Calcium-dependent protein-protein interactions induce changes in proximity relationships of Cys48 and Cys64 in chicken skeletal troponin I.

The goal of this study was to relate conformational changes in the N-terminal domain of chicken troponin I (TnI) to Ca2+ activation of the actin-myosin interaction. The two cysteine residues in this region (Cys48 and Cys64) were labeled with two sulfhydryl-reactive pyrene-containing fluorophores [N-(1-pyrene)maleimide, and N-(1-pyrene)iodoacetamide]. The labeled TnI showed a typical fluorescence spectrum: two sharp peaks of monomer fluorescence and a broad peak of excimer fluorescence arising from the formation of an excited dimer (excimer). Results obtained show that forming a binary complex of labeled TnI with skeletal TnC (sTnC) in the absence of Ca2+ decreases the excimer fluorescence, indicating a separation of the two residues. This reduction in excimer fluorescence does not occur when labeled TnI is complexed with cardiac TnC (cTnC). The latter causes only partial activation of the Ca2+-dependent myofibrillar ATPase. The binding of Ca2+ to the two N-terminal sites of sTnC causes a significant decrease in excimer fluorescence and an increase in monomer fluorescence in complexes of labeled TnI with skeletal TnC or TnC/TnT, while Ca2+ binding to site II of cTnC only causes an increase in monomer fluorescence but no change in excimer fluorescence. Thus a conformational change in the N-terminal region of TnI may be necessary for full activation of muscle contraction.     

Unloaded shortening of skinned muscle fibers from rabbit activated with and without Ca2+.

Unloaded shortening velocity (VUS) was determined by the slack method and measured at both maximal and submaximal levels of activation in glycerinated fibers from rabbit psoas muscle. Graded activation was achieved by two methods. First, [Ca2+] was varied in fibers with endogenous skeletal troponin C (sTnC) and after replacement of endogenous TnC with either purified cardiac troponin C (cTnC) or sTnC. Alternatively, fibers were either partially or fully reconstituted with a modified form of cTnC (aTnC) that enables force generation and shortening in the absence of Ca2+. Uniformity of the distribution of reconstituted TnC across the fiber radius was evaluated using fluorescently labeled sTnC and laser scanning fluorescence confocal microscopy. Fiber shortening was nonlinear under all conditions tested and was characterized by an early rapid phase (VE) followed by a slower late phase (VL). In fibers with endogenous sTnC, both VE and VL varied with [Ca2+], but VE was less affected than VL. Similar results were obtained after extraction of TnC and reconstitution with either sTnC or cTnC, except for a small increase in the apparent activation dependence of VE. Partial activation with aTnC was obtained by fully extracting endogenous sTnC followed by reconstitution with a mixture of aTnC and cTnC (aTnC:cTnC molar ratio 1:8.5). At pCa 9.2, VE and VL were similar to those obtained in fibers reconstituted with sTnC or cTnC at equivalent force levels. In these fibers, which contained aTnC and cTnC, VE and VL increased with isometric force when [Ca2+] was increased from pCa 9.2 to 4.0. Fibers that contained a mixture of a TnC and cTnC were then extracted a second time to selectively remove cTnC. In fibers containing aTnC only, VE and VL were proportional to the resulting submaximal isometric force compared with maximum Ca(2+)-activated control. With aTnC alone, force, VE, and VL were not affected by changes in [Ca2+]. The similarity of activation dependence of VUS whether fibers were activated in a Ca(2+)-sensitive or -insensitive manners implies that VUS is determined by the average level of thin filament activation and that, with sTnC or cTnC, VUS is affected by Ca2+ binding to TnC only.     

Mutation of the high affinity calcium binding sites in cardiac troponin C.

