The Troponin Tail Domain Promotes a Conformational State of the Thin Filament That Suppresses Myosin Activity*

In cardiac and skeletal muscles tropomyosin binds to the actin outer domain in the absence of Ca2+, and in this position tropomyosin inhibits muscle contraction by interfering sterically with myosin-actin binding. The globular domain of troponin is believed to produce this B-state of the thin filament (Lehman, W., Hatch, V., Korman, V. L., Rosol, M., Thomas, L. T., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593–606) via troponin I-actin interactions that constrain the tropomyosin. The present study shows that the B-state can be promoted independently by the elongated tail region of troponin (the NH2 terminus (TnT-(1–153)) of cardiac troponin T). In the absence of the troponin globular domain, TnT-(1–153) markedly inhibited both myosin S1-actin-tropomyosin MgATPase activity and (at low S1 concentrations) myosin S1-ADP binding to the thin filament. Similarly, TnT-(1–153) increased the concentration of heavy meromyosin required to support in vitro sliding of thin filaments. Electron microscopy and three-dimensional reconstruction of thin filaments containing TnT-(1–153) and either cardiac or skeletal muscle tropomyosin showed that tropomyosin was in the B-state in the complete absence of troponin I. All of these results indicate that portions of the troponin tail domain, and not only troponin I, contribute to the positioning of tropomyosin on the actin outer domain, thereby inhibiting muscle contraction in the absence of Ca2+.

Calcium binding to the thin filament protein troponin is required for cardiac and skeletal muscles to contract, and several studies indicate that this regulation involves shifting the tropomyosin position on the actin filament. When the regulatory sites of troponin do not have bound Ca 2ϩ , tropomyosin is located on the actin outer domain. In this position tropomyosin sterically interferes with much more of the myosin-binding site than it does in the presence of Ca 2ϩ , and therefore contraction is inhibited at low Ca 2ϩ concentrations. This regulatory scheme is supported by three-dimensional helical reconstruc-tions of thin filaments examined by electron microscopy with negative staining (1,2) or unstained in vitreous ice (3), and by modeling of x-ray diffraction patterns of muscle (4). Furthermore, it is consistent with the solution kinetics of myosin S1thin filament binding in the absence of ATP (5). However, it is unclear how troponin affects the position of tropomyosin on actin, and more generally the inhibitory action of troponin is not well understood at a structural level, as opposed to better understandings of tropomyosin.
Troponin consists of a relatively globular domain (including TnI 1 and TnC) and an elongated tail region, the NH 2 terminus of TnT (6,7). The inhibitory actions of troponin have long been attributed to the TnI subunit. Skeletal muscle TnI inhibits the actin-myosin ATPase rate in the absence of the other troponin subunits TnC and TnT (8 -10), and this effect requires lower TnI concentrations in the presence rather than in the absence of tropomyosin (11). Cardiac TnI has similar properties, although the inhibition is less effective (12)(13)(14). The inhibitory effects of skeletal muscle TnI can be mimicked by the so-called inhibitory peptide, residues 96 -116 (10,15), or identically by the corresponding cardiac peptide (14). The reversal of inhibition is related to Ca 2ϩ -dependent TnI-TnC interactions, elucidated in part at the atomic level (16). An additional TnI region, approximately 130 -150 residues, has also been implicated in inhibition (17)(18)(19). These and other data are consistent with an inhibitory mechanism consisting primarily of a TnI-actin interaction that is reversed by Ca 2ϩ , i.e. a localized actin-troponin interaction tethers the much longer tropomyosin on the actin outer domain in the absence, but not in the presence, of Ca 2ϩ . Indeed, our own electron microscope results show that Ca 2ϩ causes a decrease in troponin density in contact with actin (20). However, no high resolution data exists for these interactions, or for the assembled thin filament, and it remains possible that other mechanisms are also important for regulation and for determining the shifting positions of tropomyosin on the actin surface.
This report describes new and unexpected attributes of the troponin tail, i.e. the NH 2 terminus of TnT. In the absence of all other portions of troponin, including TnI, cardiac TnT-(1-153) inhibited the interaction of myosin with actin-tropomyosin filaments. Helical reconstruction of filaments indicated that the isolated troponin tail caused tropomyosin to bind preferentially to the actin outer domain, a position in which tropomyosin sterically interferes with strong myosin binding to actin. The results indicate that the troponin tail contributes directly to the inhibition of muscle contraction that occurs in the absence of calcium.
