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INTRODUCTION |
Regulation of vertebrate striated muscle thin filament activity
involves a Ca2+-sensitive change of the state of the thin
filament, which affects the interaction between actin molecules and
myosin cross-bridges (1). The thin filaments can be described as a two
state allosteric and cooperative binding system in which the
cooperative unit consists of seven actins and one tropomyosin: troponin
I and myosin heads act as negative and positive allosteric effectors
respectively (2, 3). The equilibrium between the on and off states
(Kt in the model of Geeves and Halsall (2)) is
regulated by Ca2+ through the troponin complex and
cross-bridge binding. Troponin-tropomyosin regulation of thin filament
activity is found throughout the animal kingdom, including insect
striated muscles (4-6).
Tropomyosin is a dimeric
-helical coiled-coil that spans seven
monomers along the actin filament (7). Tropomyosin molecules bind
end-to-end, thus forming a continuous strand along the actin filament.
According to current models, the tropomyosin strand may exist in two or
more locations on the surface of the actin (8-10). In one position,
corresponding to the biochemical on state, the negatively charged
tropomyosin is located over actin subdomains 3 and 4 in a "trough"
of positive charge (11, 12). The putative strong and weak myosin
binding sites on actin are exposed and the myosin cross-bridges may
cycle unimpeded. Recent work using actin mutants supports this model:
charge change mutations within the trough of actin such as E311A/R312A
in yeast (13) and K238A/E241A/E360H in Dictyostelium (14)
result in weaker tropomyosin binding and destabilization of the on
state.
In striated muscles from arthropods (Limulus) and
vertebrates (Rana) the tropomyosin strand has been shown to
be over the junction between actin domain 3 and 1 and over domain 2 in
the off state and consequently strong myosin binding sites are blocked (Refs. 8 and 10, Fig. 1). The inhibitory
component of troponin, troponin I, is a positively charged protein that
binds to actin and switches the tropomyosin to the off state. It is
noteworthy that although troponin I controls the actin-tropomyosin
state, it does not bind directly to tropomyosin, so it probably acts indirectly through actin (15). Electrostatic charges are likely to play
an important part in this interaction.

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Fig. 1.
Surface model of the actin monomer showing
the location of the carboxyl oxygens of glutamic acid 93 relative to
the positions of tropomyosin in the off and on (Ca2+ + cross-bridges) states. Actin structure was drawn from the atomic
coordinates published by Lorentz et al. (43) using Molview
molecular graphics software. The tropomyosin positions are based on the
models of Holmes (11) and Lehman et al. (10).
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The amino acids in actin involved in stabilizing the off state have not
been positively identified, but there is evidence for troponin I
contacts on domains 1 and 2 of actin (16). This is compatible with
reports that changes in the shape of domain 2 of actin are associated
with regulation (17).
A cluster of negative charge due to Asp56,
Glu57, and Glu93 is located at the bottom of
domain 2, close to the position occupied by tropomyosin when in the off
state (Fig. 1) (10). We considered the possibility that this negative
charge cluster may repulse tropomyosin, thus maintaining the on state
and that neutralization of the repulsion by inhibitory proteins may be
an essential feature of the inhibitory mechanism. To test this
hypothesis, we studied the state of actin-tropomyosin in a mutant of
the Drosophila flight muscle-specific actin gene
Act88F with a charge reversal mutation of glutamic acid 93 to lysine (E93K actin)(18, 19). The E93K mutation produces a flightless
phenotype characterized by disordered sarcomeres. However in
vitro motility assays have demonstrated that E93K actin filaments
can move over myosin and produce force, which is not very different
from the wild type (20, 21). Using in vitro motility
techniques similar to those we used to characterize tropomyosin based
regulation in striated and smooth muscles (22, 28, 32, 33), we can
demonstrate that charge reversal of amino acid 93 on actin does indeed
change the equilibrium of actin-tropomyosin toward the off state.
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EXPERIMENTAL PROCEDURES |
Actin Preparation--
Drosophila
WT1 indirect flight
muscle-specific actin was made from a rosy506
homozygous strain (rosy506 is an eye
color mutation) and E93K mutant actin from a rosy506 Act88FKM88P[Act88FE93K]
strain, which has a single copy of the E93K mutant
Act88F gene inserted into an Act88F null genome
by P-element-mediated transformation (18). Flies were grown and then
stored at
20 °C prior to processing.