Fast skeletal and cardiac troponin C (TnC) contain two high affinity Ca2+/Mg2+ binding sites within the C-terminal domain that are thought to be important for association of TnC with the troponin complex of the thin filament. To test directly the function of these high affinity sites in cardiac TnC they were systematically altered by mutagenesis to generate proteins with a single inactive site III or IV (CBM-III and CBM-IV, respectively), or with both sites III and IV inactive (CBM-III-IV). Equilibrium dialysis indicated that the mutated sites did not bind Ca2+ at pCa 4. Both CBM-III and CBM-IV were similar to the wild type protein in their ability to regulate Ca(2+)-dependent contraction in slow skeletal muscle fibers, and Ca(2+)-dependent ATPase activity in fast skeletal and cardiac muscle myofibrils. The mutant CBM-III-IV is capable of regulating contraction in permeabilized slow muscle fibers but only if the fibers are maintained in a contraction solution containing a high concentration of the mutant protein. CBM-III-IV also regulates myofibril ATPase activity in fast skeletal and cardiac myofibrils but only at concentrations 10-100-fold greater than the normal protein. The pCa50 and Hill coefficient values for Ca(2+)-dependent activation of fast skeletal muscle myofibril ATPase activity by the normal protein and all three mutants are essentially the same. Competition between active and inactive forms of cardiac and slow TnC in a functional assay demonstrates that mutation of both sites III and IV greatly reduces the affinity of cardiac and slow TnC for its functionally relevant binding site in the myofibrils. The data indicate that although neither high affinity site is absolutely essential for regulation of muscle contraction in vitro, at least one active C-terminal site is required for tight association of cardiac troponin C with myofibrils. This requirement can be satisfied by either site III or IV.     

Effect of temperature and the F27W mutation on the Ca2+ activated structural transition of trout cardiac troponin C

The Ca(2+) sensitivity of cardiac contractile element is reduced at lower temperatures, in contrast to that in fast skeletal muscle. Cardiac troponin C (cTnC) replacement in mammalian skinned fibers showed that TnC plays a critical role in this phenomenon (Harrison and Bers, (1990), Am. J. Physiol. 258, C282-8). Understanding the differences in affinity and structure between cTnCs from cold-adapted ectothermic species and mammals may bring new insights into how the different isoforms provide different resistances to cold. We followed the Ca(2+) titration to the regulatory domain of rainbow trout cTnC by NMR (wild type at 7 and 30 degrees C and F27W mutant at 30 degrees C) and fluorescence (F27W mutant, at 7 and 30 degrees C) spectroscopies. Using NMR spectroscopy, we detected Ca(2+) binding to site I of trout cTnC at high concentrations. This places trout cTnC between mammalian cTnC, in which site I is completely inactive, and skeletal TnC, in which site I binds Ca(2+) during muscle activation, and which is not as much affected by lower temperatures. This binding was seen both at 7 and at 30 degrees C. Despite the low Ca(2+) affinity, trout TnC site I may increase the likelihood of an opening of the regulatory domain, thus increasing the affinity for TnI. This way, it may be responsible for trout cTnC's capacity to function at lower temperatures.     

Influence of length on force and activation-dependent changes in troponin c structure in skinned cardiac and fast skeletal muscle

Linear dichroism of 5' tetramethyl-rhodamine (5'ATR) was measured to monitor the effect of sarcomere length (SL) on troponin C (TnC) structure during Ca2+ activation in single glycerinated rabbit psoas fibers and skinned right ventricular trabeculae from rats. Endogenous TnC was extracted, and the preparations were reconstituted with TnC fluorescently labeled with 5'ATR. In skinned psoas fibers reconstituted with sTnC labeled at Cys 98 with 5'ATR, dichroism was maximal during relaxation (pCa 9.2) and was minimal at pCa 4.0. In skinned cardiac trabeculae reconstituted with a mono-cysteine mutant cTnC (cTnC(C84)), dichroism of the 5'ATR probe attached to Cys 84 increased during Ca2+ activation of force. Force and dichroism-[Ca2+] relations were fit with the Hill equation to determine the pCa50 and slope (n). Increasing SL increased the Ca2+ sensitivity of force in both skinned psoas fibers and trabeculae. However, in skinned psoas fibers, neither SL changes or force inhibition had an effect on the Ca2+ sensitivity of dichroism. In contrast, increasing SL increased the Ca2+ sensitivity of both force and dichroism in skinned trabeculae. Furthermore, inhibition of force caused decreased Ca2+ sensitivity of dichroism, decreased dichroism at saturating [Ca2+], and loss of the influence of SL in cardiac muscle. The data indicate that in skeletal fibers SL-dependent shifts in the Ca2+ sensitivity of force are not caused by corresponding changes in Ca2+ binding to TnC and that strong cross-bridge binding has little effect on TnC structure at any SL or level of activation. On the other hand, in cardiac muscle, both force and activation-dependent changes in cTnC structure were influenced by SL. Additionally, the effect of SL on cardiac muscle activation was itself dependent on active, cycling cross-bridges.     