Binding of TnT-  to Actin-Tropomyosin-Troponin or TnT-(1-153) bind very tightly to the thin filament, making their affinities difficult to measure. Therefore, the relative affinity of TnT-(1-153) for actin-tropomyosin was measured by its ability to displace 3 H-labeled whole troponin from the thin filament. Bound and free 3 H-troponin were separated by ultracentrifugation as previously described (26). By fitting the data to Equation 1 in Ref. 22, the affinity of TnT-(1-153) (or unlabeled whole troponin in control experiments) for actin-tropomyosin was measured, relative to the affinity of the 3 H-labeled whole troponin. The conditions used were: 25°C, 7 M actin, 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 DTT, 0.3 mg/ml bovine serum albumin, and 0.1 mM CaCl 2 ( Fig. 1), 0.5 mM EGTA, or 7 M myosin S1. CaCl 2 was also present in experiments including myosin S1, but had no effect on troponin-thin filament binding under these conditions (29).
Actin-activated MgATPase Assays-The thin filament-activated myosin S1 MgATPase rate was measured by serial determinations of [ 32 P]P i released from [␥-32 P]ATP (30). Rates were linear with time during the initial 10 min used for the assay. Unless whole troponin was present, the Ca 2ϩ concentration had no effect on the results. In the absence of actin, the myosin S1 MgATPase rate was negligible. The conditions used were: 25°C, 5 mM Tris-HCl (pH 7.5), 1 mM DTT, 3. Binding of Myosin S1 to the Thin Filament-Myosin S1-ADP binding to the thin filament was measured as previously described (31) using pyrene-labeled actin (32), monitoring the decrease in fluorescence intensity as increasing S1 was added. The fitted parameters were K S and L. The unitless equilibrium constants Y and L ϫ K T were fixed at 97 and 2.49, respectively (28). This method produced curves matching the data, which could alternatively be accomplished with other assumptions (i.e. some assumptions were required, because the data was not sufficient to determine uniquely all the equilibrium constants (K S , L, Y, and L ϫ K T ). The choice to selectively fit K S and L to assess the effects of TnT-(1-153) was based on the following: 1) a stabilization of the B-state would directly affect L, which is defined as the equilibrium constant for tropomyosin to shift from the actin inner domain to the B-state, outer domain position (with no change in actin itself); 2) K S is the affinity of myosin S1 for M-state actin monomers, which depends upon experimental conditions; 3) L ϫ K T describes a change in actin (to the active or M-state) that primarily depends on tropomyosin, not troponin; 4) Y is a cooperativity term principally (albeit not entirely; see "Discussion") attributable to tropomyosin flexibility.
Motility Measurements and Analysis-Rhodamine-phalloidin-labeled F-actin was prepared as described previously (25) and used within 2 weeks. For motility solutions containing the un-regulated actin, the actin solution was diluted to 8 nM in the motility chamber. For thin filaments containing tropomyosin, 8 M actin was incubated with 2 M tropomyosin or 2 M tropomyosin/TnT-(1-153) for at least 24 h before use. For the motility solution, the solution was diluted to 8 nM actin, 2 nM tropomyosin or 8 nM actin, 2 nM tropomyosin, 2 nM TnT-(1-153) in the motility chamber and tropomyosin or tropomyosin/TnT-(1-153) were added to the motility solution to 2 M. This assured that the actin filament was labeled with the regulatory proteins.