A myofibrillar preparation was made from 50 g (50,000) of flies
according to the method of Saide et al. (23). After a high salt extraction (20 mM KPO4, 500 mM
KCl, pH 7.0) on ice for 10 min, the myofibril pellet was dehydrated by
resuspending first in 6 volumes of ice-cold 50% acetone, then in 6 volumes of ice-cold 100% acetone, and finally allowed to air dry
overnight at room temperature.
Actin extraction from the acetone powder was performed essentially
after the methods of Bullard (24) and Pardee and Spudich (25) in ACEX
(2 mM Tris-Cl, 0.2 mM CaCl2, 0.2 mM ATP, 1 mM dithiothreitol, pH 8.0) at 4 °C
for 60 min. After spinning the extract at 43,000 × g
for 15 min at 4 °C, a tenth of the extract volume of 10 × polymerization buffer (50 mM Tris-Cl, 500 mM
KCl, 20 mM MgCl2, 10 mM ATP, pH
8.0) was added and polymerization allowed to proceed for 2 h at
4 °C. Solid KCl was added to a final concentration of 800 mM. After incubation for 10 min at 37 °C to dissociate the contaminating Drosophila thin filament proteins, the
preparation was spun at 90,000 rpm for 20 min at 4 °C in a Beckman
TLA100.3 rotor. The F-actin pellet was resuspended in 3 ml of ACEX,
homogenized, and then dialyzed for 24 h in 3 × 1-liter
changes of ACEX. The optical density at A280 to
A310, with an extinction coefficient of 0.62 cm2/mg, was used to estimate the molarity of the G-actin
stock.
Anion exchange chromatography employing a 1-ml Mono Q column (Amersham
Pharmacia Inc.) was used to isolate the indirect flight muscle-specific
actin isoform from the mixture of the six Drosophila isoforms constituting the G-actin stock. Before application to the
column, the actin stock was clarified by first spinning at 90,000 rpm
for 20 min in a Beckman TLA100.3 rotor and then filtering through
0.2-mm filters (Millipore). A 28-min segmented gradient from 0 to 500 mM NaCl in 20 mM MOPS, pH 6.5, was then used to resolve the actin mixture into four different isoform peaks (Fig. 2).

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Fig. 2.
Separation of Drosophila whole
fly actin extracts by FPLC on Mono Q. A, ACT88F (wild type)
flies; B, Act88F-E93K flies. A and B
in the wild type profile are positively identified as as ACT88F actin,
since they are absent in the KM88 ACT88F null mutant
chromatogram. Peak A is arthrin, and peak B is
ACT88F-mod+ (posttranslationally altered products of the
Act88F gene; the nature of mod+ is unknown (45)). In the
E93K profile peaks A and B have moved as a result
of the amino acid substitution. The positions of peaks C and
D are not altered and represent products of other actin
genes. Peak B represents pure WT or E93K flight
muscle-specific actin. C, wild type actin 88F on a
two-dimensional gel. It shows one major spot and a minor one of the
same mass. There is no arthrin.
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The peak B fractions from chromatographic runs of WT (or E93K) actin
were pooled and polymerized as before for 2 h at room temperature
and then spun at 100,000 rpm for 10 min at 4 °C in a Beckman
TLA100.3 rotor. Six separate paired preparations of WT and E93K actin
were prepared; yield was 30-100 µg per preparation. E93K does not
polymerize as readily as WT actin, and in one preparation it was
necessary to add heavy meromyosin at 1:500 mol/mol in order to promote
polymerization. The F-actin pellets were resuspended in ACEX to a final
concentration of about 5 µM actin.
