From the Departments of Internal Medicine and
Biochemistry, University of Iowa, Iowa City, Iowa 52242, the
¶ Department of Physiology and Biophysics, Boston University
School of Medicine, Boston, Massachusetts 02118, and the
Department of Cell Biology, University of Massachusetts Medical
School, Worcester, Massachusetts 01655
Received for publication, February 5, 2001, and in revised form, March 14, 2001
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ABSTRACT |
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Familial hypertrophic cardiomyopathy (FHC)
is caused by missense or premature truncation mutations in proteins of
the cardiac contractile apparatus. Mutant proteins are incorporated
into the thin filament or thick filament and eventually produce
cardiomyopathy. However, it has been unclear how the several,
genetically identified defects in protein structure translate into
impaired protein and muscle function. We have studied the basis of FHC
caused by premature truncation of the most frequently implicated thin
filament target, troponin T. Electron microscope observations showed
that the thin filament undergoes normal structural changes in response
to Ca2+ binding. On the other hand, solution studies
showed that the mutation alters and destabilizes troponin binding to
the thin filament to different extents in different regulatory states, thereby affecting the transitions among states that regulate myosin binding and muscle contraction. Development of hypertrophic
cardiomyopathy can thus be traced to a defect in the primary mechanism
controlling cardiac contraction, switching between different
conformations of the thin filament.
Familial hypertrophic cardiomyopathy
(FHC)1 is caused by missense
or premature truncation mutations in proteins of the cardiac contractile apparatus (1-4). Despite the varied functions of these
many proteins, the clinical and histological manifestations of FHC
define a common syndrome involving thickening of one or more parts of
the left ventricular wall, myocyte disarray, fibrosis, and a variety of
cardiac symptoms including sudden death (reviewed in Ref. 5).
Cardiomyopathic mutations have been described for thick filament
proteins, as well as for every thin filament component except troponin
C (TnC), i.e. for Both the thin filament and the thick filament are dynamic interacting
protein assemblies. Large structural transitions in myosin produce the
cross-bridge stroke that results in muscle contraction (7, 8).
Similarly, changes in thin filament structure are critical for
Ca2+ regulation of contraction (9-11). FHC mutations in
either filament presumably act by altering filament structure or
dynamics, although no direct structural examination of FHC mutants has
been reported. However, critical insights into the basis of FHC have
come from mapping myosin mutations onto the atomic model of the myosin
head (12, 13). Similarly for the thin filament, mutations can be mapped
on to the atomic structures of the components where these are
available. However, a full understanding of thin filament mutations has
not been possible because of the lack of an atomic model of the thin
filament as a whole and because no direct structural studies have been performed.
Our recent elucidation of thin filament molecular structure by
three-dimensional reconstruction of electron micrographs approaches such a model and has provided essential structural insights into the
thin filament regulatory mechanism (14, 15). These studies show that
tropomyosin adopts three distinct positions on actin depending on
Ca2+ binding to troponin and myosin binding to actin (10).
In the absence of Ca2+, tropomyosin is localized on the
periphery of the filament, where it sterically inhibits actin-myosin
interaction, thereby causing relaxation (9, 16). Activation results
from a two-step movement of tropomyosin away from the myosin binding
site, the first induced by Ca2+, partially switching on the
thin filament, and the second by myosin head binding leading to full
activation (10). Our structural experiments have also enabled
functional changes to be correlated with perturbations of regulatory
transitions in thin filament structure. In this paper, we correlate the
structural and functional effects of a FHC mutation in TnT to
characterize the disease at the molecular level. We examine a
28-residue COOH-terminal truncation of cardiac TnT, similar to protein
resulting from a FHC splice site mutation at the beginning of intron 15 (2). Heterozygotes for this mutation experience ~25% mortality by
age 25, similar to the mortality associated with other TnT mutations
(2). In transgenic animal models expressing the mutant protein, both
systolic and diastolic function are compromised (17). Moreover, in a variety of in vitro experimental systems, thin filaments
containing this mutation exhibit impaired regulation of actin-myosin
interactions reflected in reduced inhibition of actomyosin ATPase
activity in the absence of Ca2+, diminished activation of
myosin cycling in the presence of Ca2+ (6, 18, 19), and
diminished force (18, 20). Despite the obviously altered control
mechanisms, the present report shows that tropomyosin adopts normal
positions on the actin filament, both in the presence and in the
absence of Ca2+. The origin of the thin filament functional
abnormalities is instead shown to be due to weakened binding of
troponin to the thin filament to different extents in the three
regulatory states, thereby affecting the transitions among these states
that control myosin binding and regulate contraction. Development of
hypertrophic cardiomyopathy due to this mutation can thus be traced to
a defect in the energetics of thin filament conformational switching.
