory peptide is dissociated.
TnC + TnI·A  TnC’ + TnI·A TnC’·TnI + A
KTnC
KBlocked
14
eqn 1
In the absence of calcium the equilibrium lies to the left (KTnC = 0.1, see Figure 7) and TnI is bound to
actin. In the presence of calcium the equilibrium lies to the right (KTnC = 10) and the switch peptide is
bound to TnC. If TnI is coupled to Tm as in Figure 4 then the right hand side represents Tm in the
blocked or B-position on actin and the left hand side is the C- state.
Figure. 7: Cooperative model of calcium binding to the regulatory domain of TnC. This
is a classic Monod, Wyman and Changeux (1965, JMB, 12, 88-118) cooperative model in
which TnC exists in 2 conformations: Open and Closed. The Closed form has a low affinity
for calcium (106 M-1) and the open form binds calcium 10 times more tightly (107 M-1). In the
absence of calcium, TnC is predominantly in the closed conformation ([TnC’]/[TnC] = K’Tn0 =
1) and with one calcium bound the TnC is a 50:50 mixture of the two conformations. When
both calcium ions bind, the open form predominates ([TnC’Ca2]/[TnCa2] =10. Note that since
the TnI signal peptide binds to the open conformation, the presence of TnI will increase the
proportion of TnC in the open conformation and increase the apparent affinity of TnC for
calcium. Redrawn from Geeves & Lehrer 2002(68)
Note that this is a balanced set of equilibria, the switching from TnI·A to TnC’·I is driven by the
binding of 2 calcium ions and this switches the TnC’/TnC ratio from 0.1 to 10, therefore,
energetically, the switch cannot go from 100% in the OFF or B-state and to 100 % in the ON or Cstate. The relationship between the value of KBlocked and the availability of actin sites (assuming they
are switched in groups of 7 by a single TnI) is shown in Figure 8. This illustrates the key factor with
coupled equilibria. If the value of KB is too small, the system cannot be turned on by two calcium
ions. If it is too large, then it cannot be turned off by 2 calcium ions. Thus, if we are limited to a
switch of 2 calcium ions that can alter the equilibrium between two forms of TnC by 100 fold, the
actin filament can be switched from 10% on to 90% on at the maximum and the reality is probably
near 20 to 80%. If strong myosin binding to actin allows an additional degree of activation (because
each TnI switched on makes 7 actins available) the system will be a more efficient switch. Even for
the simple model of coupled equilibria illustrated by eqn 1 and Figure 7, it should be clear that by
binding to actin, myosin competes with both TnI and Tm for sites on actin and therefore can activate
the filament in the presence or absence of calcium. This assumes that the myosin binding energy is
high enough to overcome the inhibition by TnI and Tm. This is true for the rigor myosin complex but
the system is again carefully poised for myosin in the presence of ATP. There is a large body of
evidence that rigor-like myosin can activate the filament but myosin heads turning over ATP in the
steady state can also alter the degree of activation (below).
15
Figure. 8: Plot of the fraction of actin sites available to bind myosin as a function of
KBlocked with and without calcium. Note the greatest calcium sensitivity of the available
actin sites occurs when KBlocked has a value between 1 and 10.
5.2 Myosin binding to actin.
The fraction of actin sites available for myosin to bind in regulated filaments can be addressed
directly by measuring how fast myosin heads bind to actin. In simple kinetic theory, the rate of
actin binding to myosin is given by –d[A]/dt = k[A][M], where k is the second order rate constant for
the reaction. If the reaction is followed under conditions where excess actin (ratio [A]:[M ] > 5:1) is
mixed with myosin heads (or S1, the motor domain), then the observed reaction takes the form of
an exponential where the observed exponential rate constant , kobs, is defined as kobs = k.[A] where
k is the second order rate constant for the reaction. This reaction is well defined for pure actin
filaments. If actin.Tm or actin.TmTn (in the presence of calcium) filaments are used instead then the
value of kobs changes little, indicating that neither k nor the availability of the actin sites has changed
(59, 61). Thus Tm in the Closed position, as defined by the structural studies, does not slow the rate
constant of binding nor the availability of actin sites. However, if calcium is removed from the
system then an exponential process is observed, as before, but the value of kobs is reduced to 30% of
its value with calcium (Figure 9A). As calcium is varied, the value of kobs varies and a Hill plot of kobs
vs calcium concentration (or pCa) yields a mid-point at pCa ~6.1 and a Hill co-efficient of ~1.4 (Fig
9C)(86-87). This result is predicted from the model described above. It simply shows that
availability of actin sites decrease as calcium is lowered and this occurs with a Hill coefficient
compatible with the behaviour of a calcium binding switch with 2 calcium binding sites. The
availability of actin sites goes from being mostly all available at high calcium to ~30% available at low
calcium. Note that the Hill coefficient of ~1.4 requires at least 2 calciums to bind to activate the
system. The value does not put an upper limit on the communication between adjacent Tns along
the filament but it does not require any longer range interactions.