The control of myocardial contraction with skeletal fast muscle troponin C

The present study describes experiments on the myocardial trabeculae from the right ventricle of Syrian hamsters whose troponin C (TnC) moiety was exchanged with heterologous TnC from fast skeletal muscle of the rabbit. These experiments were designed to help define the role of the various classes of Ca2+-binding sites on TnC in setting the characteristic sensitivities for activations of cardiac and skeletal muscles. Thin trabeculae were skinned and about 75% of their troponin C extracted by chemical treatment. Tension development on activations by Ca2+ and Sr2+ was found to be nearly fully blocked in such TnC extracted preparations. Troponin C contents and the ability to develop tension on activations by Ca2+ and Sr2+ was permanently restored after incubation with 2-6 mg/ml purified TnC from either rabbit fast-twitch skeletal muscle (STnC) or the heart (CTnC, cardiac troponin C). The native (skinned) cardiac muscle is characteristically about 5 times more sensitive to activation by Sr2+ than fast muscle, but the STnC-loaded trabeculae gave response like fast muscle. Attempts were also made to exchange the TnC in psoas (fast-twitch muscle) fibers, but unlike cardiac muscle tension response of the maximally extracted psoas fibers could be restored only with homologous STnC. CTnC was effective in partially extracted fibers, even though the uptake of CTnC was complete in the maximally extracted fibers. The results in this study establish that troponin C subunit is the key in setting the characteristic sensitivity for tension control in the myocardium above that in the skeletal muscle. Since a major difference between skeletal and cardiac TnCs is that one of the trigger sites (site I, residues 28-40 from the N terminus) is modified in CTnC and has reduced affinity for Ca2+ binding, the possibility is raised that this site has a modulatory effect on activation in different tissues and limits the effectiveness of CTnC in skeletal fibers.     

Isometric force redevelopment of skinned muscle fibers from rabbit activated with and without Ca2+.

Fiber isometric tension redevelopment rate (kTR) was measured during submaximal and maximal activations in glycerinated fibers from rabbit psoas muscle. In fibers either containing endogenous skeletal troponin C (sTnC) or reconstituted with either purified cardiac troponin C (cTnC) or sTnC, graded activation was achieved by varying [Ca2+]. Some fibers were first partially, then fully, reconstituted with a modified form of cTnC (aTnC) that enables active force generation and shortening in the absence of Ca2+. kTR was derived from the half-time of tension redevelopment. In control fibers with endogenous sTnC, kTR increased nonlinearly with [Ca2+], and maximal kTR was 15.3 +/- 3.6 s-1 (mean +/- SD; n = 26 determinations on 25 fibers) at pCa 4.0. During submaximal activations by Ca2+, kTR in cTnC reconstituted fibers was approximately threefold faster than control, despite the lower (60%) maximum Ca(2+)-activated force after reconstitution. To obtain submaximal force with aTnC, eight fibers were treated to fully extract endogenous sTnC, then reconstituted with a mixture of a TnC and cTnC (aTnC:cTnC molar ratio 1:8.5). A second extraction selectively removed cTnC. In such fibers containing aTnC only, neither force nor kTR was affected by changes in [Ca2+]. Force was 22 +/- 7% of maximum control (mean +/- SD; n = 15) at pCa 9.2 vs. 24 +/- 8% (mean +/- SD; n = 8) at pCa 4.0, whereas kTR was 98 +/- 14% of maximum control (mean +/- SD; n = 15) at pCa 9.2 vs. 96 +/- 15% (mean +/- SD; n = 8) at pCa 4.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiac troponin T isoforms affect the Ca(2+) sensitivity of force development in the presence of slow skeletal troponin I: insights into the role of troponin T isoforms in the fetal heart

In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca(2+) sensitivity of force development compared with cTnT3- and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca(2+) was released from site II of cTnC in the cTnI.cTnC complex (122/s) was 12.5-fold faster than for the ssTnI.cTnC complex (9.8/s). Addition of cTnT3 to the cTnI.cTnC complex resulted in a 3.6-fold decrease in the Ca(2+) dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI.cTnC complex resulted in a 1.9-fold increase in the Ca(2+) dissociation rate from site II of cTnC. The rate at which Ca(2+) dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca(2+) dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle.     