In Vitro Motility Assay--
The in vitro motility
assay was performed as described by Fraser and Marston (22) using 100 µg/ml skeletal muscle HMM on cover glasses coated with silicone by
soaking in 0.2% dichloromethylsilane in chloroform. F-actin was
labeled with rhodamine-phalloidin (
) as described by Kron et
al. (26). F-actin·
-tropomyosin and actin·
-tropomyosin-troponin complexes were formed at 10 × assay concentration: 100 nM actin·
, 150 nM tropomyosin, and 0-600 nM troponin were
mixed in 50 mM KCl, 25 mM imidazole-HCl, 4 mM MgCl2, 1 mM EDTA, 5 mM dithiothreitol, pH 7.4 (buffer A), and incubated for
30-60 min. The complexes were diluted 10-fold immediately prior to
infusion into the motility cell. We have demonstrated that tropomyosin
does not dissociate during the experiment under these conditions and
that additional tropomyosin in buffers C and D is not necessary
(22).
A flow cell was prepared from a freshly siliconized coverslip and a
microscope slide as described by Kron et al. (26). Assay components and buffers were infused into the flow cell at 30-60-s intervals. Two 50-µl aliquots of HMM at 100 µg/ml were infused in
buffer A (50 mM KCl, 25 mM imidazole-HCl, 4 mM MgCl2, 1 mM EDTA, 5 mM dithiothreitol, pH 7.4) to provide a coating of
immobilized HMM on the coverslip. This was followed by 2 × 50 µl of buffer B (buffer A + 0.5 mg/ml bovine serum albumin) then
2 × 50 µl of 10 nM actin·
± associated
tropomyosin-troponin in buffer A. 50 µl of buffer C (buffer B + 0.1 mg/ml glucose oxidase, 0.02 mg/ml catalase, 3 mg/ml glucose, 0.5%
methylcellulose, ± troponin at assay concentration) and 50 µl of
buffer D (buffer C + 1 mM ATP) were then infused.
Ca2+ concentration was varied by incorporating
Ca2+·EGTA buffers in the final assay buffers C and D.
The movement of actin·
-tropomyosin filaments over the immobilized
skeletal muscle HMM was observed under a Zeiss epifluorescence microscope (63×/1.4 objective) with a DAGE-SIT-68 camera and recorded on video tape. Videos were digitized, and the movement was analyzed to
determine fraction of filaments moving and velocity of motile filaments
using either the automatic tracking program described by Marston
et al. (27) or the manual tracking procedure in the cases
where filaments were not easily detected (22, 28).
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RESULTS AND DISCUSSION |
Comparison of Rabbit Skeletal and Wild Type Drosophila Flight
Muscle Actin Filament Motility--
Actin filament movement over
immobilized heavy meromyosin was studied in an in vitro
motility assay. With this assay it is possible to measure a number of
parameters in place of the single parameter of ATPase. These are
fraction of actin filaments moving, velocity of the filaments that are
moving, and the number of filaments attached to myosin per unit area.
It has been shown that Ca2+ can regulate all three of these
parameters independently through troponin-tropomyosin (22, 28, 29).
In previous work it has been shown that addition of arthropod
(Limulus) tropomyosin to actin increases filament velocity
(4), but this is not observed with vertebrate striated muscle
tropomyosin. We therefore decided to test the Drosophila
actin with vertebrate smooth muscle tropomyosin (from sheep aorta) as
well as with vertebrate striated muscle tropomyosin (from rabbit).
Smooth muscle tropomyosin tends to activate actomyosin ATPase, while
skeletal muscle tropomyosin is usually inhibitory (30, 31). In the
in vitro motility assay smooth muscle tropomyosin gives an
increase in filament velocity similar to Limulus tropomyosin
(4, 32, 33). The increased velocity has been suggested as indicating
that actin-tropomyosin is predominantly in the on state, since smooth
muscle tropomyosin has a higher Kt and size of
cooperative unit compared with skeletal muscle tropomyosin (28, 30,
31)
The regulation of Drosophila flight muscle actin filament
movement by troponin and tropomyosin has not been studied before, and
it is possible that Drosophila actin could have regulatory characteristics quite different from vertebrate actin. Table
I compares Drosophila actin
and the well studied rabbit striated muscle actin filaments (22, 28,
33). The two types of actin filaments moved at the same velocities with
a high proportion of the filaments motile. Smooth muscle tropomyosin
increased the velocity of both types of actin filaments by around 25%,
while skeletal muscle tropomyosin had no effect on velocity, although cosedimentation experiments showed it did bind under the conditions of
the motility assay (22). The fraction of filaments motile was not
affected by either tropomyosin.