16
Figure. 9: Calcium dependence of the rate constant for myosin S1 binding to actin. A.
Excess actin.TmTn binding S1 as the calcium concentration is varied. The transients are
fitted to single exponentials and the kobs values plotted in C. 2.5 M actin and 1 M skTm.Tn
vs 0.25 M S1. B. Excess S1 binding to actin.TmTn as calcium concentration is varied. The
half time of the reaction is plotted in C. 2.5 M S1 vs and 0.25 M actin +1 M skTm.Tn S1.
C. Dependence of the kobs values from A and the half time values from B on pCa. The
curves are fitted to a Hill equation with mid points of 6.02 & 6.09 and a Hill coefficient of 1.5
and 2.8 respectively. Reproduced from Boussouf et al 2007(121).
17
The same experiment with cardiac TnC has a similar mid-point, a smaller Hill coefficient (~1) and a
larger fraction available in the absence of calcium (87). This is compatible with the cardiac TnC ,
which has only a single calcium binding site, being unable to turn the actin filament off as effectively
as skTnC. The smaller Hill coefficient is again consistent with limited cooperativity between Tn
molecules along the filament. Several studies using probes on cardiac Tn do report the Hill
coefficient for calcium binding to the filament is greater than 1(88). A value greater than 1 does
require communication between adjacent TnCs. However, the data needs to be examined carefully
to define the precision with which the Hill coefficient is defined since the value is often not much
greater than 1. A useful control is to measure the Hill coefficient of the isolated Tn or TnC compared
to the value in the filament. A significant increase in the Hill coefficient for the filament over that
measured for isolated Tn does predict longer range cooperativity between Tn’s. Note though, if TnI
can interact with Tm on the parallel actin strand, or if actins communicate across the filament, then
the structural unit becomes (A7TmTn)2 and the Hill coefficient can be as high as 2, without requiring
cooperativity along the filament.
The cooperativity of myosin binding to actin gives a different picture. Myosin in solution can bind to
each actin in the A7Tm unit which could lead to very high values of the apparent Hill coefficient. In
practice Hill coefficients are not used and a more explicit model is required.
Simple equilibrium
binding data has been discussed above and has been interpreted in terms of different models by
McKillop & Geeves, Tobacmann and Chalovich(20, 59, 72). The experimental data do not differ
significantly between the three groups, but the models predict different degrees of cooperativity
because cooperativity is defined in different ways. What emerges is the apparent degree of
cooperativity can be greater for the system in the presence of calcium than in its absence. The
exact degree will depend on the details of the model used. For McKillop & Geeves 3-state model,
the apparent number of actins turned on by one strongly bound myosin increases from 7 in the
absence of calcium to ~12 in the presence of calcium i.e. an increase from a single structural unit to
a value close to 2 structural units.
Estimates of the unit size that are less dependent on detailed model fitting use different approaches.
Ishii & Lehrer (89) introduced the use of the excimer fluorescence of a pyrene label attached to both
halves of the Tm dimer to monitor the Closed to M-state transition of thin filaments and used both
equilibrium and kinetic measurement to define how many myosins are required to bind to a filament
to induce the fluorescence change. This was 1 myosin head per 7 actins in the absence of calcium
and one per 12-14 actins in either the presence of calcium or in the absence of Tn. The kinetic data
also established that the movement of Tm between the closed and open state was very fast > 1000
s-1. This cooperative unit size is dependent on the assumption of random binding of myosin heads to
actin, but if the binding is highly cooperative, then random binding will not be a valid assumption
and the method could over-estimate the number of myosin heads required to change the filament
state. The approach of Geeves and Lehrer(29) avoids this problem (see Figure 10). A fully
decorated thin filament (myosin bound to every actin site) is rapidly mixed with ATP. By comparing
the light scattering signal (which monitors ATP induced myosin dissociation from actin) with excimer
fluorescence (which monitors Tm position) it is possible to estimate how many myosin heads
dissociate from actin before the Tm moves. In this case the assumption of random ATP binding to
myosin sites along the filament is experimentally verified. ATP binding and dissociation of myosin
from actin is independent of the presence of Tm and Tn on the filament and also independent of the
presence of calcium. The answer again is that on average of 6- 7 myosin dissociate from a filament
before Tm moves back to the C-state without calcium and 12-14 myosin heads when calcium is
present.
