Calcium regulates troponin-tropomyosin binding in the reconstituted thin filament

Steady-state fluorescence anisotropy technique was used to determine the binding constant of troponin for IAEDANS-labeled tropomyosin under various conditions. In the absence of actin, Ca does not affect the binding between troponin and tropomyosin. The presence of actin greatly strengthens troponin-tropomyosin binding in the absence of Ca. However, Ca weakens troponin-tropomyosin binding by about 2.5-fold in the reconstituted filament. It is suggested that the Ca-regulated binding may serve as a molecular switch for the troponin molecule to get “on” and “off” the actin-myosin interaction site regulating muscle contraction-relaxation cycles.     

Cooperative interactions between troponin molecules bound to the cardiac thin filament

Striated muscle thin filaments contain many troponin molecules, which contact each other indirectly via tropomyosin and actin. Such allosteric interactions between troponin molecules may be responsible for cooperative Ca2+ binding to the regulatory sites of the cardiac thin filament (Tobacman, L. S., and Sawyer, D. S. (1990) J. Biol. Chem. 265, 931-939). To test whether thin filament-bound troponin molecules interact, we studied the competitive binding of troponin and troponin T-troponin I (an inhibitory complex lacking the Ca2+ binding subunit troponin C) to actin-tropomyosin. The relative affinities of these two forms of troponin for the thin filament depended upon their relative concentrations. Under conditions where total binding was saturated, each form binds with greater apparent affinity to sites that have similar neighbors. A theoretical model for competitive binding of two ligands to interacting sites on a linear lattice was developed and fit to the data. Surprisingly, energetically unfavorable interactions occurred between adjacent troponin and troponin T-troponin I molecules not only in the presence of Ca2+, but also in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and/or myosin subfragment 1. Removal of Ca2+ strengthened the affinity of troponin for the thin filament less than 50%. These results suggest that, even in the absence of myosin, long range allosteric interactions occur between troponin molecules. The detailed involvement of tropomyosin and actin in these interactions remains to be established.     

Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant.

Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.     

Equilibrium distribution of skeletal actin-tropomyosin-troponin states, determined by pyrene-tropomyosin fluorescence

Actin-tropomyosin-troponin has three structural states, but the functional properties of regulation can be explained with models having two functional states. As a step towards assigning functional properties to all the structural states, we examined fluorescent probes that monitor changes in troponin and tropomyosin. Tropomyosin labeled with pyrene-iodoacetamide is thought to reflect the transition to the most active state, whereas N-((2-iodoacetoxy)ethyl)-N-methyl)-amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I is thought to monitor the transition to any state other than the inactive state. The fraction of actin in an active state determined from pyrene excimer fluorescence agreed with that calculated from light-scattering measurements of myosin subfragment 1 (S1)-ADP to regulated actin in both the presence and absence of Ca2+ over a range of ionic strength conditions. The only exceptions were conditions where the binding of S1-ADP to actin was too strong to measure accurately. Pyrene-tropomyosin excimer fluorescence was Ca2+ dependent and so reflected the change in population caused by both Ca2+ binding and S1-ADP binding. Pyrene labeling of tropomyosin did not cause a large perturbation of the transition among states of regulated actin. Using pyrene-tropomyosin fluorescence we were able to extend the ionic strength dependence of the parameters describing the co-operativity of binding of S1-ADP to actin as low as 0.1 M. The probes on tropomyosin and troponin I had different responses to Ca2+ and S1-ADP binding. These different sensitivities can be explained by an intermediate between the inactive and active states of regulated actin.     