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Table I
Comparison of the motility of rabbit skeletal muscle and Drosophila
flight muscle (ACT88F) actins
Values are means ± S.E.
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In previous experiments we have demonstrated in our system that
addition of up to 16 nM troponin at Ca9 results in a marked decrease in the fraction of filaments moving accompanied by a modest
decrease in the velocity of the filaments that remain motile (22, 28).
Addition of skeletal muscle troponin to wild type 88F actin-skeletal
muscle tropomyosin filaments produced the same decrease in the fraction
of filaments motile (half-maximal effect at about 4 nM
troponin) and the same small decrease in velocity of motile filaments
(16% at 16 nM troponin) as was observed previously with
skeletal muscle actin filaments (compare Fig.
3 with Fig. 3 in Ref. 22). This behavior
was observed irrespective of the type of tropomyosin used and is
characteristic of actin-tropomyosin filaments in the off state (28).
Different technical approaches to measuring troponin control of
filament motility have produced differing patterns of results in some
experimental circumstances (these are discussed in Ref. 28); however,
all the published analyses agree that reduction in the proportion of
filaments that are motile is a characteristic feature of
actin-tropomyosin filaments switched off by troponin or other
inhibitory proteins (22, 32, 46, 47). At pCa5 troponin did not alter
the fraction of filaments motile or the velocity.

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Fig. 3.
Regulation of wild type Drosophila
flight muscle actin filament movement by skeletal muscle
troponin-tropomyosin. The effect of 0-16 nM skeletal
muscle troponin on skeletal muscle actin- -tropomyosin filament
velocity (upper panel) and fraction of filaments motile
(lower panel) was measured at pCa9 (closed
circles) and pCa5 (open circles) at 25 °C. The
movement of all filaments over a 0.65-s time interval was determined in
10 fields of view, and the data were plotted as a frequency histogram
of velocities. There were two populations of filaments, moving and not
moving. Any movement of less than 1 µm/s was defined as nonmotile
according to the protocols described in Ref. 27. Moving filaments had a
Gaussian distribution of velocities with a S.D. about 0.3 times mean
velocity (S.D. is constant when the filament number exceeds 400 and is
primarily determined by Brownian motion (27)). The fraction of
filaments moving and the mean velocity of the moving filaments are
plotted here. Each point represents a single measurement using 500 filament velocity values.
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We conclude that the motility parameters of actin from rabbit and
Drosophila are indistinguishable, and they are regulated by
tropomyosin and troponin in the same way. This justifies the use of
mammalian regulatory proteins in our investigation of mutant Drosophila actin.
Comparison of Wild Type and E93K Mutant Actin--
Although the
E93K mutation has a flightless phenotype E93K actin, filaments have
been reported to be capable of movement in the in vitro
motility assay and of exerting force (20, 21). We found that wild type
and E93K actin moved at almost the same velocity at 30 °C. At
28 °C wild type moved 26% faster than E93K actin, and it has been
reported that at 23 °C wild type moved 50% faster than E93K actin
(20). Thus our results are in broad agreement with previous experiments
using E93K actin and confirm that the mutation does not seriously
compromise the actin-myosin interaction. Some differences are to be
expected, since we have used a different surface (siliconized glass),
and we performed experiments at higher ionic strength than previously
and used 0.5% methyl cellulose (20, 21). We have found that silica surfaces seem to provide less resistance to movement than
nitrocellulose and also that the addition of methylcellulose to the
medium enables weakly bound filaments to remain attached to the myosin
surface (28). Thus the conditions we have adopted would minimize
differences in motility performance between wild type and mutant
actin.
When smooth or skeletal muscle tropomyosin were added to wild type
actin filaments, the fraction of filaments that were motile did not
change. In contrast, when either smooth or striated muscle tropomyosin
was added to the mutant E93K actin filaments, the fraction of filaments
motile was greatly reduced (Fig. 4).
Experiments were done with six paired preparations of wild type and
E93K actin. The fraction of wild type actin filaments motile was
79 ± 3.6% (S.E., n = 7) and of E93K actin
71 ± 2% (S.E., n = 7). Tropomyosin progressively
reduced the fraction of E93K actin filaments motile reaching 14 ± 2% (n = 5) with 16 nM skeletal muscle
tropomyosin and 17 ± 2% (n = 3) with 16 nM smooth muscle tropomyosin; in contrast, tropomyosin did
not affect WT actin filament motility (Fig. 4).