Nitrocellulose-coated coverslips and flow cells were prepared as previously described (25). An HMM solution (35-300 g/ml) was injected into the chamber. The density of HMM on the motility chamber surface was computed from studies showing that HMM binds to the surface according to a binding isotherm whose apparent K m was 168 Ϯ 45 g of HMM ml Ϫ1 and was saturated at 3850 Ϯ 30 g of HMM m Ϫ2 of motility surface (34). Two min after adding the HMM, the solution was replaced by 50 l of 0.5 mg/ml bovine serum albumin in assay buffer (25 mM MOPS, 25 mM KCl, 2 mM MgCl 2 , 2 mM EGTA (pCa 9), 1 mM DTT, pH 7.4). One minute later 50 l of 8 nM RhP-actin, 8 nM RhP-phalloidinactin and 2 M tropomyosin, or the 8 nM RhP-actin and 2 M tropomyosin/TnT-(1-153) fragment were introduced and allowed to incubate for 2 min. Finally to initiate and sustain motility, 50 l of a motility solution was introduced into the flow cell. It contained 25 mM MOPS (pH 7.4), 26.1 mM KCl, 2 mM MgCl 2 , 2 mM EGTA, 1 mM Na 2 -ATP, 0.15% methyl cellulose, and 20 mM DTT, and photobleaching protective agents (35): 14 mM glucose, 240 units of glucose oxidase/ml, and 9 ϫ 10 3 units of catalase/ml (both enzymes were from Sigma). The ionic strength (50 mM) and pCa of the activating solution were calculated as previously described (36). If the actin contained either tropomyosin or the tropomyosin/TnT fragment, it was added to the motility solution to 2 M.
Quantification of the thin filament sliding speed was performed using a Motion Analysis System (Santa Rosa, CA). Data was acquired and analyzed as previously described (25) and were expressed as the mean Ϯ S.E. In these analyses filaments not moving at a uniform sliding speed were rejected. However, the results were qualitatively the same if, instead, all filaments were averaged, including those moving at non-uniform speeds.
Electron Microscopy-Bovine cardiac or rabbit skeletal muscle tropomyosin (4.6 M) were combined with TnT-(1-153) (4.5 M) and then with F-actin (25 M) in a solution of 100 mM NaCl, 0.2 mM EGTA, 3 mM MgCl 2 , 1 mM NaN 3 , 1 mM DTT, 5 mM sodium phosphate/Pipes buffer (pH 7.1) at room temperature (ϳ25°C). After an ϳ10 min incubation the sample was diluted 20-fold and applied to carbon-coated electron microscope grids and negatively stained as described before (37). Electron micrograph images of filaments were recorded on a Philips CM120 electron microscope at ϫ60,000 magnification under low-dose conditions (ϳ12 e Ϫ /Å 2 ). Micrographs were digitized using an Eikonix 1412 scanner at a pixel size corresponding to 0.7 nm in the filaments. Well stained regions of filaments were selected and straightened as before (38,39). Helical reconstruction was carried out by standard methods (40 -42) as previously described (43,44). The reconstruction had a resolution (45) of 2.5 to 3.0 nm; the positional accuracy of the method is ϳ0.5 nm (46).

The Troponin Tail Region Binds Tightly to the Thin Filament in Both the Presence and
Absence of Myosin S1-The significance of the isolated troponin tail for myosin-thin filament interactions can be assessed most directly by studying a tail construct that binds tightly and specifically to the thin filament. As shown in a recent report (22), cardiac TnT-(1-153) promotes the binding of tropomyosin to actin (as also does TnT-(70 -170) (47)) almost as strongly as does whole troponin. Furthermore, representative data in Fig. 1 show that this same TnT fragment binds to actin-tropomyosin tightly enough to displace whole troponin from the thin filament. Because whole troponin binds to actin-tropomyosin with an affinity of 3 ϫ 10 8 M Ϫ1 in the presence of Ca 2ϩ (48) (the Fig. 1 conditions), these competitive binding data imply that TnT-(1-153) binds to the thin filament with affinity of 5 ϫ 10 7 M Ϫ1 . This quantitative result is consistent with the previous, more qualitative conclusion that the tightest association between troponin and the thin filament involves an interaction between the tropomyosin COOH terminus and the NH 2 -terminal half of TnT (reviewed in Ref. 49); in the current experiments, the TnT NH 2 terminus is the only portion of troponin that is present, and it binds tightly to actin-tropomyosin. In part, the basis for this is revealed in a recent crystallographic study (50), in which COOH-terminal tropomyosin residues were observed to destabilize the core of the coiled-coil, creating a TnT recognition site. On the other hand, the structural basis for tight binding of the tropomyosin-TnT complex to actin is unknown. Table I, column 3, states the averaged results of Fig. 1 plus two identical experiments, and of similarly replicated experiments performed either with myosin S1 also bound to the thin filament, or else with CaCl 2 replaced by EGTA. Ca 2ϩ weakens the binding of troponin to the thin filament about 2-fold (48,51), but should have no comparable effect on TnT-(1-153), which does not bind Ca 2ϩ . Consistent with this prediction, Table I shows that TnT-(1-153) competed with troponin slightly more effectively when Ca 2ϩ was bound to troponin (0.152 versus 0.096), and Ca 2ϩ had no discernable effect on TnT-(1-153) itself (K ϭ 5 ϫ 10 7 M Ϫ1 regardless of Ca 2ϩ ). Table I also shows that decoration of actin-tropomyosin with myosin S1 had similar effects on thin filament binding of either whole troponin or the isolated troponin tail, weakening the affinities of each by an order of magnitude. However, note that TnT-(1-153) bound to the thin filament with significant affinity, 4 ϫ 10 6 M Ϫ1 , even when filaments were fully decorated with myosin S1.