The Delta 14 mutation of human cardiac troponin T enhances ATPase activity and alters the cooperative binding of S1-ADP to regulated actin

The complex of tropomyosin and troponin binds to actin and inhibits activation of myosin ATPase activity and force production of striated muscles at low free Ca(2+) concentrations. Ca(2+) stimulates ATP activity, and at subsaturating actin concentrations, the binding of NEM-modified S1 to actin-tropomyosin-troponin increases the rate of ATP hydrolysis even further. We show here that the Delta14 mutation of troponin T, associated with familial hypertrophic cardiomyopathy, results in an increase in ATPase rate like that seen with wild-type troponin in the presence of NEM-S1. The enhanced ATPase activity was not due to a decreased incorporation of mutant troponin T with troponin I and troponin C to form an active troponin complex. The activating effect was more prominent with a hybrid troponin (skeletal TnI, TnC, and cardiac TnT) than with all cardiac troponin. Thus it appears that changes in the troponin-troponin contacts that result from mutations or from forming hybrids stabilize a more active state of regulated actin. An analysis of the effect of the Delta14 mutation on the equilibrium binding of S1-ADP to actin was consistent with stabilization of an active state of actin. This change in activation may be important in the development of cardiac disease.     

Myosin-Va binds to and mechanochemically couples microtubules to actin filaments

Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (approximately 1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 microM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.     

Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T

Striated muscle contraction is regulated by Ca2+ binding to troponin, which has a globular domain and an elongated tail attributable to the NH2-terminal portion of the bovine cardiac troponin T (TnT) subunit. Truncation of the bovine cardiac troponin tail was investigated using recombinant TnT fragments and subunits TnI and TnC. Progressive truncation of the troponin tail caused progressively weaker binding of troponin-tropomyosin to actin and of troponin to actin-tropomyosin. A sharp drop-off in affinity occurred with NH2-terminal deletion of 119 rather than 94 residues. Deletion of 94 residues had no effect on Ca2+-activation of the myosin subfragment 1-thin filament MgATPase rate and did not eliminate cooperative effects of Ca2+ binding. Troponin tail peptide TnT1-153 strongly promoted tropomyosin binding to actin in the absence of TnI or TnC. The results show that the anchoring function of the troponin tail involves interactions with actin as well as with tropomyosin and has comparable importance in the presence or absence of Ca2+. Residues 95-153 are particularly important for anchoring, and residues 95-119 are crucial for function or local folding. Because striated muscle regulation involves switching among the conformational states of the thin filament, regulatory significance for the troponin tail may arise from its prominent contribution to the protein-protein interactions within these conformations.     

Analysis of troponin-tropomyosin binding to actin. Troponin does not promote interactions between tropomyosin molecules.

The binding of tropomyosin to actin and troponin-tropomyosin to actin was analyzed according to a linear lattice model which quantifies two parameters: Ko, the affinity of the ligand for an isolated site on the actin filament, and gamma, the fold increase in affinity when binding is contiguous to an occupied site (cooperativity). Tropomyosin-actin binding is very cooperative (gamma = 90-137). Troponin strengthens tropomyosin-actin binding greatly but, surprisingly, does so solely by an 80-130-fold increase in Ko, while cooperativity actually decreases. Additionally, troponin complexes containing TnT subunits with deletions of either amino acids 1-69 (troponin70-259) or 1-158 (troponin159-259) were examined. Deletion of amino acids 1-69 had only small effects on Ko and y, despite this peptide's location spanning the joint between adjacent tropomyosins. Ca2+ reduced Ko by half for both troponin and troponin70-159 and had no detectable effect on cooperativity. Troponin159-259 had much weaker effects on tropomyosin-actin binding than did troponin70-259 and had no effect at all in the presence of Ca2+. This suggests the importance of Ca(2+)-insensitive interactions between tropomyosin and troponin T residues 70-159. Cooperativity was slightly lower for troponin159-259 than tropomyosin alone, suggesting that the globular head region of troponin affects tropomyosin-tropomyosin interactions along the thin filament.     

Phosphorylation of an actin.tropomyosin.troponin complex from human skeletal muscle.

A human skeletal actin.tropomyosin.troponin complex was phosphorylated in the presence of [gamma-32 P]ATP, Mg2+, adenosine 3':5'-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 microM cyclic AMP. In the presence of 10(-7) M Ca2+ and protein kinase 0.1 mole of [32P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [32P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca2+, 5.10(-5) M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [32P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [32P]phosphate. Increasing the Ca2+ concentration did not significantly decrease the [32P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstitute human skeletal actomyosin made with the [32P] phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca2+.     