18
Figure. 10: Pyrene labelled Tm monitors Tm change of position on the actin surface.
An actin filament (actin pyrene-Tm) is fully decorated with myosin heads such that every
actin has a myosin bound to it. Rapid addition of ATP induces dissociation of the myosin
heads which can be followed by a change in the light scattering signal. The light scattering
monitors each head that dissociates and the signal can be described by a single
exponential (kobs = k[ATP] where k is the 2nd order rate constant for ATP binding). A
fluorescent label on Tm shows a major lag in the signal change which can be modelled as a
number of myosin which need to dissociate before the myosin binds. The fitted line to the
fluorescence signal represents the best fit of a single exponential to the last 50% of the
transient (kFl). Increasing the ATP concentration ( A vs B) by a factor of 5 increases the kobs
for light scattering kLS buy a factor of 5 as expected but the relationship between the light
scattering and the fluorescence (kFl) remains unchanged (C). This shows the lag is cause by
the stoichiometric relationship between the two signals, not how fast Tm moves. In the case
of A·Tm filament on average 7 myosins need to dissociate before the pyrene-Tm signal
changes. For an actin·TmTn filament 12-14 myosin need to dissociate. D) Model of the ATP
induced myosin dissociation from the M.A.Tm complex monitored by light scattering (ΔLS)
19 in fluorecence (ΔFl ) of a label fluorescent
and Tm movement for a monitored by the change
label attached to tropomyosin. Adapted from Geeves & Lehrer 1994(29).
In summary, the results show that calcium binding to TnC in the thin filament has a modest
cooperativity that is compatible with little interaction between adjacent TnC’s – but long-range
interactions are not excluded. Strong myosin binding to thin filaments is cooperative and in the
absence of calcium a single myosin binding can activate ~ 7 actins or one A7TmTn structural unit. In
the presence of calcium this is increased to 12-14 actins or close to 2 structural units if this occurs
along the filament. The difference in the absence of calcium is presumably due to the presence of
strong TnI-actin contacts every 7th actin site which limits the transmission of the movement of Tm
over longer distances.
The myosin binding cooperativity discussed so far is that for a strongly bound myosin making a rigortype interaction. The degree of cooperativity as defined here does not change if ADP is bound to
myosin even though the ADP reduces the affinity of myosin for actin ~ 50-100 fold. Thus it is not the
affinity to actin but the type of interaction with actin that is important. The situation when myosin is
interacting with filaments in the presence of ATP is more complex and is dealt with in the next
section.
Other probes, either fluorescent or spin probes, on specific thin filament proteins have revealed the
relationship between calcium binding on the one hand and myosin binding on the other and
specifically how fast the information is transmitted between TnC and myosin through the filament.
In all cases the protein conformation changes are very fast and are unlikely to limit the rates of
activation or relaxation of the filament.(61, 90-94)
6. THE MYOSIN ATPase
The studies outlined in the biochemical section used rigor-type myosin binding to probe the state of
the actin filament. One criticism of such an approach is that the binding of the myosin heads in the
absence of ATP myosin may in some way be fundamentally different to that of the active myosin
cross bridge, e.g. it differs by more than just the lifetime of the cross-bridge. This is possible but
seems unlikely. Everything we know about the actomyosin interface comes from structural studies
of the rigor type actomyosin cross-bridge so it is not possible to say with certainty how different the
active cross-bridge will appear.
Studies of the ATPase are difficult for several technical reasons. Firstly, the affinity of a myosin head
for actin is very weak in the presence of ATP, and full activation of the ATPase cannot be achieved at
accessible actin concentrations (~100 M) at physiological ionic strength. The affinity of myosin for
actin can be increased by reducing ionic strength but this has other consequences. Each step of the
ATPase cycle can be altered by changing salt concentration not just the affinity for actin. For
example, the ATP hydrolysis step is slowed down at low salt and may be close to the Vmax of the
ATPase (95-96). Similarly the maximum rate constant for myosin binding to actin is slowed down at
low ionic strength (97). The regulatory system is also salt dependent and full regulation can be lost at
the low ionic strengths used for full actin activation (86). The standard ATPase assay uses a small
S1 concentration (<1 M) and large actin concentrations, typically > 20 M to give full activation.