Protein Purification--
Rabbit fast skeletal muscle actin and
myosin subfragment 1 were purified to homogeneity as described
previously (14). Cardiac tropomyosin and troponin subunits were
purified (14) from bovine heart obtained at a local slaughterhouse.
Bovine cardiac TnT containing a 28-residue COOH-terminal truncation was
expressed in DE3 cells using the pET3d-based expression vector, as
described previously (6), as was wild type recombinant TnT. In humans,
the FHC-inducing splice site mutation in cardiac TnT, intron 15 G1A, results in two truncated proteins, one missing 14 COOH-terminal residues, and the other in which the 28 COOH-terminal
residues are replaced by seven novel residues. In some experiments (as
indicated), TnT was carboxymethylated on Cys39 using
[3H]iodoacetic acid (Amersham Pharmacia Biotech). Labeled
and unlabeled troponins were reconstituted by combining TnI, TnC, and
TnT under denaturing conditions in a 1:1:1 mixture, followed by
sequential dialysis, and G100 chromatography monitored by
SDS-polyacrylamide gel electrophoresis (6).
Effect of the Mutation on Troponin's Affinity for the Thin
Filament--
Troponin binds very tightly to the thin filament, making
the affinity difficult to measure directly. Therefore, the effect of
the mutation on this process was determined by competition (21).
Unlabeled control or mutant troponin was used to displace radiolabeled
control troponin from the thin filament. Increasing concentrations of
unlabeled troponin were added to labeled thin filaments, and
displacement was measured by determining the supernatant radioactivity
after thin filament sedimentation in a TLA100 ultracentrifuge at 35,000 rpm for 30 min. Data were analyzed as in Hinkle et al. (21),
to determine the value for KR, i.e.
the ratio of the affinity of the competing troponin for the thin
filament, relative to the thin filament affinity of control
[3H]troponin. Conditions: 25 °C, 7 µM
actin, 7 µM myosin S1, 3 µM tropomyosin, 1 µM 3H-labeled troponin, 10 mM
Tris (pH 7.5), 300 mM KCl, 3 mM
MgCl2, 0.2 mM dithiothreitol, 0.3 mg/ml
bovine serum albumin, 0.5 mM EGTA, and either 0 or 0.6 mM CaCl2. These high ionic strength conditions
were used to impair troponin-tropomyosin polymerization, which
otherwise interferes with binding measurements (22). Competing unlabeled troponin was added to samples at concentrations ranging between 0 and 4 µM.