Effects of Cardiac Tropomyosin, Troponin, and the Isolated Troponin Tail on Myosin S1-Thin Filament MgATPase Activity-In the presence of Ca 2ϩ and assessed using subsaturating actin concentrations, cardiac troponin-tropomyosin increases the actin-myosin S1 MgATPase rate (21), and this effect is independent of the actin or myosin S1 isoforms (52)(53)(54)(55). This is opposite to results with skeletal muscle troponin-tropomyosin, which decreases the MgATPase rate, regardless of actin concentration, by causing a 60% drop in the value of V max observed in the presence of high actin concentrations (56). To better understand these opposite findings, the effect of cardiac troponin-tropomyosin on the MgATPase rate was measured over a range of actin concentrations. As shown in Table II, the activating effect of the cardiac regulatory proteins is because of an increase in the apparent affinity (1/K app ) of myosin S1 for actin, combined with an unchanged (rather than decreased) V max of about 25 s Ϫ1 .
In contrast to the effects of tropomyosin plus troponin, cardiac tropomyosin alone decreased not only the K app , but also the V max , with an 8-fold effect. This alteration in V max agrees with previous data employing skeletal muscle tropomyosin, which decreases the V max by 5-fold (56). The troponin tail fragment enhanced this inhibitory effect of the tropomyosin, decreasing the V max by another 60%, i.e. from 2.6 to 1.0 s Ϫ1 , which is less than 5% of the V max observed for either unregulated actin or actin-tropomyosin-troponin-Ca 2ϩ . This inhibitory effect of tropomyosin-TnT-(1-153) is similar to that produced by adding EGTA to fully regulated thin filaments: a drop in V max by 96%, with little effect on K app (21). These data imply a significant inhibitory effect of the troponin tail region on myosin ATPase activity, in the absence of any other portion of troponin. The cardiac troponin tail enhances the inhibitory actions of cardiac tropomyosin.
Effect of the Troponin Tail Region on in Vitro Motility-To determine whether the troponin tail region affects the mechanical function of myosin, as well as having the enzymatic effects shown above, in vitro motility experiments were performed (Fig. 2). In parallel with the myosin S1 ATPase results, TnT-(1-153) decreased the heavy meromyosin-propelled sliding speed of actin-tropomyosin filaments, and this inhibitory effect was much larger than the effect of tropomyosin alone. (The illustrated effects of tropomyosin alone resemble findings in an earlier report (31).) For all tested concentrations of heavy meromyosin that were attached to the sliding surface, the observed thin filament sliding speed was decreased when TnT-(1-153) was added to the actin-tropomyosin. Furthermore, both the threshold concentration of HMM required to induce movement, and the HMM concentration that produced 50% of maximal speed were increased when TnT-(1-153) was present. Qualitatively, these data suggest that the troponin tail weakens or inhibits the transition toward a myosin-thin filament attachment that is required for sliding to proceed. However, this inhibition is not as profound as that produced by whole troponin in the absence of Ca 2ϩ , which prevents all filament sliding under conditions comparable with Fig. 2 (25,36). This suggests that full inhibition requires TnI-actin interactions that are absent when the troponin tail is studied in isolation.