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Fig. 4.
In vitro motility analysis of
Drosophila wild type actin and E93K actin filament movement
over skeletal muscle HMM. Left panels, the effect of sheep
aorta tropomyosin on actin· filament movement, measured at
30 °C. Right panels, effect of skeletal muscle
tropomyosin on actin· filament movement measured at 25 °C.
Upper panels, actin filament velocity, µm/s. Lower
panels, fraction of actin filaments motile. Closed
symbols, wild type 88F actin; open symbols, E93K actin.
Six paired preparations of actin were used, and the results were
pooled. Each point represents the mean and S.E. of three to six
measurements using up to four separate preparations.
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Skeletal and smooth muscle tropomyosin had different effects upon
filament velocity. The skeletal muscle tropomyosin had no effect on
velocity of the wild type actin as observed previously with rabbit
actin (Table I) and slightly decreased velocity of E93K actin filaments
(Fig. 4). The decrease was greater at 25 °C (up to 37%, Fig. 4)
than at 27 °C (up to 19%). Smooth muscle tropomyosin enhanced the
velocity of wild type actin filaments by 25% at 30 °C (Fig. 4,
Table I), but this was not observed with E93K actin filaments,
instead velocity declined in the same way as it did with skeletal muscle tropomyosin (Fig. 4).
It will be noted that the the large decrease in the fraction of E93K
actin filaments motile and the small decrease in velocity when
tropomyosin was added (Fig. 4) resembles the effect of adding troponin
at pCa9 to wild type actin-tropomyosin filaments (Fig. 3). As has been
discussed, this effect is characteristic of actin-tropomyosin filaments
in the off state; thus it appears that the E93K mutation in actin
causes tropomyosin to bind to actin only in the off state (32).
To confirm that the "switch off" of E93K actin filament movement
was due to the tropomyosin-mediated mechanism, we added NEM-S-1 to E93K
actin-tropomyosin. NEM-S-1 forms strong bonds to actin, even in the
presence of ATP, thus it switches actin-tropomyosin to the on state
independently of regulatory proteins (29, 34). We have shown previously
that adding NEM-S-1 to actin-tropomyosin resulted in an increase in
filament motility (28). When NEM-S-1 was mixed with E93K actin-smooth
muscle tropomyosin filaments, we observed that it restored both the
fraction of filaments motile and their velocity to the level observed
with E93K actin when tropomyosin was absent (Fig.
5). This reversal of inhibition required less than 0.4 NEM-S-1 per actin, thus confirming that it was acting cooperatively upon tropomyosin in the same way as it does with wild
type striated muscle actin-tropomyosin (28, 34). E93K actin-tropomyosin
filaments were also reactivated by adding troponin in the presence of
Ca2+ (Fig. 5). This indicates that the effect of the E93K
mutation is to shift the on/off equilbrium of the thin filament toward the off state to a certain degree, but the Ca2+-activated
troponin complex is capable of shifting the equilibrium back to the on
state. In contrast a deletion mutation in tropomyosin has been shown to
produce thin filaments that cannot be activated by Ca2+ and
troponin (36), although they can be switched on by rigor S-1.

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Fig. 5.
Restoration of the motility of E93K
actin· -tropomyosin filaments by the addition of NEM-S-1 or
troponin at pCa5. The motility of 10 nM E93K
actin· + 20 nM smooth muscle tropomyosin was measured
at 25 °C. Left, NEM-S-1 was prepared (44) and added after
the E93K actin· -smooth muscle tropomyosin in buffers C and D. Right, troponin was mixed with E93K actin· -smooth
muscle tropomyosin in buffer A and applied to the motility cell.
Troponin and pCa5 Ca/EGTA buffer was present in buffers C and D. Filament movement in buffer D was analyzed as described in the legend
to Fig. 3. The fraction of actin· -tropomyosin filaments motile and
their velocity increased back to the level obtained with pure E93K
actin (shown as open circles and dotted
lines).