The reactivity of sulfhydryl groups of bovine cardiac troponin C

Bovine cardiac troponin C (cTnC) contains 2 cysteine residues, Cys-35 located in the nonfunctional Ca2+-binding loop I and Cys-84 in the N-terminal segment of the central helix. We have studied the reactivity of Cys residues in cTnC with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). The latter compound fluoresces only when reacted with the protein. The reaction with DTNB followed second order kinetics with respect to DTNB, the rate constants being 3.37 s-1 M-1 and 1.82 s-1 M-1 in the presence and absence of Ca2+, respectively. These rates are much slower than the rate of reaction with Cys-98 of skeletal TnC (sTnC) or with the urea-denatured cTnC, indicating that both Cys residues are partly buried within the structure of the protein. The increase in reactivity was induced by binding of Ca2+ to the single low affinity Ca2+ binding site (site II). The fluorescence increase upon reaction of cTnC with CPM in the absence of Ca2+ could be fitted with a single exponential equation indicating that both cysteine residues are equally available to the reagent. The reaction in the presence of Ca2+ was biphasic. Analysis of CNBr fragments of cTnC labeled with CPM under various conditions indicated that in the presence of Ca2+ the reactivity of Cys-84 is increased while that of Cys-35 is slightly decreased. This finding is consistent with the model of Herzberg et al. (Herzberg, O., Moult, J., and James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644) and the data of Ingraham and Hodges (Ingraham, R. H., and Hodges, R. S. (1988) Biochemistry 27, 5891-5898), suggesting that the Ca2+-induced conformational change in the N-terminal half of TnC involves separation of the helix C from the central helix, thereby increasing the accessibility of Cys-84. The slow overall kinetics, however, indicates that the structure in the vicinity of Cys residues is relatively compact regardless of Ca2+. We interpret the increase in reactivity towards CPM as consistent with a Ca2+-induced exposure of a hydrophobic pocket in the vicinity of Cys-84.     

Calcium-binding properties of troponin C in detergent-skinned heart muscle fibers

In order to obtain information with regard to behavior of the Ca2+ receptor, troponin C (TnC), in intact myofilament lattice of cardiac muscle, we investigated Ca2+-binding properties of canine ventricular muscle fibers skinned with Triton X-100. Analysis of equilibrium Ca2+-binding data of the skinned fibers in ATP-free solutions suggested that there were two distinct classes of binding sites which were saturated over the physiological range of negative logarithm of free calcium concentration (pCa): class I (KCa = 7.4 X 10(7) M-1, KMg = 0.9 X 10(3) M-1) and class II (KCa = 1.2 X 10(6) M-1, KMg = 1.1 X 10(2) M-1). The class I and II were considered equivalent, respectively, to the Ca2+-Mg2+ and Ca2+-specific sites of TnC. The assignments were supported by TnC content of the skinned fibers determined by electrophoresis and 45Ca autoradiograph of electroblotted fiber proteins. Dissociation of rigor complexes by ATP caused a downward shift of the binding curve between pCa 7 and 5, an effect which could be largely accounted for by lowering of KCa of the class II sites. When Ca2+ binding and isometric force were measured simultaneously, it was found that the threshold pCa for activation corresponds to the range of pCa where class II sites started to bind Ca2+ significantly. We concluded that the low affinity site of cardiac TnC plays a key role in Ca2+ regulation of contraction under physiological conditions, just as it does in the regulation of actomyosin ATPase. Study of kinetics of 45Ca washout from skinned fibers and myofibrils revealed that cardiac TnC in myofibrils contains Ca2+-binding sites whose off-rate constant for Ca2+ is significantly lower than the Ca2+ off-rate constant hitherto documented for the divalent ion-binding sites of either cardiac/slow muscle TnC or fast skeletal TnC.     

Binding of cardiac troponin-I147-163 induces a structural opening in human cardiac troponin-C.