The Troponin Tail Region Increases the Cooperativity of Myosin S1-ADP Binding to the Thin Filament-Tropomyosin sterically interferes with strong binding of myosin to actin (i.e. binding observed in the absence of ATP), and TnT-(1-153) strengthens tropomyosin binding to actin. Therefore, the inhibitory properties of this TnT fragment could be because of tighter attachment of tropomyosin in a position that interferes with myosin. To test this, myosin S1-ADP binding to pyrenelabeled actin was examined in the presence of tropomyosin alone, or tropomyosin plus TnT-(1-153) (Fig. 3). Inclusion of the troponin tail region had little effect, with the notable exception of the initial portions of the S1-binding curves, at low myosin S1-ADP concentrations. From this region of the curves, it is apparent that myosin binding to the thin filament was more cooperative when the troponin tail was present. TnT-(1-153) inhibited the initial binding of myosin to myosin-free or nearly myosin-free filaments. This is consistent with an effect of the troponin tail region that consists of stabilizing tropomyosin in a position on actin that interferes with strong myosin binding. Once tropomyosin is displaced from this position by the binding of a relatively small number of myosin heads, the troponin tail has little effect on additional myosin binding. These conclusions are supported by the quantitative analysis of Fig. 3; from curve fitting using the model in Refs. 28 and 31, the equilibrium constant for tropomyosin to shift to the actin outer domain (in the model, this equals L 7 ) increased about 5-fold, and the myosin binding constant (K S ) was unaffected.
Effect of the Troponin Tail Region on the Position of Tropomyosin on the Actin Surface-The most straightforward extrapolation from the above results is that TnT-(1-153) causes tropomyosin to bind tightly to the actin outer domain, where it inhibits myosin cycling by steric interference. However, tropomyosin sterically interferes with myosin not only when the tropomyosin is on the actin outer domain (the B-state, characteristic of actin-troponin-tropomyosin filaments in the absence of Ca 2ϩ (1, 2)), but also sterically interferes, albeit to a lesser extent, when the tropomyosin is on the outer edge of the actin inner domain (the C-state, which is the predominant state in the presence of troponin plus Ca 2ϩ ) (4,44). Only when tropomyosin shifts further onto the actin inner domain (the M-state) is there no steric interference between tropomyosin and myosin (44). Therefore, the ATPase, in vitro motility, and myosin binding data in the current study could result from the troponin tail strengthening association of tropomyosin with actin in either the B-state or the C-state position. As precedents for the latter possibility, specific mutations in either tropomyosin (33) or actin (55) result in C-state filaments that are profoundly inhibitory, even more so than the current results with TnT- (1-153). Furthermore, if the B-state depends upon TnI-actin interactions, then actin-tropomyosin-TnT-(1-153) filaments should not be in the B-state.
To assess these issues, thin filaments were examined by electron microscopy with helical reconstruction (Fig. 4). Fila-  -(1-153) to actin-tropomyosin The relative affinity of TnT-(1-153) for actin-tropomyosin, normalized to the affinity of radiolabeled troponin, was determined by competition as exemplified in Fig. 1. These data were converted to absolute affinity values (column 4), using published data for the thin filament affinity of troponins (column 1). Thin filament states B, C, or M (57) refer to the predominant azimuthal position of Tm on actin (A), as determined experimentally in the presence of either TnT-(1-153) or troponin (Tn). g Strongly bound myosin S1 sterically prevents tropomyosin from binding in any position other than the M-state (4,44); it is presumed that TnT-(1-153) does not alter this.

FIG. 2. Effect of the troponin tail region on in vitro motility.
