Effects of a Cardiomyopathy-causing Troponin T Mutation on Thin Filament Function and Structure*

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)(2)(3)(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 ␣-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.
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 Ca 2ϩ 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 Ca 2ϩ binding to troponin and myosin binding to actin (10). In the absence of Ca 2ϩ , 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 Ca 2ϩ , 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 ϳ25% 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 Ca 2ϩ , diminished activation of myosin cycling in the presence of Ca 2ϩ (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 Ca 2ϩ . 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.

EXPERIMENTAL PROCEDURES
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 G 1 A, 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 Cys 39 using [ 3 H]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 K R , i.e. the ratio of the affinity of the competing troponin for the thin filament, relative to the thin filament affinity of control [ 3 H]troponin. Conditions: 25°C, 7 M actin, 7 M myosin S1, 3 M tropomyosin, 1 M 3 H-labeled troponin, 10 mM Tris (pH 7.5), 300 mM KCl, 3 mM MgCl 2 , 0.2 mM dithiothreitol, 0.3 mg/ml bovine serum albumin, 0.5 mM EGTA, and either 0 or 0.6 mM CaCl 2 . 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 M.
Electron Microscopy and Three-dimensional Reconstruction of Thin Filaments Containing Mutant Troponin-Thin filaments were reconstituted by mixing F-actin (24 M) first with cardiac tropomyosin (8 M) and then troponin (8 M, 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 MgCl 2 , 0.5 mM EGTA, 1 mM dithiothreitol, 10 mM sodium phosphate buffer (pH 7.1). Filaments were allowed to incubate at room temperature (ϳ25°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 Ca 2ϩ by a comparable 20-fold dilution in the same buffer lacking both KCl and EGTA but containing 0.1 mM CaCl 2 . 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 ϫ 60,000 magnification under low dose conditions (ϳ12 e Ϫ /Å 2 ). 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.
Measurement of Myosin S1 Binding to Thin Filaments-To measure myosin S1-ADP binding to control and mutant thin filaments, actin was labeled on Cys 374 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 M actin, 0.4 M tropomyosin, 0.4 M control or mutant troponin, 20 mM imidazole (pH 7.5), 150 mM KCl, 3 mM MgCl 2 , 2 mM ADP, 0.2 mg/ml bovine serum albumin, 25 units of hexokinase, 1 mM glucose, 20 M P 1 ,P 5 -di(adenosine 5Ј)-pentaphosphate, 0.5 mM EGTA, with or without CaCl 2 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 Ca 2ϩ 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-Ca 2ϩ 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 Ca 2ϩ -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 Ca 2ϩ , the mutant troponin has only 22% the normal affinity for the thin filament, and in the presence of Ca 2ϩ 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 Ca 2ϩ 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 Ca 2ϩ , so any effects were not due to possible nonspecific fila-ment 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 Ca 2ϩ . In filaments examined in the absence of Ca 2ϩ , 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 Ca 2ϩ , tropomyosin moved to the outer edge of the 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 ϳ40 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. "   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 K R ϭ 0.9, where K R 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  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 Ca 2ϩ and Ca 2ϩ -free conditions (14,37,38). Since tropomyosin was found in the normal blocking and Ca 2ϩ -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 Ca 2ϩ -sensitive regulation of cardiac contraction. To address this, the effect of the mutation on myosin S1 binding to the thin filament was examined, since Ca 2ϩ -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 Ca 2ϩ , 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 Ca 2ϩ , 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 Ca 2ϩ , corresponding to incomplete diastolic relaxation in the intact heart: myosin binding is not suppressed, so cycling continues even at low Ca 2ϩ . 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 Ca 2ϩ that blocks the myosin binding site, a second position in the presence of Ca 2ϩ 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)(42)(43)(44)(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 Ca 2ϩ ("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).

DISCUSSION
The most commonly observed functional effect of cardiomyopathic mutations in the thin filament has been an increase in Ca 2ϩ sensitivity, i.e. a decrease in the Ca 2ϩ concentration needed for activation. In the heart, elevated Ca 2ϩ sensitivity may both increase cardiac force in systole and impair relaxationdependent cardiac filling during diastole. Both TnI and tropomyosin mutations increase the apparent Ca 2ϩ 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 Ca 2ϩ sensitivity. Although some assays showed that myosin cycling on actin was inhibited effectively by Ca 2ϩ 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 Ca 2ϩ . 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 Ca 2ϩ as it does in the absence of the mutation. Contrary to the expectation that tropomyosin, at low Ca 2ϩ , 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 Ca 2ϩ , 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 Ca 2ϩ . 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 Ca 2ϩ . 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 Ca 2ϩ . 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 Ca 2ϩ -induced activation involves perturbations of myosin cross-bridge kinetics, despite unaltered equilibria of myosin binding and tropomyosin position on actin in the presence of Ca 2ϩ . 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 acti- vation 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)(53)(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 filamentbased 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 Ca 2ϩ -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 Ca 2ϩ -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, regu-lation was defective (current study), and when cooperativity was abnormally increased, myosin cycling was impaired despite tropomyosin localization in the normal Ca 2ϩ -induced position (14,15).
The primary functions of the regulatory proteins are to inhibit myosin cycling in the absence of Ca 2ϩ 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 Ca 2ϩ , 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 Ca 2ϩ and, in a different position, in the presence of myosin. This complexity in activation arises because Ca 2ϩ binding to troponin does not truly activate the system: Ca 2ϩ 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 Ca 2ϩ 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.
FIG. 5. Binding of myosin S1-ADP to normal and mutant thin filaments in the absence of Ca 2؉ . 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 Ϯ 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 Ca 2ϩ . 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 Ca 2ϩ . Dashed lines are best fit theoretical curves. These results (and data in the presence of Ca 2ϩ , 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 Ca 2ϩ ) and in the stability of the Ca 2ϩ state of the thin filament (in the presence of Ca 2ϩ ). This corresponds to effects of the mutation and of Ca 2ϩ on the equilibrium constant (here defined as K T ) for the tropomyosin strand to shift to the fully active, inner domain position on actin. In the absence of Ca 2ϩ , K T equaled 0.557 Ϯ 0.009 for wild type filaments and 0.658 Ϯ 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 K T1 7 /K T2 7 , i.e. (0.658/0.557) 7 ϭ 3.2. This result is in good agreement with results shown in Table I, measuring relative troponin affinities (i.e. K R values), where a comparable 3.2-fold effect of the mutant was observed. In that case troponin binding in the active state (Ca 2ϩ and myosin) is destabilized a factor of 0.71, in the low Ca 2ϩ 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 Ϯ 0.04 ϫ 10 6 M Ϫ1 (see "Experimental Procedures"), a value comparable with that previously obtained under slightly different conditions (35).