Three-dimensional reconstruction of thin filaments containing mutant tropomyosin

Interactions of the components of reconstituted thin filaments were investigated using a tropomyosin internal deletion mutant, D234, in which actin-binding pseudo-repeats 2, 3, and 4 are missing. D234 retains regions of tropomyosin that bind troponin and form end-to-end tropomyosin bonds, but has a length to span only four instead of seven actin monomers. It inhibits acto-myosin subfragment 1 ATPase (acto-S-1 ATPase) and filament sliding in vitro in both the presence and absence of Ca(2+) (, J. Biol. Chem. 272:14051-14056) and lowers the affinity of S-1.ADP for actin while increasing its cooperative binding. Electron microscopy and three-dimensional reconstruction of reconstituted thin filaments containing actin, troponin, and wild-type or D234 tropomyosin were carried out to determine if Ca(2+)-induced movement of D234 occurred in the filaments. In the presence and absence of Ca(2+), the D234 position was indistinguishable from that of the wild-type tropomyosin, demonstrating that the mutation did not affect normal tropomyosin movement induced by Ca(2+) and troponin. These results suggested that, in the presence of Ca(2+) and troponin, D234 tropomyosin was trapped on filaments in the Ca(2+)-induced position and was unable to undergo a transition to a completely activated position. By adding small amounts of rigor-bonded N-ethyl-maleimide-treated S-1 to mutant thin filaments, thus mimicking the myosin-induced "open" state, inhibition could be overcome and full activation restored. This myosin requirement for full activation provides support for the existence of three functionally distinct thin filament states (off, Ca(2+)-induced, myosin-induced; cf.;, J. Mol. Biol. 266:8-14). We propose a further refinement of the three-state model in which the binding of myosin to actin causes allosteric changes in actin that promote the binding of tropomyosin in an otherwise energetically unfavorable "open" state.     

Reciprocal coupling between troponin C and myosin crossbridge attachment

The attachment of cycling myosin crossbridges to actin and the resultant muscle contraction are regulated in skeletal muscle by the binding of Ca2+ to the amino-terminal, regulatory sites of the troponin C (TnC) subunit of the thin filament protein troponin. Conversely, the attachment of crossbridges to actin has been shown to alter the affinity of TnC for Ca2+. In this study, fluorescently labeled TnC incorporated into reconstituted thin filaments was used to investigate the relationship between crossbridge attachment to actin and structural changes in the amino-terminal region of TnC. Fluorescence intensity changes were measured under the following conditions: saturating [Ca2+] in the absence of crossbridges, rigor crossbridge attachment in the presence and absence of Ca2+, and cycling crossbridge attachment. The percent of heavy meromyosin crossbridges associated with the thin filaments under these conditions was also determined. The results show that, in addition to the binding of Ca2+ to TnC, the attachment of both rigor and cycling crossbridges to actin alters the structure of TnC near the regulatory, Ca2+-specific sites of the molecule. A differential coupling between weakly versus strongly bound crossbridge states and TnC structure was detected, suggesting a possible differential regulation of these states by conformational changes in TnC. These findings illustrate a reciprocal coupling, via thin filament protein interactions, between structural changes in TnC and the attachment of myosin crossbridges to actin, such that each can influence the other, and indicate that TnC is not simply an on-off switch but may exist in a number of different conformations.     

An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin

Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca(2+) to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca(2+) showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.     

Mutations in actin subdomain 3 that impair thin filament regulation by troponin and tropomyosin.

Thin filament-mediated regulation of striated muscle contraction involves conformational switching among a few quaternary structures, with transitions induced by binding of Ca(2+) and myosin. We establish and exploit Saccharomyces cerevisiae actin as a model system to investigate this process. Ca(2+)-sensitive troponin-tropomyosin binding affinities for wild type yeast actin are seen to closely resemble those for muscle actin, and these hybrid thin filaments produce Ca(2+)-sensitive regulation of the myosin S-1 MgATPase rate. Yeast actin filament inner domain mutant K315A/E316A depresses Ca(2+) activation of the MgATPase rate, producing a 4-fold weakening of the apparent Ca(2+) affinity and a 50% decrease in the MgATPase rate at saturating Ca(2+) concentration. Observed destabilization of troponin-tropomyosin binding to actin in the presence of Ca(2+), a 1.4-fold effect, provides a partial explanation. Despite the decrease in apparent MgATPase Ca(2+) affinity, there was no detectable change in the true Ca(2+) affinity of the thin filament, measured using fluorophore-labeled troponin. Another inner domain mutant, E311A/R312A, decreased the MgATPase rate but did not change the apparent Ca(2+) affinity. These results suggest that charged residues on the surface of the actin inner domain are important in Ca(2+)- and myosin-induced thin filament activation.     