Electron Microscopy and Three-dimensional Reconstruction of Thin
Filaments Containing Mutant Troponin--
Thin filaments were
reconstituted by mixing F-actin (24 µM) first with
cardiac tropomyosin (8 µM) and then troponin (8 µM, prepared as above from wild type troponin I and C and
mutant TnT) in a solution of 250 mM KCl (used to prevent
thin filament aggregation that tends to be induced by troponin), 3 mM MgCl2, 0.5 mM EGTA, 1 mM dithiothreitol, 10 mM sodium phosphate
buffer (pH 7.1). Filaments were allowed to incubate at room temperature
(~25 °C) for 5-10 min before making a 20-fold dilution with
additional buffer lacking KCl such that the final KCl concentration was
12.5 mM. Samples of reconstituted filaments were also
treated with Ca2+ by a comparable 20-fold dilution in the
same buffer lacking both KCl and EGTA but containing 0.1 mM
CaCl2. Diluted filaments were then applied to carbon-coated
electron microscope grids and negatively stained as described
previously (23). Electron micrograph images were recorded on a Philips
CM120 electron microscope at × 60,000 magnification under low
dose conditions (~12 e Measurement of Myosin S1 Binding to Thin Filaments--
To
measure myosin S1-ADP binding to control and mutant thin filaments,
actin was labeled on Cys374 with
N-(1-pyrenyl)iodoacetamide, which is sensitive to bound S1
(34). Steady state fluorescence intensity was monitored during titration of myosin S1 in 1.8-ml stirred, water-jacketed samples at
25 °C. Excitation and emission wavelengths were set at 368 and 407 nm, respectively, using an SLM 8000 spectrofluorometer. The conditions
were 1 µM actin, 0.4 µM tropomyosin, 0.4 µM control or mutant troponin, 20 mM
imidazole (pH 7.5), 150 mM KCl, 3 mM MgCl2, 2 mM ADP, 0.2 mg/ml bovine serum
albumin, 25 units of hexokinase, 1 mM glucose, 20 µM
P1,P5-di(adenosine
5')-pentaphosphate, 0.5 mM EGTA, with or without CaCl2 added to 0.6 mM. Fluorescence data were
analyzed as in Ref. 14, with an 80% decrease in fluorescence
representing 100% saturation of actin with myosin. Data were modeled
as described previously (35), to estimate the effect of the mutation on
the equilibria between switched on and off states. In consideration of
Table I, this analysis assumed that the mutation selectively alters the
free energy for formation of the myosin-blocking state and the
Ca2+ state of the thin filament, to degrees determined by
curve-fitting of the myosin S1 binding data. All other parameters (35)
were held constant.
Effect of Cardiomyopathic TnT Truncation on Stability of Different
Thin Filament Conformations--
Ca2+ controls muscle
contraction by reversibly binding to the globular domain of troponin,
which includes TnC, TnI, and a portion of TnT that contains the 28 residues removed by the FHC splice site mutation (reviewed in Ref. 36).
The interaction of troponin's globular domain with actin and
tropomyosin is Ca2+-sensitive and is believed crucial for
regulation. Previously, we showed that truncation of TnT's 28 COOH-terminal residues weakens troponin binding to thin filaments (6).
In the absence of Ca2+, the mutant troponin has only 22%
the normal affinity for the thin filament, and in the presence of
Ca2+ its affinity is 43% that of control troponin (Table
I) (6). We show here that, in contrast,
the mutation has little effect on troponin binding when myosin is also
bound to the thin filament (Fig. 1). In
averages of multiple experiments such as that shown in Fig. 1, binding
of troponin to filaments decorated with myosin subfragment 1 (S1) was
only slightly diminished by the FHC TnT mutation. The affinity was
almost 75% that of the labeled control troponin, considerably greater
than the values obtained in the absence of myosin S1 (Table I). Thus
the mutation has different effects on thin filament stability under
different conditions, suggesting that it could affect the equilibrium
constants among the various thin filament conformations and therefore
transitions among thin filament states.
Electron Microscopy and Three-dimensional Reconstruction of Thin
Filaments Containing Mutant Troponin--
Although the above
measurements provide thermodynamic information on thin filament
stability, they do not determine how the TnT mutation might affect
filament structure. For example, the position of tropomyosin could be
abnormal in the presence of the mutant TnT, especially in the absence
of both Ca2+ and myosin, when troponin binding to the thin
filament is particularly weak. Electron microscopy was performed to
determine the structural impact of the mutant TnT on thin filaments
reconstituted with otherwise normal troponin subunits and tropomyosin.
Thin filaments in electron micrographs of negatively stained samples
containing normal and mutant troponin (Fig.
2) were well dispersed in both the
presence and absence of Ca2+, so any effects were not due
to possible nonspecific filament aggregation caused by the mutation. In
three-dimensional reconstructions of thin filaments reconstituted using
mutant TnT, the position of tropomyosin was readily identified in
helical projection and cross-section (Fig.