Rhodamine-phalloidin-labeled thin filaments were applied to surfaces with variable densities of attached heavy meromyosin, and the speed of continuously sliding filaments was measured. Tropomyosin had relatively small, inhibitory effects on its own, but substantial sliding inhibition was observed when TnT-(1-153) was also present. Each symbol is the average of between 207 and 522 filaments, with S.E. values no larger than the symbols, and S.D. values between 0.3 and 1.3 m/s. The motility chamber solutions contained sufficient concentrations (indicated) of tropomyosin and/or TnT-(1-153) to saturate the actin filaments (see "Materials and Methods"). ments containing tropomyosin plus the troponin tail contained additional density, corresponding to the tropomyosin strand, which is not observed in reconstructions of bare actin filaments (3,57). Regardless of whether the tropomyosin was from skeletal muscle or from cardiac muscle, this additional density contacted with the actin outer domain in indistinguishable, B-state positions. This can also be seen in helical projections and Z-sections of the reconstructions (Fig. 5). For comparison, it is significant that in the absence of any portion of troponin, skeletal muscle tropomyosin binds in the C-state position (57) (at the outer edge of the inner domain), and cardiac tropomyosin localizes to the same B-state position (57) now observed when TnT-(1-153) was present. Therefore, the cardiac troponin tail does not shift the predominant position of the cardiac tropomyosin, but rather strengthens its binding to actin. In contrast, the cardiac troponin tail shifts the position of skeletal muscle tropomyosin on actin, from the C-state to the B-state position. DISCUSSION A principal observation in this report is that, in the presence of tropomyosin, the TnT NH 2 terminus inhibits the interactions between myosin and the thin filament, suppressing strong myosin-actin binding, the maximal actomyosin ATPase rate, and the rate of filament sliding. Previous studies have clearly indicated that the inhibitory properties of troponin involve the globular, TnI-containing region of the troponin complex (for review, see Refs. 49 and 58). However, the current results indicate that striking inhibition is produced independently by another region of the extended troponin molecule: the troponin tail fragment TnT-(1-153) examined in this report. This unexpected result has implications both for the function of the troponin tail region, and for the overall mechanism of thin filament-mediated regulation.
The first functional characterizations of the troponin tail region employed rabbit fast skeletal muscle TnT chymotryptic fragment TnT-(1) (59 -61), now known to consist of residues 1-165 of the predominant fast skeletal isoform (Protein Information Resource code TPRBTS). TnT-(1) does not interact with TnC or TnI, and extends away from the globular region of troponin to form an elongated structure that binds to a long segment of tropomyosin (62). TnT-(1) increases the cooperativity with which myosin S1 converts actin-tropomyosin filaments to the M-or active state (63). In a new report by Maytum and colleagues, (84) TnT-(1) was found to be inhibitory to myosin S1, similar to the actions of the cardiac construct TnT-(1-153) examined in the present article. Similarly, Ohtsuki and coworkers (65,66) found that TnT-(1) inhibited both superprecipitation and actomyosin ATPase rates in the presence of tropomyosin, TnI, and TnC. This agreement supports the generality of our findings that the troponin tail, in the absence of the troponin I-containing globular domain, has substantial inhibitory effects.
On the other hand, the detailed studies of Maytum et al. (84) suggest that TnT-(1) stabilizes the C-state of the thin filament, rather than the B-state now observed with the cardiac troponin tail fragment. Thus, the two troponin tail fragments appear to act by different mechanisms. One possible explanation is that selected experimental methods differ in the two studies: TnT-(1) has not been examined by electron microscopy, nor have the effects of TnT-(1-153) on S1-thin filament binding kinetics been determined. These methods generally agree, but not always (57). However, the most likely explanation for the discrepancy is that structural differences between the peptides result in different behavior. TnT-(1-153) of the current study shares high homology to skeletal TnT-(1) only in one region: cardiac TnT residues 74 -153 are 80% homologous to skeletal muscle TnT residues 54 -133. The more NH 2 -terminal parts of the peptides are not homologous, and TnT-(1) contains 31 conserved residues at its COOH terminus that are not included in TnT-(1-153). These dissimilarities may explain why the cardiac TnT fragment inhibits myosin S1 ATPase activity more than does skeletal muscle TnT-(1), and also why the cardiac fragment stabilizes the B-state rather than the C-state. (Note that these two functional differences are distinct; the degree of ATPase inhibition does not always correlate with the presence of the B-state (33,55,57,67).) Unfortunately, a cardiac TnT fragment similar to skeletal muscle TnT-(1) has poor solubility (22), making the importance of the additional COOH-terminal residues in skeletal muscle TnT-(1) difficult to test.