The ratio of S1 to actin sits is thus very low < 1:20 and little myosin induced activation will be
observed. This is a problem if full activation requires both myosin and calcium binding. The
alternative is to seed the system with inactive but strongly binding myosin heads (eg NEM-treated
S1(98)) or to use limiting ATP concentrations to lengthen the lifetime of the AM complex. All of
these are effective but use rigor type myosin cross-bridge bridges to affect the actin structure. Thus
precise measurement of the Vmax of the ATPase and the interpretation of the experimental data is
complex.
The fact that strongly bound myosin cross bridges accelerate the ATPase in the presence of thin
filament was first noted by Murray & Weber (99) and this was followed by more detailed
20
measurements by Lehrer & Morris(11). These show that low ATP concentrations result in an
acceleration of the ATPase. Simple kinetic theory predicts that the ATPase rate should increase
linearly with the S1 concentration but the activation is greater than expected, indicative of
cooperative activation. Both of these effects are absent if a pure actin filament is used. Both
observations provide evidence for a myosin induced activation of the thin filament. The
interpretation is that the short-lived strongly-bound A·M·ADP or A·M states of the cross-bridge cycle
can hold Tm in the on or M-position and thereby allow other myosin heads access to an active actin
site nearby. The difficulty in a more detailed interpretation is that the life-time of the attached
cross-bridge is not well defined in the steady-state and few detailed models of the reaction have
been attempted because the system is under-defined. Attempts have been made to model the
mechanism in the muscle fibre(100-101) where there is a fixed stoichiometry between myosin heads
and actin sites and it is possible to define the fraction of the time in the cross-bridge cycle that each
myosin remains attached to actin. The fibre studies demonstrate that treatments that lengthen the
life-time of the myosin attachment to actin (e.g. increase ADP. Pi or reduced ATP
concentrations(102-103)) activate the filament while treatments that shorten the lifetime (e.g.
added phosphate) deactivate the filament. In vitro motility assays are an experimental system that
lies between muscle fibre and solution experiments in terms of the complexity and it is possible that
this may provide a route to improving or understanding of the regulatory system in the presence of
ATP (see section 7).
Current ATPase assays often use a fixed actin and myosin concentration and study the effects of the
presence of Tm & Tn and calcium on the measured ATPase rate. These provide very useful
information about the interacting components and have been widely used to compare isoforms of
Tn, the effects of mutations in one or more of the thin filament components or of phosphorylation
of Tn. However, because they use a fixed actin and S1 concentration a detailed molecular
interpretation of the effects of the changes in Tn is not always possible. The approach does
however have the great advantage of simplicity and reproducibility which makes it an essential tool
in defining the type of effects that mutations or phosphorylation events in Tn have on the
mechanism. A common misinterpretation of such assays is caused by the observation that addition
of Tm and or Tn in the presence of calcium can accelerate the ATPase compared to actin alone. This
leads to the use of the term “potentiation” of the ATPase by Tm and or Tn. While this is
undoubtedly true under the conditions of the assays it does not mean that the Vmax of the ATPase
is potentiated. In fact, in most cases the activation is probably caused by an increase in the apparent
affinity of myosin for actin. Few cases of an increase of Vmax have been established because of the
difficulty in measuring the Vmax, as outlined above.
White and his collaborators (104)have made the best attempt to address the complex issue of
regulation of the ATPase in the steady-state by using rapid double-mixing experiments, but even
here low salt concentrations are needed to achieve full activation of the thin filament. They
incubated S1 with ATP for 2 s to allow a steady-state mixture of M.ATP and M·ADP·Pi to form and
then mixed this rapidly with a large excess of thin filaments and excess ATP. By using either a
fluorescently labelled nucleotide in the first mix, or the fluorescent phosphate binding protein assay
(105) they were able to establish that the thin filament accelerated the release of Pi and nucleotide
and that this activation was much greater in the presence of calcium but the presence of some rigor
strong binding bridges is required to give the maximum acceleration. The experimental design is
quite complex, and defining exactly what is present in the solution after mixing with thin filaments is
not simple. Despite these complications, the experiment supports the general view outlined here
that actin binding is affected by calcium, and that full activation requires the presence of some
strongly bound myosin heads. Questions remain on the degree of cooperativity and if calcium
regulated events other than myosin binding.