3) and in surface view (Fig.
4), both in the presence and absence of
Ca2+. In filaments examined in the absence of
Ca2+, tropomyosin was positioned at the inner aspect of the
outer domain of actin in close contact with actin subdomains 1 and 2. In contrast, in the presence of Ca2+, tropomyosin
moved to the outer edge of the inner domain of actin over subdomains 3 and 4, exposing most of the actin residues believed to interact with
myosin. This regulatory movement of tropomyosin was indistinguishable
from that observed in our previous work with cardiac muscle thin
filaments containing wild type troponin examined under
Ca2+ and Ca2+-free conditions (14, 37,
38). Since tropomyosin was found in the normal blocking and
Ca2+-induced positions in these filaments, the effects of
the TnT mutation on inhibition and activation of myosin S1-thin
filament MgATPase rates and on troponin-thin filament binding were not due to aberrant tropomyosin position.
Myosin S1-ADP Binding to Control and FHC Mutant Thin
Filaments--
The above structural results leave unanswered the
question of how TnT truncation alters Ca2+-sensitive
regulation of cardiac contraction. To address this, the effect of the
mutation on myosin S1 binding to the thin filament was examined, since
Ca2+-dependent control of this process is
central to how troponin and tropomyosin regulate contraction (10, 36,
39). Our results show, as shown previously, that myosin binding to
control thin filaments is very cooperative in the absence of
Ca2+, resulting in a sigmoidal binding curve (Fig.
5, squares) (35, 40, 41).
Virtually identical results were found for thin filaments containing
the mutant TnT (triangles), with one important exception, namely, a much lower cooperativity in myosin binding to actin. As is
evident when viewed with an expanded scale (see inset of Fig. 5) the binding was less sigmoidal when mutant TnT was present. This indicates a defect in inhibition of cross-bridge binding to the
thin filament in the absence of calcium. However, only the initial
portions of the binding curves differ in Fig. 5; once the filament was
30-40% saturated, the results were similar for mutant and control
samples. Little or no cooperativity was evident in the presence of
Ca2+, and therefore no significant effect of the TnT
mutation on S1 binding was detected (data not shown).
The above results and interpretation qualitatively explain the impaired
inhibition of myosin cycling induced by the mutation at low
Ca2+, corresponding to incomplete diastolic relaxation in
the intact heart: myosin binding is not suppressed, so cycling
continues even at low Ca2+. The myosin binding data were
further assessed by quantitative curve-fitting. Cooperative myosin S1
binding to the thin filament is a complex process involving the
following features: (i) tropomyosin adopts a predominant position on
the actin filament in the absence of Ca2+ that
blocks the myosin binding site, a second position in the presence of Ca2+ that exposes much but not all of the
myosin binding site on actin, and a fully active position in
the presence of myosin in which the binding site is fully exposed (10);
(ii) despite the above, tropomyosin and myosin reciprocally promote
rather than weaken each other's binding to actin (41-45), suggesting
a conformational change on the thin filament binding surface. (14, 46);
(iii) shifts in tropomyosin strand position tend to persist over
contiguous sections of the actin filament (10, 35). Data displayed in Fig. 5 for the mutant and control filaments were fitted to a recent model incorporating these features (35), generating values for the
equilibrium constant between the low Ca2+ ("blocked")
and fully active states of a thin filament. The presence of the mutant
TnT caused a 3-fold enhancement of the transition from the blocked to
the active state, i.e. filaments with the mutation were less
"switched off" and therefore would tend to equilibrate more toward
"switched on" states. The magnitude of this effect is in good
agreement (details in Fig. 5 legend) with the measured effect of the
mutation on the binding of troponin to actin, suggesting that the two
processes, although distinct, are energetically coupled. Furthermore,
this means that the decrease in the cooperativity of myosin binding
caused by the mutant is quantitatively explained by significantly
greater destabilization of the blocked state than the active state of
the filament. Comparable conclusions were reached if the binding data
were analyzed by a related kinetic model detailing thin filament
transitions proposed by McKillop and Geeves (47) (analysis not shown).