Comparative studies on the functional roles of N- and C-terminal regions of molluskan and vertebrate troponin-I

Vertebrate troponin regulates muscle contraction through alternative binding of the C-terminal region of the inhibitory subunit, troponin-I (TnI), to actin or troponin-C (TnC) in a Ca(2+)-dependent manner. To elucidate the molecular mechanisms of this regulation by molluskan troponin, we compared the functional properties of the recombinant fragments of Akazara scallop TnI and rabbit fast skeletal TnI. The C-terminal fragment of Akazara scallop TnI (ATnI(232-292)), which contains the inhibitory region (residues 104-115 of rabbit TnI) and the regulatory TnC-binding site (residues 116-131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin Mg-ATPase. However, it did not interact with TnC, even in the presence of Ca(2+). These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin. On the other hand, ATnI(130-252), which contains the structural TnC-binding site (residues 1-30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC. Moreover, the ternary complex consisting of this fragment, troponin-T, and TnC activated the ATPase in a Ca(2+)-dependent manner almost as effectively as intact Akazara scallop troponin. Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnC-binding site to the inhibitory region of TnI. Together with the observation that corresponding rabbit TnI-fragment (RTnI(1-116)) shows similar activating effects, these findings suggest the importance of the TnI N-terminal region not only for maintaining the structural integrity of troponin complex but also for Ca(2+)-dependent activation.     

Regulation of muscle contraction: bindings of troponin and its components to actin and tropomyosin.

The bindings of troponin components to actin and tropomyosin has been studied by cosedimentation with actin and affinity chromatography. It is shown that troponin binds to actin and tropomyosin in the presence and absence of calcium but the binding to actin is sensitive to ionic strength. Troponin-I + C binds to actin-tropomyosin in the absence of calcium but not to actin or tropomyosin alone. Troponin-I binds to actin and the binding is improved in the presence of tropomyosin even though troponin-I does not bind to tropomyosin alone. Troponin-C does not bind to actin or tropomyosin. The results suggest that the binding of troponin by actin is influenced by tropomyosin. A model of regulation by troponin is proposed.     

Troponin-tropomyosin: an allosteric switch or a steric blocker?

The interaction of myosin subfragment 1 (S1) with actin-tropomyosin-troponin (regulated actin) is highly nucleotide dependent. The binding of S1 or S1-ADP (but not S1-ATP nor N,N'-rho-phenylenedimaleimide-modified S1-ATP) to regulated actin activates ATP hydrolysis even in the absence of Ca(2+). Investigations with S1 and S1-ADP have led to the idea that some actin sites are directly blocked toward the binding of S1 either by tropomyosin or troponin. The blocked state is thought to occur only at ionic strengths greater than 50 mM. The question is whether nonactivating S1 binding is blocked under the same conditions. We show that troponin inhibits binding of the nonactivating state, N,N'-rho-phenylenedimaleimide-S1-ATP, to actin but only when tropomyosin is absent. A lag in the rate of binding of activating S1 to actin (an indicator of the blocked state) occurs only in the presence of tropomyosin. Thus, tropomyosin inhibits binding of rigor S1 but not S1-ATP-like states. No evidence for an ionic strength-dependent change in the mechanism of regulation was observed either from measurements of the rate of activating S1 binding or from the equilibrium binding of nonactivating S1 to actin. At all conditions examined, N,N'-rho-phenylenedimaleimide-S1-ATP bound to regulated actin in the absence of Ca(2+). These results support the view of regulation in which tropomyosin movement is an allosteric switch that is modulated by activating myosin binding but that does not function solely by regulating myosin binding.     