Subtle differences in the amino acid sequence of either tropomyosin or actin can lead to a change in the equilibrium position of tropomyosin on actin (57). Therefore, it is not surprising that different fragments of TnT in either the presence or the absence of S1 may perturb M-state to C-state to B-state equilibria to different extents. Interestingly, very different properties were reported for an NH 2 -terminal fragment of skeletal muscle TnT that is still longer than TnT-(1), by 27 conserved residues. Unlike the results with both cardiac TnT-(1-153) and skeletal TnT-(1), skeletal fragment TnT-(1-191) causes a modest increase in the myosin-actin-tropomyosin ATPase rate (68), similar to findings with holo-TnT (69,70). Although the regulatory effects of TnT-(1-191) (and holo-TnT) were not examined in detail, the available data suggest that different regions of the troponin tail have distinct effects on the thin filament, and sometimes opposite effects on myosin-thin filament interactions. The same conclusions can be inferred from more indirect approaches to these issues: deletional (71,72) or mutational (36,52,(73)(74)(75)(76)(77) analyses of the TnT NH 2 terminus. However, interpretation of some of the earlier data is complicated by the high likelihood of a non-native structure for the TnT COOH terminus in the absence of TnI and TnC (78). Also, despite the near-native folding implied by the tight and specific binding of TnT-(1-153) to the thin filament, it is possible that the troponin tail peptide properties are affected by non-native structure. These several considerations, and the complex nature of thin filament activation, require that interpretations be made with caution.
Nevertheless, when considered in sum the current and previous reports suggest that the troponin tail produces specific interactions that stabilize tropomyosin binding to actin in the B-state position, and separate interactions that stabilize the C-state. When studied as an isolated peptide, this can result in an equilibrium favoring one or the other state, depending on the construct. This conclusion advances understanding of the regulatory mechanism when it is combined with previous results, which have shown that when Ca 2ϩ binds to troponin, selected TnI sites move further from actin (79 -81) and there is decreased troponin density near actin (20). Ca 2ϩ -mediated detachment of part of TnI from actin has been generally viewed as critical for the switching of the thin filament out of the B-state. The current work does not refute this viewpoint, but rather shows that it is incomplete; it is now clear that Ca 2ϩ must also reverse other interactions that strongly stabilize the B-state, interactions involving TnT-(1-153).
Because both TnT-(1-153) and the inhibitory peptide of TnI promote formation of the B-state, it is unclear how the thin filament switches to the more activated C-state in the presence of Ca 2ϩ . We have suggested previously (48,51,57,82) that the B to C transition includes an active process, and involves more than a passive release of TnI from actin. TnT may contribute to this (48), as also proposed by Potter et al. (69). The present work supports the more detailed conclusion that the C-state is stabilized by Ca 2ϩ -triggerred actions of portions of troponin other than TnT- . In analogy to the quaternary structure behavior of allosteric systems in general, the tropomyosintroponin tail complex presumably interacts broadly with alternative sites on actin, as the complex shifts its position on the actin surface. The stabilization of the B-state by the cardiac troponin tail, in the absence of TnI, suggests significant gaps in our understanding of thin filament activation, and reinforces the importance of further work on the structure of the actintroponin-tropomyosin complex.
Finally, it is notable that the properties of tropomyosin-TnT-(1-153) on the MgATPase rate V max , sliding speed, and the cooperativity of myosin S1 binding to actin in each case exaggerate similar effects produced by tropomyosin alone. The simplest explanation, as stated above, is that these effects are because of greater stabilization of the B-state when TnT-(1-153) is present. However, this need not be the only mechanism, and there may be several functional differences between tropomyosin alone and tropomyosin in complex with the troponin tail. For example, TnT and tropomyosin have extended regions of contact with each other when co-crystallized (62), suggesting that when tropomyosin binds to the troponin tail, the resultant complex may be less flexible than is implied by studies of tropomyosin alone (83). A full understanding of regulation will require the explication of the cooperative transitions among the several states of the thin filament, active and inactive. The flexibility of the tropomyosin-troponin strand is a critical aspect of regulation because of its potential to alter the cooperativity of these transitions, i.e. the longitudinal propagation of conformational change along the thin filament (28,64). We predict that the troponin tail region will prove important for this cooperative aspect of thin filament function.