21
7. IN VITRO MOTILITY ASSAYS & REGULATION
In vitro motility assays (106)provide an alternative route to explore the regulation of actin filament
activity. In these assays the measurements are made of the speed at which actin filaments move
over a surface coated with myosin or myosin motor domains. The basic requirements for the
motility assay were defined by Homsher et al (107)and include the density of myosin on a surface,
the surface attachment, and the ionic strength. Once the assay was established it was readily
applied to regulated actin filaments.(108-110) The concentration of actin filaments in the assay is
very low (nM) and initial concerns were that the regulated filament would disassemble at these
concentrations. Therefore a large excess of Tm and Tn were added to the assay to ensure the
regulatory components remains attached. It was subsequently shown that once the filaments are
assembled (at M concentration) that the disassembly is very slow and the filaments can be simply
diluted in to the assay conditions (110). This greatly simplifies the assay and allows other factors to
be explored such as the addition of other actin binding proteins which provide a frictional drag to
mechanically load the filaments.
The results show that, as for velocity measurements of skinned fibres, regulated filament tend to
move or not move, the velocity of an individual filament is less well regulated but the number of
filaments moving is calcium sensitive. At face value this is compatible with a simple steric blocking
model where calcium controls access to the actin. However, this observation can be unpicked a little
more thoroughly.
The assay depends upon the definition of smooth movement. It is not
uncommon for filament to go through a stop –start pattern and the velocity measured for a filament
will depend upon how long and how frequent the stop periods are during an observation of a
filament. If an assay is used that counts only “smoothly moving” filaments then the data can be
binned into 3 groups, smooth movement, non-motile and intermediate. In this case the number of
non-motile filaments shows a classic pCa curve while the velocity of smoothly moving filaments is
calcium independent until the numbers of moving filaments becomes very small. The number of
intermediately motile filaments will also be calcium dependent and by taking a longer observation
window and scoring these filaments as motile at the average velocity of the period of observation
the overall average velocity will also show calcium dependence. The calcium dependence of velocity
will depend upon the exactly how each filament is scored.
Understanding exactly what is happening in the assays is of interest. The observation that the
velocity of smoothly moving filaments is not calcium dependent suggests that the myosin sees either
an on or off state filament, if it is on then the myosin completes its power stroke as normal giving
“normal” velocity. The issue becomes one of defining how many myosin heads are required to
produce continuous smooth movement. There is clearly some dependence of the assay on the
density of myosins on a surface but inhomogeneity of the surface and variability in the availability of
myosins on a surface do make interpretation difficult. A recent study by Baker (111) makes an
interesting attempt to take the analysis of such data much further. The work proposes a model in
which myosin cross-bridges constitutively activate the thin filament, and the length of time a
filament is active is modelled from the kinetics of cross-bridge association and dissociation.
In vitro motility velocities have also reported that addition of Tm or TmTn can increase the velocity
of actin filaments (110, 112). This observation has been compared to the “potentiation” of the
ATPase rates by Tm and TmTn. In the motility assays the origin of the increase in velocity has not
been well defined. This may be the result of an increase in the myosin cycling rate or there could be
other possibilities. As in the ATPase assays, the increase could be the result an increase in the
apparent affinity of myosin for actin at the sub-maximal actin concentrations available to myosin on
the surface. It could also be the result of a change in fibre properties such as an increase in stiffness
of the filament allowing a more efficient coupling of the myosin power stroke to actin movement.
22
8. FIBRES & MYOFIBRILS
The study of thin filament regulation in muscle fibres is beyond the scope of this review. But
ultimately we cannot expect to understand how the regulatory system works without being able to
describe the activation and relaxation process in muscle fibres. Studies of the regulatory system in
myofibrils skinned muscle fibres and intact muscle continue to reveal more about the complexities
of the system. The hope is that the simpler in vitro studies will provide the framework which defines
what is and what is not possible for the protein components to achieve. The ability to work in
myofibrils (113-114) and to collect mechanical and optical data from contracting myofibrils should
bring the both methods and interpretations of solution and muscle fibre work much closer together.