The most commonly observed functional effect of cardiomyopathic
mutations in the thin filament has been an increase in Ca2+
sensitivity, i.e. a decrease in the Ca2+
concentration needed for activation. In the heart, elevated
Ca2+ sensitivity may both increase cardiac force in systole
and impair relaxation-dependent cardiac filling during
diastole. Both TnI and tropomyosin mutations increase the apparent
Ca2+ affinity of the thin filament (48, 49), and there are
several reports (Refs. 6, 18, and 50, for example) that TnT missense mutations can cause similar effects. Some TnT mutations also produce other functional abnormalities including decreased force (reviewed in
Ref. 6). A variety of phenotypic effects is not unexpected considering
the complexity of troponin-mediated regulation of contraction.
Both the present and previous work suggest that cardiac relaxation is
altered by the TnT mutation examined here, but by a mechanism other
than enhanced Ca2+ sensitivity. Although some assays showed
that myosin cycling on actin was inhibited effectively by
Ca2+ removal (6, 19), the predominance of the published
data suggest that the TnT mutation impairs the ability of the
regulatory proteins to shut off myosin cycling in the
absence of Ca2+. This conclusion is supported
directly by measurements of force production (18) and actomyosin and
actomyosin S1 MgATPase rates (6, 18) and is indirectly supported by
altered diastolic function observed in intact hearts (17). Defective
interactions between the mutant TnT and the inhibitory TnI subunit (19)
could be related to these instances of compromised regulation. Our
studies here provide new mechanistic insights into these observations, showing that myosin binding to the thin filament is not as effectively inhibited by troponin containing the mutant TnT as by wild type troponin and that the cooperative switching on and off of the filament
is disrupted by the mutant.
Our reconstructions of thin filaments containing the mutant TnT show
that tropomyosin occupies the same positions at high and low
Ca2+ as it does in the absence of the mutation. Contrary to
the expectation that tropomyosin, at low Ca2+, should
sterically interfere with myosin cross-bridge attachment, steric
blocking apparently is ineffective in mutant filaments judging from the
incomplete suppression of actomyosin ATPase activity (6) and from
direct measurement of myosin S1-actin binding (Fig. 5). This is readily
explained by our observation that tropomyosin is less tightly held by
troponin in its inhibitory position, presumably causing less steric
hindrance of myosin binding to actin. Our data as a whole show that it
is the energetics of thin filament conformational transitions that are
altered by the mutant TnT. We suggest that, at low Ca2+,
the mutant filament remains fully or almost fully in the blocked conformation with tropomyosin covering myosin binding sites on actin.
Despite this, the mutation, in effect, lowers the energetic barrier for
the cooperative thin filament transition to the active state leading to
partial activation and cross-bridge cycling even in the absence of
Ca2+. By destabilizing the blocked state more than the
active state, the mutation diminishes the cooperativity of myosin
binding and causes defective inhibition of myosin cycling in the
absence of Ca2+. Defective inhibition would be of obvious
importance in heterozygous patients, consistent with the dominant
inheritance of this disorder. At the purified protein level, mixtures
of wild type and mutant TnT produce intermediate functional behavior
(51), except in the presence of troponin concentrations too low to
saturate the thin filament. A more complex pattern could exist in
vivo, where both direct and indirect effects of the mutation can
occur. The present study addresses the direct effects of the mutation,
which are ultimately responsible for the cascade of pathological events in the hearts of affected individuals.