The interaction of troponin-C (TnC) with troponin-I (TnI) plays a central role in skeletal and cardiac muscle contraction. We have recently shown that the binding of Ca2+ to cardiac TnC (cTnC) does not induce an "opening" of the regulatory domain in order to interact with cTnI [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216-18221; Spyracopoulos et al. (1997) Biochemistry 36, 12138-12146], which is in contrast to the regulatory N-domain of skeletal TnC (sTnC). This implies that the mode of interaction between cTnC and cTnI may be different than that between sTnC and sTnI. In sTnI, a region downstream from the inhibitory region (residues 115-131) has been shown to bind the exposed hydrophobic pocket of Ca2+-saturated sNTnC [McKay, R. T., et al. (1997) J. Biol. Chem. 272, 28494-28500]. The present study demonstrates that the corresponding region in cTnI (residues 147-163) binds to the regulatory domain of cTnC only in the Ca2+-saturated state to form a 1:1 complex, with an affinity approximately six times weaker than that between the skeletal counterparts. Thus, while Ca2+ does not cause opening, it is required for muscle regulation. The solution structure of the cNTnC.Ca2+.cTnI147-163 complex has been determined by multinuclear multidimensional NMR spectroscopy. The structure reveals an open conformation for cNTnC, similar to that of Ca2+-saturated sNTnC. The bound peptide adopts a alpha-helical conformation spanning residues 150-157. The C-terminus of the peptide is unstructured. The open conformation for Ca2+-saturated cNTnC in the presence of cTnI (residues 147-163) accommodates hydrophobic interactions between side chains of the peptide and side chains at the interface of A and B helices of cNTnC. Thus the mechanistic differences between the regulation of cardiac and skeletal muscle contraction can be understood in terms of different thermodynamics and kinetics equilibria between essentially the same structure states.     

Modulation of the interaction between the two halves of troponin C by the other troponin subunits

The interactions between troponin subunits have been studied by intrinsic fluorescence and electron spin resonance (ESR) spectroscopy. The tryptophan fluorescence of troponin T (TnT) and troponin I (TnI) when complexed with troponin C (TnC) undergoes a Ca2+-dependent transition. The midpoints of such spectral changes occur at pCa approximately equal to 6, suggesting that the conformational change of TnT and TnI is induced by Ca2+ binding to the low-affinity sites of TnC. When TnC is labelled at Cys-98 with a maleimide spin probe (MSL), the spin signal is sensitive to Ca2+ binding to both the high and the low-affinity sites of TnC in the presence of either or both of the other two troponin subunits. Since Cys-98 is located in the vicinity of one of the high-affinity sites, these results are indicative of a long-range interaction between the two halves of the TnC molecule. Our earlier kinetic studies [Wang, C.-L. A., Leavis, P. C. & Gergely, J. (1983) J. Biol. Chem. 258, 9175-9177] have shown such interactions in TnC alone. Since the ESR spectral change associated with metal binding to the low-affinity sites is only observed when MSL-TnC is complexed with TnT and/or TnI, this long-range interaction within TnC appears to be mediated through the other troponin subunits.     

Fast skeletal muscle skinned fibers and myofibrils reconstituted with N-terminal fluorescent analogues of troponin C

Glycerinated rabbit fast skeletal muscle fibers were chemically skinned with 1% Brij 35 and partially depleted of endogenous troponin C subunit (TnC) by exposure of the fibers to EDTA (Zot, H. G., and Potter, J. D. (1982) J. Biol. Chem. 257, 7678-7683). The TnC-depleted fibers exhibited a decrease in maximal tension that was mostly restored by readdition of TnC or by the addition of the fluorescent 5-dimethylaminonaphthalene-1-sulfonyl aziridine analogue, TnCDanz. TnCDanz is known to undergo an increase in fluorescence intensity when Ca2+ binds to the two low affinity Ca2+-specific regulatory sites of TnC. Steady-state fractional fluorescence and tension changes were measured simultaneously as a function of Ca2+. The Ca2+ sensitivity of the fluorescence curve was about 0.6 log unit greater than the tension curve. This difference in sensitivity could be explained if separate conformational states of TnC, brought about by Ca2+ binding to the Ca2+-specific sites, produce the fluorescence and tension changes. TnC-depleted fibers were also reconstituted with the fluorescent 2-[(4'-iodoacetamido)analino]naphthalene-6-sulfonic acid analogue, cardiac TnCIaans, which undergoes an increase in fluorescence intensity when Ca2+ binds to the single Ca2+- specific regulatory site. The steady-state fractional fluorescence and tension curves for fibers reconstituted with cardiac TnCIaans had nearly the same Ca2+ sensitivity. The steady-state fractional fluorescence of myofibrils reconstituted with TnCDanz was found to have a greater sensitivity to Ca2+ than the simultaneously measured ATPase. In all cases paired fractional fluorescence and activity curves tended to have parallel dependence on Ca2+. These procedures make it possible to study the Ca2+ binding properties of the Ca2+- specific sites in intact myofibrils and skinned fibers; the results presented suggest that the Ca2+ affinity of the Ca2+-specific sites of troponin are reduced in the thin filament compared to that of troponin in solution.     