Most of the fibre studies do show that full activation of the myosin filament requires both myosin
heads and calcium as seen in solution(8, 115). It remains unclear if the contribution of the two
effects is the same in fibres and in protein solutions. The major discrepancy between fibre work and
solution studies is in the degree of cooperativity. There remain consistent reports of much greater
cooperativity in muscle fibres than observed in solution.(12-13) Whether this is a real difference in
the properties of the system brought about by missing element in the solution studies or if it
reflects the limitations of the different experimental systems remains to be defined. Two recent
studies have challenged to consensus on the role of myosin cross-bridges in cooperatively activating
the thin filament (13, 116). Both have used blebbistatin to reduce the force generated by a muscle
fiber by >50% and show that there are only marginal differences in both the calcium required for
50% activation and the cooperativity of activation. This is not predicted if the force holding strongly
bound cross-bridges are the agents of cooperative activation. One issue to resolve is the mode of
action of blebbistatin as this does not block myosin binding to actin but prevents the major myosin
cleft from closing. It remains possible that the blebbistain inhibited myosin can bind to actin but not
generate force. This binding mode may be inhibited by the blocked state of the thin filament and
could therefore remain calcium sensitive. Solution studies of blebbistatin inhibited myosin binding
to actinTmTn will be needed to resolve this issue.
Most actin filaments in the cell body of non-muscle cells appear to be stabilised by Tm binding.
What role this Tm plays in regulating myosin traffic along these stable filaments remains to be
defined. In some case there is evidence from cell microscopy, as well as biochemical and structural
evidence that non-muscle Tm can prevent some classes of myosin interacting with actin filaments.
There are a large number of different Tm isoforms in a mammalian cell as well as a large number of
different myosin isoforms. Understanding which isoforms are present at the same time in an area of
a cell and if the Tm present has any role in regulating the myosin activity remains poorly understood.
What is clear to-date is that in general no equivalent of Tn has been identified in non-muscle thin
filaments.
The focus of this review has been on the regulatory system of fast skeletal muscle. There are
significant changes in the isoforms of Tn for different muscle types with muscle and developmental
specific isoforms of TnC, TnI and TnT. The reader is referred to the reviews by Perry(1, 3-4) for
details. In most cases the properties of the different isoforms have not been as well defined as for
the fast muscle system – with the exception of the cardiac muscle isoforms of Tn. Differences
between isoforms are likely to be one of degree rather than any fundamental difference in
properties. The switching of isoforms is often not a single subunit but of whole Tn and can correlate
with changes of the myosin isoform between muscle types. This probably reflects the delicate
balance between each of the players in the allosteric activation-deactivation process. A stronger,
longer-lived myosin·ADP complex, expected in a slow or cardiac muscle myosin, will be more
effective at activating a muscle fibre and harder to turn off – so different Tn components may be
required to keep the calcium sensitivity in the physiological range. The balance between different
components in the regulatory system have been demonstrated by two interesting examples of a
23
mutation in one part of the regulatory system being compensated by a mutation in a different part
of the regulatory pathway (117-118). Of significance is the rescue of a TnI mutation which appears
to give a thin filament that is constitutively active by a myosin mutation that on its own does not
activate as well. The presence of both mutations results in a fly with near normal functioning
muscle.
While there have been studies of different Tn isoforms there have been few studies to date of
regulation in solution using a slow or cardiac myosin isoform. This is likely to be significantly
different in how it interacts with the thin filament regulatory system and remains a major gap in our
knowledge.
Studies of cardiac thin filaments have also revealed a much more complex tuneable regulatory
system. Phosphorylation of TnI and TnT at multiple sites are important modulators of the regulation
system. These have been well defined for their effect on calcium sensitivity but less so for the effects
on the structural and biochemical states of the thin filament.
Recent years has also seen a huge growth in the discovery of mutants in the sarcomeric proteins
which are associated with cardio myopathies. Many of these are located in the thin filament
proteins. A recently emerging paradigm for these studies is that mutations in the TmTn complex
that associated with DCM cause an decrease calcium sensitivity of the thin filament whereas HCM
linked mutations are caused by a increased sensitivity(119-120). While this provides a testable
hypothesis for Tm & Tn mutations it seems unlikely that all mutations will fall into such a simple
categories.
The next few years promises much more progress in understanding the mechanism of regulation.
The recent publication of a 6 Å structure of an actin filament(65) suggests that it will soon be
possible the define at atomic resolution the interface between actin and myosin, actin and TmTn
and if either protein induces a change in the actin structure. Understanding in detail how binding
to actin changes the myosin structure will enable us to define if the Tm positions on actin simply
control access of actin to myosin or if there are additional effects such as limiting the rate of Pi
release. The availability of the high resolution structures will also enable the role of mutations in
any of the proteins involved to be given a molecular interpretation.
24
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