The thin filament reconstructions show a normal position for
tropomyosin in the presence of calcium, and our binding experiments show no effect of the mutation on the binding of myosin to thin filaments in the presence of Ca2+. Both observations seem
consistent with normal activity once filaments are switched on, yet a
number of studies indicate that not only relaxation but also myosin
cycling is altered by the mutation (6, 18-20). This suggests that
alteration of Ca2+-induced activation involves
perturbations of myosin cross-bridge kinetics, despite unaltered
equilibria of myosin binding and tropomyosin position on actin in the
presence of Ca2+. Interestingly, a mutually induced
increase in binding of myosin and tropomyosin to thin filaments is
thought to be accompanied by an actin or actin surface conformational
change related to normal activation of ATPase rate and force (14, 35,
46). It is possible that TnT also plays a part in this process, which is aberrant when the FHC mutant is present, as suggested by experiments indicating that TnT or certain TnT fragments themselves alter thin
filament activation (52-54).
In addition to elucidating the mechanism of TnT-related FHC, our
results provide key insights into muscle regulatory mechanisms in
general. An interesting pattern is emerging from the current and
previous studies in which tropomyosin shifts among its normal positions
on actin, but thin filament-based regulation is profoundly abnormal
(14, 15, 55). In the present study, the inhibited state was
destabilized by the mutant TnT even though tropomyosin occupied the
normal steric blocking position and displayed normal Ca2+-induced movement. Moreover, in two previous
investigations examining actin (15) and tropomyosin (14) mutations
unrelated to cardiomyopathy, myosin binding and cycling were
inhibited despite normal Ca2+-induced switching of
tropomyosin away from the steric blocking position on actin. These
results taken as a whole indicate that tropomyosin movement is not
sufficient for relief of inhibition and that tropomyosin position on
the outer domain is not sufficient to produce full inhibition. However,
an excellent correlation has held in all these studies between
cooperative inhibition of myosin binding and inhibition of myosin
cycling. When cooperativity in binding was suppressed, regulation was
defective (current study), and when cooperativity was abnormally
increased, myosin cycling was impaired despite tropomyosin localization
in the normal Ca2+-induced position (14, 15).
The primary functions of the regulatory proteins are to inhibit
myosin cycling in the absence of Ca2+ and to release this
inhibition in its presence. Normal inhibition of actin-myosin
interaction requires tight binding of the regulatory proteins to actin
in the absence of Ca2+, tight enough to hold tropomyosin in
a position that sterically interferes with myosin binding. Similarly,
normal activation requires tight troponin-tropomyosin binding to actin
both in the presence of Ca2+ and, in a different position,
in the presence of myosin. This complexity in activation arises because
Ca2+ binding to troponin does not truly activate the
system: Ca2+ does not cause tropomyosin to move far enough
over the actin surface to permit the strong myosin-actin binding that
is part of the myosin cross-bridge cycle. The stepwise regulatory
effects of both Ca2+ and myosin binding depend on the
dynamics and affinities of all components, including the correct
stabilities of all three states of the thin filament. Disrupting
any of these processes, as we have shown here in the case of a
cardiomyopathic TnT mutation, can lead to defective conformational
switching, defective regulation of contraction, and ultimately
devastating clinical consequences.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tropomyosin and cardiac actin and
troponin I and T (TnI, TnT). TnT appears to be the most frequent thin
filament target, and mutations in TnT are associated with relatively
high mortality despite only modest cardiac hypertrophy (2).
Experimentally, TnT mutations produce physiological dysfunction in
transgenic animals and in cultured cells and altered function of
purified proteins assessed in vitro (reviewed in Ref. 6). However, the underlying mechanisms leading to these dysfunction(s) remain poorly understood.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/Å2). Micrographs
were digitized using either Eikonix model 1412 or Imacon Flextight
Precision II scanners at a pixel size corresponding to 0.7 nm in the
filaments. Regions of filaments were selected and straightened as
described previously (24, 25). Helical reconstruction was
carried out by standard methods (26-28) as described previously (10,
29). While actin and tropomyosin contributions are readily delineated
in reconstructions, densities due to troponin are not apparent (see
Ref. 30). Resolution (31) in all reconstructions was between 2.5 and
3.0 nm; comparison of reconstructions made from images digitized on the
respective scanners showed no obvious differences at this resolution.
Tropomyosin and actin densities displayed in reconstructions were
significant (32, 33) at equal to or greater than 99.95% confidence levels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Effect of a cardiomyopathy-inducing TnT mutation on troponin binding to
thin filaments
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Fig. 1.