Coupled expression of troponin T and troponin I isoforms in single skeletal muscle fibers correlates with contractility

Striated muscle contraction is powered by actin-activated myosin ATPase. This process is regulated by Ca(2+) via the troponin complex. Slow- and fast-twitch fibers of vertebrate skeletal muscle express type I and type II myosin, respectively, and these myosin isoenzymes confer different ATPase activities, contractile velocities, and force. Skeletal muscle troponin has also diverged into fast and slow isoforms, but their functional significance is not fully understood. To investigate the expression of troponin isoforms in mammalian skeletal muscle and their functional relationship to that of the myosin isoforms, we concomitantly studied myosin, troponin T (TnT), and troponin I (TnI) isoform contents and isometric contractile properties in single fibers of rat skeletal muscle. We characterized a large number of Triton X-100-skinned single fibers from soleus, diaphragm, gastrocnemius, and extensor digitorum longus muscles and selected fibers with combinations of a single myosin isoform and a single class (slow or fast) of the TnT and TnI isoforms to investigate their role in determining contractility. Types IIa, IIx, and IIb myosin fibers produced higher isometric force than that of type I fibers. Despite the polyploidy of adult skeletal muscle fibers, the expression of fast or slow isoforms of TnT and TnI is tightly coupled. Fibers containing slow troponin had higher Ca(2+) sensitivity than that of the fast troponin fibers, whereas fibers containing fast troponin showed a higher cooperativity of Ca(2+) activation than that of the slow troponin fibers. These results demonstrate distinct but coordinated regulation of troponin and myosin isoform expression in skeletal muscle and their contribution to the contractile properties of muscle.     

Heterogeneity of contractile proteins. A continuum of troponin-tropomyosin expression in mammalian skeletal muscle

Comparison of the myofibrillar proteins from several adult rabbit skeletal muscles has led to the identification of multiple forms of fast and slow troponin T. In Briggs et al. (Briggs, M. M., Klevit, R., and Schachat, F. H. (1984) J. Biol. Chem. 259, 10369-10375) two species of rabbit fast skeletal muscle troponin T (TnT), TnT1f and TnT2f, were characterized. Here, the distribution of these fast TnT species and the alpha- and beta- tropomyosin (Tm) subunits is characterized in fast muscles and in single muscle fibers. Evidence is also presented for two forms of slow skeletal muscle TnT. The presence of each fast TnT species is associated with the presence of a different Tm dimer: TnT1f with alpha beta-Tm and TnT2f with alpha 2-Tm. Histochemical analysis shows that expression of the fast TnT-Tm combinations is not due to differences in the distribution of fast-twitch glycolytic and fast-twitch oxidative-glycolytic fiber types. The absence of a correlation between histochemical typing and the composition of the thin filament Ca2+-regulatory complex is more apparent in individual fast muscle fibers where both fast TnT-Tm combinations appear to be expressed in a continuum. The implications of these observations for mammalian skeletal muscle fiber diversity are discussed.     

The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase

The cardiac troponin (Tn) complex, consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca2+-binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca2+ concentration. Cardiac Tn binds 3 mol Ca2+/mol and contains two Ca2+-binding sites with a binding constant of 3 X 10(8) M-1 and one binding site with a binding constant of 2 X 10(6) M-1. In the presence of 4 mM MgC12, the binding constant of the sites of higher affinity is reduced to 3 X 10(7) M-1, while Ca2+ binding to the site at the lower affinity is unaffected. The two high affinity Ca2+-binding sites of cardiac Tn are analogous to the two Ca2+-Mg2+ sites of skeletal Tn, while the single low affinity site is similar to the two Ca2+-specific sites of skeletal Tn (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4625-5633). The Ca2+-binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca2+/mol and contains two sites with a binding constant of 1 X 10(7) M-1 and a single site with a binding constant of 2 X 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the high affinity sites of TnC and Tn, the binding constants for Mg2+ were 0.7 and 3.0 X 10(3) M-1, respectively. The Ca2+ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison by the Ca2+-binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca2+] and at millimolar [Mg2+] suggests that activation of the ATPase occurs over the same range of [Ca2+] where the Ca2+-specific site of cardiac Tn binds Ca2+.     