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Molecular Interpretation of the Effect of the E93K
Mutation--
Recent structural work has indicated that tropomyosin is
loosly associated with actin and that its location may be dependent upon the charge distribution on the surface of actin. The negatively charged tropomyosin lies over domains 3 and 4 of actin in a trough of
positive charge when in the on state (8, 10). Changes in the surface
charge of actin in this region by chemical modification (e.g. of Lys238 by pentane dione (35)) or by
mutations (E311A/R312A (13) and K238A/E241A/E360H (14)) lead to a
reduction in tropomyosin binding affinity and modification of
regulatory behavior. In addition mutations in tropomyosin can also
modulate regulation (33, 36)
The likely position of tropomyosin when in the off state has only
recently been visualized by electron microscopy and three-dimensional reconstructions. In both vertebrates and arthropods tropomyosin is seen
to have moved across the face of the actin monomer so that it is over
the junction between domains 1 and 3 and over domain 2 at the top of
the actin monomer, covering the putative myosin binding site (8, 10)
(Fig. 1). Although this description provides an explanation of how the
steric blocking model of regulation could work, it provides no clues as
to why tropomyosin is located in this position in the presence of
inhibitory proteins.
Several lines of evidence point to a possible involvement of domain 2 of actin in transmitting a signal from regulatory protein to
tropomyosin. Actin monomers within actin filaments are flexible and
conformational changes within the monomer are mainly in the region of
domain 2 (38, 39). Furthermore detailed modeling of x-ray diffraction
patterns from active and relaxed muscles have been interpreted in terms
of a movement of domain 2 (17). Interestingly in this model the
distance between domain 2 and tropomyosin is halved when the muscle is
switched off, but there are only minor changes in the distance between
tropomyosin and the other three domains.
Since it is proposed that tropomyosin is stabilized in the on position
by actin surface charges in domains 3 and 4, it seems reasonable to
propose that charges on the surface of domains 1 and 2 contribute to
stabilizing tropomyosin in the off position. It has long been
appreciated that in inhibitory proteins such as troponin I and
caldesmon inhibitory function is related to concentrations of positive
charge (the inhibitory peptide of troponin I, residues 104-115,
contains 4 arginines and 2 lysines (40, 41)). There are complementary
clusters of negative charged amino acids on the surface of actin, the
most notable of which is at the bottom of domain 2 (Asp56,
Glu57, Glu93). This negatively charged patch is
close to tropomyosin in the off state (10) as illustrated in Fig. 1.
The critical nature of this region to muscle function is highlighted by
the fact that the E93K mutation produces a flightless phenotype, and
the E93A/R95A mutation in yeast is lethal (37). Mutations in this
region affect sarcomere assembly (18), and this may be a consequence of
the poor polymerizability of E93K actin or due to an altered
interaction with tropomyosin. The motility parameters of E93K actin are
not greatly different from WT, but the interaction of E93K with
tropomyosin is altered as shown by these experiments (Fig. 4). The
observation that a charge reversal mutation of glutamic acid 93 to
lysine results in tropomyosin binding to actin in the off state, as
defined by the motility parameters (low fraction of filaments motile
with little change in velocity) suggests one aspect of actin structure that may be important for regulation. Alterations of other amino acids
in this charged cluster (D56A/E57A) seem to have similar effects upon
actin-tropomyosin state (48), but charge changes at the nearby surface
helix (E99A/E100A) or in the middle of domain 1 (D24A/D25A) have no
effect upon regulation (49), this indicating a specific role for this
site.
We propose that electrostatic charge on the surface of domain 2 of
actin plays a critical role in determining the equilibrium between
states of actin-tropomyosin that is a central feature of the steric
blocking mechanism of actin filament regulation (8, 42). Concentrated
negative charge in this region, as found in wild type actin, promotes
the on state, and the absence or reversal of such charge, as found with
the E93K actin mutant and actin-troponin I complex, permits tropomyosin
to move toward domain 2 and thus promotes the off state. It is possible
that the ability of structurally unrelated proteins such as troponin and caldesmon to inhibit actin-tropomyosin interaction with myosin may
simply be due to neutralizing negative charge on the surface of actin,
thus permitting tropomyosin to move to the off position on actin. It
would also be compatible with the general observation that inhibitory
proteins bind to actin rather than to tropomyosin.
We thank Anne Lawn for excellent
technical assistance in purifying the actins.