Binding of normal and mutant troponins to
myosin S1-decorated thin filaments. The effect of the mutation was
measured by competition, i.e. by the mutation's effect on
the ability of troponin to displace radiolabeled wild type troponin
from the thin filament. Three unlabeled troponins were compared in this
representative experiment: FHC troponin (triangles),
reconstituted from truncated TnT and cardiac TnI and TnC; wt troponin
(squares), reconstituted from recombinant TnT and cardiac
TnI and TnC; native troponin (circles), isolated from bovine
hearts as a ternary complex. The dashed line shows the
predicted competitive behavior when the labeled and unlabeled troponins
have nearly equal affinity for the thin filament. The line
is calculated for KR = 0.9, where
KR is a measure of the relative affinity of
(unlabeled) troponin for the thin filament, comparing mutant and
control unlabeled troponins relative to labeled control troponin (see
"Experimental Procedures"). Since a single curve describes all
three data sets, the mutation has little effect. See line 1 of Table I
for a comparison based upon multiple experiments such as the one shown
here.
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Fig. 2.
Electron micrograph of negatively stained
thin filaments containing mutant troponin T. Representative
reconstituted thin filaments containing F-actin, tropomyosin, and
troponin made from wild type troponin I and C and mutant troponin T are
shown. Note that the filaments are well dispersed and separated from
each other. Also note the regularly spaced bulges that appear on the
filaments at ~40 nm intervals (marked by dashes on one
filament). These periodic bulges are a manifestation of troponin. In
many cases, globular ends of troponin are particularly well resolved on
either side of filaments (marked by arrowheads). On a
neighboring filament, a tropomyosin strand is visible (marked by
arrow). Narrower filaments representing bare F-actin
filaments and not displaying troponin bulges or tropomyosin strands are
occasionally observed (one example marked by open arrows).
Note the difference in diameter of this filament from the rest. The
lack of troponin and tropomyosin on the latter is supported by
three-dimensional reconstruction and apparently results from infrequent
"all-or-none" cooperative dissociation of troponin and tropomyosin
from filaments under the conditions used for the electron microscopy.
The scale bar represents 100 nm. Experimental conditions are
given under "Experimental Procedures."
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Fig. 3.
Helical projections (a,
b) and transverse sections (z-sections)
(c, d) of maps of three-dimensional
reconstructions of negatively stained F-actin tropomyosin-troponin
complexes containing mutant troponin T. Helical projections were
formed by projecting component densities down the long pitch actin
helices (i.e. along the n = 2 helical
tracks) onto a plane perpendicular to the thin filament axis; hence,
the resulting projections show axially averaged positions of
tropomyosin relative to actin made to appear bilaterally symmetrical
about actin's central axis. In contrast, transverse sections show the
position of tropomyosin at a given axial level along filaments and
connectivity to specific subdomains of actin. Because adjacent actin
monomers on either side of the filament axis are staggered, sectioning
through the center of actin subdomains 1 and 3 of one actin monomer
results in sectioning through subdomains 2 and 4 of the other.
a and c, EGTA-treated filaments, actin subdomains
1-4 are labeled in c. b and d,
Ca2+-treated F-actin. Note the tropomyosin density
(arrows) associated with subdomains 1 and 2, i.e.
on the outer domain of actin monomers of EGTA-treated filaments, and on
subdomains 3 and 4, i.e. on the inner domain of in
Ca2+-treated filaments. Sections shown are at the same
axial position in each reconstruction, and the actin monomers in
projections and sections have the same relative orientation.
Reconstructions were generated by averaging a data set containing 20 EGTA-treated filaments and another having 8 Ca2+-treated
filaments. The average phase residuals ( ± S.D.), measuring
the relative fitting between individual filaments in each set and the
averaged data, were 60.1 ± 5.0° and 60.6 ± 7.2° for
EGTA- and Ca2+-treated filaments, respectively. The average
up-down phase residuals (
± S.D.), measuring relative
filament polarity, were 19.5 ± 5.6° and 17.1 ± 7.0°,
respectively. The densities contributing to actin and tropomyosin in
the maps shown were statistically significant at confidence levels
greater than 99.95%.