The C-terminus of troponin T is essential for maintaining the inactive state of regulated actin

Striated muscle contraction is regulated by the actin binding proteins tropomyosin and troponin. Defects in these proteins lead to myopathies and cardiomyopathies. Deletion of the 14 C-terminal residues of cardiac troponin T leads to hypertrophic cardiomyopathy. We showed earlier that regulated actin containing Δ14 TnT was more readily activated than wild-type regulated actin. We suggested that the equilibria among the inactive (blocked), intermediate (closed or calcium), and active (open or myosin) states was shifted to the active state. We now show that, in addition, such regulated actin filaments cannot enter the inactive or blocked state. Regulated actin containing Δ14 TnT had ATPase activities in the absence of Ca2+ that were higher than wild-type filaments but far below the fully active rate. The rapid dissociation of S1-ATP from regulated actin filaments containing Δ14 TnT and acrylodan-labeled tropomyosin did not show the fluorescence increase characteristic of moving to the inactive state. Replacing wild-type TnI with S45E TnI, that favors the inactive state, did not restore the fluorescence change. We conclude that TnT has a previously unrecognized role in forming the inactive state of regulated actin.     

Characterization of carbethoxylated actin

The carbethoxylation of histidine residues in G-actin impairs actin polymerization. The histidine residue essential for polymerization was identified as histidine-40 [Hegyi, G., Premecz, G., Sain, B., & Mühlrad, A. (1974) Eur. J. Biochem. 44, 7-12]. Non-polymerizable actin was separated from the polymerizable fraction after partial carbethoxylation. The non-polymerizable actin recovered the ability to polymerize following addition of phalloidin. Taking into account the evidence that phalloidin does not bind to G-actin in the absence of salt, the results indicate that the actin monomer undergoes a conformational change and subsequently binds phalloidin before polymerization. The resulting polymers activated S1 ATPase activity as effectively as control F-actin. In the presence of tropomyosin and troponin, a strong inhibition of actin-activated ATPase activity was observed in the absence of Ca2+, although no inhibition was observed in the presence of Ca2+. These results indicate that His-40 is not directly involved in a myosin binding site nor in a tropomyosin-troponin binding site.     

The effects of platelet tropomyosin on the ATPase activities of muscle actomyosin subfragment 1 in the absence and presence of troponin, its components, and calmodulin

The effect of platelet tropomyosin on the ATPase activity of a muscle actin-myosin subfragment 1 system has been examined in 30 mM KCl, 5 mM MgCl2, 2 mM ATP, 0.1 mM EGTA, 2 mM Tris, pH 7.8. Whereas muscle tropomyosin inhibits the activity by 60%, the platelet protein had no effect. Addition of muscle troponin in the absence of Ca2+ to the system inhibited the activity by up to 80% irrespective of whether muscle or platelet tropomyosin was used. The release of this inhibition by the addition of Ca2+ was much less in the case of platelet tropomyosin. This may result from the fact that platelet tropomyosin aggregates poorly in a head-to-tail manner and interacts only weakly with muscle troponin-T. In the presence of troponin-I and platelet tropomyosin, inhibition of the ATPase activity was 80%. This inhibition was largely released by the addition of troponin-C irrespective of the presence of Ca2+. The addition of brain calmodulin, however, released the inhibition in the presence of calcium but not in its absence. These effects can be correlated with the binding or lack of binding of the platelet tropomyosin to the actin filament.     

Lead-binding properties of intestinal calcium-binding proteins

The bovine and chick vitamin D-induced intestinal calcium-binding proteins (CaBP) bind lead. Bovine CaBP binds 2 atoms of lead/molecule, and chick CaBP binds 4 atoms of lead per molecule and these values are identical to those for calcium binding. 45Calcium-displacement studies indicate significantly higher affinities for lead than for calcium for both proteins. All evidence indicates that lead is bound to the 4 high affinity calcium-binding sites on chick CaBP and to the corresponding 2 high affinity sites on bovine CaBP, and that binding of lead to sulfhydryl groups is, relatively, not significant. Calmodulin, troponin C, and oncomodulin also bind lead with high affinities and in preference to calcium, indicating that lead binding is a general property of proteins belonging to the troponin C superfamily of calcium-binding proteins.