Isolation and sequence of a cDNA clone for rabbit fast skeletal muscle troponin C. Homology with calmodulin and parvalbumin

The binding of Ca2+ to troponin C (TnC) regulates skeletal muscle contraction. We have isolated a full-length cDNA clone for fast skeletal muscle TnC from a neonatal rabbit skeletal muscle library and determined its nucleic acid sequence. The amino acid sequence deduced from this clone matches the previously reported amino acid sequence (Collins, J. H., Greaser, M. L., Potter, J. D., and Horn, M. J. (1977) J. Biol. Chem. 252, 6356-6362) except at the amino terminus. According to the nucleotide sequence, the first 2 residues of TnC are threonine-aspartic acid, which is the reverse of the order reported previously. The isolation of the adult form of TnC from a neonatal library suggests that there may be no developmental isoforms of fast TnC. The protein coding region of the fast TnC clone has 67% homology with the reported nucleotide sequence for chicken slow TnC (Putkey, J. A., Carroll, S. L., and Means, A. R. (1987) Mol. Cell. Biol. 7, 549-1553). The homologies between the nucleotide sequences of TnC, calmodulin, and parvalbumin provide evidence that all three proteins were derived from a common precursor molecule which had four Ca2+-binding sites.     

Using lanthanide ions to align troponin complexes in solution: order of lanthanide occupancy in cardiac troponin C

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.     

Interactions of troponin subunits: free energy of binary and ternary complexes

We have determined the free energy of formation of the binary complexes formed between skeletal troponin C and troponin T (TnC.TnT) and between troponin T and troponin I (TnT.TnI). This was accomplished by using TnC fluorescently modified at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine for the first complex and TnI labeled at Cys-133 with the same probe for the other complex. The free energy of the ternary complex formed between troponin C and the binary complex TnT.TnI [TnC.(TnT.TnI)] was also measured by monitoring the emission of 5-(iodoacetamido)eosin attached to Cys-133 of the troponin I in TnT.TnI. The free energies were -9.0 kcal.mol-1 for TnC.TnT, -9.2 kcal.mol-1 for TnT.TnI, and -8.7 kcal.mol-1 for TnC.(TnT.TnI). In the presence of Mg2+ the free energies of TnC.TnT and TnC.(TnT.TnI) were -10.3 and -10.9 kcal.mol-1, respectively; in the presence of Ca2+ the corresponding free energies were -10.6 and -13.5 kcal.mol-1. Mg2+ and Ca2+ had negligible effect on the free energy of TnT.TnI. From these results the free energies of the formation of troponin from the three subunits were found to be -16.8 kcal.mol-1, -18.9 kcal.mol-1, and -21.6 kcal.mol-1 in the presence of EGTA, Mg2+, and Ca2+, respectively. Most of the free energy decrease caused by Ca2+ binding to the Ca2+-specific sites is derived from stabilization of the TnI-TnC linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that both Ca(2+)-specific sites of skeletal muscle TnC are required for full activity

To investigate the role of the Ca2(+)-specific (I and II) sites of fast skeletal muscle troponin C (TnC) in the regulation of contraction, we have produced two TnC mutants which have lost the ability to bind Ca2+ at either site I (VG1) or at site II (VG2). Both mutants were able to partially restore force to TnC-depleted skinned muscle fibers (approximately 25% for VG1 and approximately 50% for VG2). In contrast, bovine cardiac TnC (BCTnC), which like VG1 binds Ca2+ only at site II, could fully reactivate the contraction of TnC-depleted fibers. Higher concentrations of both mutants were required to restore force to the TnC-depleted fibers than with wild type TnC (WTnC) or BCTnC. VG1 and VG2 substituted fibers could not bind additional WTnC, indicating that all of the TnC-binding sites were saturated with the mutant TnC's. The Ca2+ concentration required for force activation was much higher for VG1 and VG2 substituted fibers than for WTnC or BCTnC substituted fibers. Also, the steepness of force activation was much less in VG1 and VG2 versus WTnC and BCTnC substituted fibers. These results suggest cooperative interactions between sites I and II in WTnC. In contrast, BCTnC has essentially the same apparent Ca2+ affinity and steepness of force activation as does WTnC. Thus, cardiac TnC must have structural differences from WTnC which compensate for the lack of site I, while in WTnC, both Ca2(+)-specific sites are probably crucial for full functional activity.