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Fig. 4.
Surface views of reconstruction of mutant
thin filaments showing the positions of tropomyosin strands on
actin. In EGTA (a), tropomyosin is associated with the
inner edge of the outer actin domain of actin. Note the interaction of
tropomyosin with actin subdomain-1 (single white arrowheads)
and bridge of density over the neighboring subdomain-2, while
subdomains-3 and -4 remain unobstructed (black cross). After
Ca2+ treatment (b), tropomyosin is associated
with the outer edge of the inner actin domain of actin. Note that
tropomyosin now interacts with subdomain-3 (double white
arrowheads) while bridging over subdomain-4 and that here
subdomains-1 and -2 (black asterisk) are unobstructed.
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Fig. 5.
Binding of myosin S1-ADP to normal and mutant
thin filaments in the absence of Ca2+.
Actin-troponin-tropomyosin complexes were formed using pyrene actin,
cardiac tropomyosin, and either wild type (squares) or
mutant (triangles) cardiac troponins. Steady state
fluorescence intensity was used to monitor myosin S1 binding to the
thin filament. The inset shows the initial portion of the
same data shown in the full figure, but using an expanded
abscissa. For each troponin, three independent titrations
were performed, with averaged results plotted. The S.E. of the points
shown were 0.013 ± 0.003, i.e. approximately the size
of the symbols in the inset. The mutation caused a marked
decrease in the cooperativity of cross-bridge attachment in the absence
of Ca2+. The difference in the initial portion of the
curves shows that the mutant troponin was poorly effective in
suppression of myosin binding to the thin filament in the absence of
Ca2+. Dashed lines are best fit theoretical
curves. These results (and data in the presence of Ca2+,
not shown) were fitted to a model of myosin S1-thin filament binding
(35), attributing effects of the mutation to alteration in the
stability of the myosin-blocking state of the thin filament (in the
absence of Ca2+) and in the stability of the
Ca2+ state of the thin filament (in the presence of
Ca2+). This corresponds to effects of the mutation and of
Ca2+ on the equilibrium constant (here defined as
KT) for the tropomyosin strand to shift to the
fully active, inner domain position on actin. In the absence of
Ca2+, KT equaled 0.557 ± 0.009 for wild type filaments and 0.658 ± 0.019 for mutant filaments,
demonstrating a greater tendency for myosin binding and switching on of
mutant filaments. When expressed on a per regulatory unit basis (seven
actins, one tropomyosin, one troponin), this equates to a 3.2-fold
effect of the mutation, since
KT17/KT27,
i.e. (0.658/0.557)7 = 3.2. This result is in
good agreement with results shown in Table I, measuring relative
troponin affinities (i.e. KR values),
where a comparable 3.2-fold effect of the mutant was observed. In that
case troponin binding in the active state (Ca2+ and myosin)
is destabilized a factor of 0.71, in the low Ca2+ blocked
state is destabilized by a factor of 0.22, and the ratio of these two
factors equals 3.2. The myosin affinity for active state thin filaments
was determined to be 2.72 ± 0.04 × 106
M 1 (see "Experimental
Procedures"), a value comparable with that previously obtained under
slightly different conditions (35).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL-63774 (to L. T.), HL-36153 (to W. L.), and AR-34711 (to R. C.) and by Shared Instrumentation Grant RR08426 (to R. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ All three laboratories contributed equally to this project.
** To whom correspondence should be addressed: Depts. of Internal Medicine and Biochemistry, 200 Hawkins Dr., S.E. 610-GH, The University of Iowa, Iowa City, IA 52242. Tel.: 319-356-3703; Fax: 319-356-3086; E-mail: larry-tobacman@uiowa.edu.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M101110200
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ABBREVIATIONS |
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The abbreviations used are: FHC, familial hypertrophic cardiomyopathy; Tn, troponin.
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REFERENCES |
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