Localization of the two tropomyosin-binding sites of troponin T

Troponin T (TnT) binds to tropomyosin (Tm) to anchor the troponin complex in the thin filament, and it thus serves as a vital link in the Ca²⁺ regulation of striated muscle contraction. Pioneer work three decades ago determined that the T1 and T2 chymotryptic fragments of TnT each contains a Tm-binding site. A more precise localization of the two Tm-binding sites of TnT remains to be determined. In the present study, we tested serial deletion constructs of TnT and carried out monoclonal antibody competition experiments to show that the T1 region Tm-binding site involves mainly a 39 amino acids segment in the N-terminal portion of the conserved middle region of TnT. We further employed another set of TnT fragments to locate the T2 region Tm-binding site to a segment of 25 amino acids near the beginning of the T2 fragment. The localization of the two Tm-binding sites of TnT provided new information for the structure-function relationship of TnT and the anchoring of troponin complex on muscle thin filament.     

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.     

Modulation of troponin T molecular conformation and flexibility by metal ion binding to the NH2-terminal variable region

Troponin T (TnT) plays an allosteric signal transduction role in the actin thin-filament-based Ca(2+)-regulation of striated muscle contraction. Developmentally regulated alternative RNA splicing produces TnT isoforms differing in their NH(2)-terminal structure. Physical property variations of the NH(2)-terminal hypervariable region of TnT may have a role in tuning the Ca(2+)-sensitivity and overall cooperativity of the muscle. We have previously demonstrated that metal ion or monoclonal antibody binding to the NH(2)-terminal region can modulate the epitopic conformation and troponin I and tropomyosin binding affinity of TnT. To further establish the molecular basis of this conformational and functional modulation, we have characterized the NH(2)-terminal variable region-originated secondary conformational effect in TnT using fluorescence spectral analysis. The chicken fast skeletal muscle TnT isoform, TnT8e16, containing a cluster of transition-metal ion binding sites (Tx) in the NH(2)-terminal variable region was used in this study. TnT8e16 was titrated for Cu(II) binding-induced changes in fluorescence intensity and anisotropy of the COOH-domain Trp residues (W234, W236, and W285), which demonstrated considerable environmental sensitivity in TnT denaturation studies. Nonlinear Stern-Volmer plots of Trp quenching indicated a metal ion binding-induced conformational change in TnT. Fluorescence anisotropy changes upon metal ion binding indicated a decrease in the mobility of the Trp residues and an increase in the flexibility of fluorescein-labeled Cys263 in the COOH domain. These data support a model that the alternatively spliced NH(2)-terminal variable region of TnT modulates conformation and flexibility of other domains of the protein.     

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.     

The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin

Analysis of two recombinant variants of chicken striated muscle alpha-tropomyosin has shown that the structure of the amino terminus is crucial for most aspects of tropomyosin function: affinity to actin, promotion of binding to actin by troponin, and regulation of the actomyosin MgATPase. Initial characterization of variants expressed and isolated from Escherichia coli has been published (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Fusion tropomyosin contains 80 amino acids of a nonstructural influenza virus protein (NS1) on the amino terminus. Nonfusion tropomyosin is a variant because the amino-terminal methionine is not acetylated (unacetylated tropomyosin). The affinity of tropomyosin labeled at Cys190 with N-[14C]ethylmaleimide for actin was measured by cosedimentation in a Beckman Airfuge. Fusion tropomyosin binds to actin with an affinity slightly greater than that of chicken striated muscle alpha-tropomyosin (Kapp = 1-2 X 10(7) versus 0.5-1 X 10(7) M-1) and more strongly than unacetylated tropomyosin (Kapp = 3 X 10(5) M-1). Both variants bind cooperatively to actin. Troponin increases the affinity of unacetylated tropomyosin for actin (+Ca2+, Kapp = 6 X 10(6) M-1; +EGTA, Kapp = 2 X 10(7) M-1), but the affinity is still lower than that of muscle tropomyosin for actin in the presence of troponin (Kapp much greater than 10(8) M-1). Troponin has no effect on the affinity of fusion tropomyosin for actin indicating that binding of troponin T to the over-lap region of the adjacent tropomyosin, presumably sterically prevented by the fusion peptide in fusion tropomyosin, is required for troponin to promote the binding of tropomyosin to actin. The role of troponin T in regulation and the mechanisms of cooperative binding of tropomyosin to actin have been discussed in relation to this work.     

Effects of troponin on thermal unfolding of actin-bound tropomyosin

Differential scanning calorimetry (DSC) was used to study the effect of troponin (Tn) and its isolated components on the thermal unfolding of skeletal muscle tropomyosin (Tm) bound to F-actin. It is shown that in the absence of actin the thermal unfolding of Tm is expressed in two well-distinguished thermal transitions with maxima at 42.8 and 53.8°C. Interaction with F-actin affects the character of thermal unfolding of Tm leading to appearance of a new Tm transition with maximum at about 48°C, but it has no influence on the thermal denaturation of F-actin stabilized by aluminum fluoride, which occurs within the temperature region above 70°C. Addition of troponin leads to significant increase in the cooperativity and enthalpy of the thermal transition of the actin-bound Tm. The most pronounced effect of Tn was observed in the absence of calcium. To elucidate how troponin complex affects the properties of Tm, we studied the influence of its isolated components, troponin I (TnI) and troponin T (TnT), on the thermal unfolding of actin-bound Tm. Isolated TnT and TnI do not demonstrate cooperative thermal transitions on heating up to 100°C. However, addition of TnI, and especially of TnT, to the F-actin–Tm complex significantly increased the cooperativity of the thermal unfolding of actin-bound tropomyosin.     

Troponin T core structure and the regulatory NH2-terminal variable region

The conserved central and COOH-terminal regions of troponin T (TnT) interact with troponin C, troponin I, and tropomyosin to regulate striated muscle contraction. Phylogenic data show that the NH2-terminal region has evolved as an addition to the conserved core structure of TnT. This NH2-terminal region does not bind other thin filament proteins, and its sequence is hypervariable between fiber type and developmental isoforms. Previous studies have demonstrated that NH2-terminal modifications alter the COOH-terminal conformation of TnT and thin filament Ca2+-activation, yet the functional core structure of TnT and the mechanism of NH2-terminal modulation are not well understood. To define the TnT core structure and investigate the regulatory role of the NH2-terminal variable region, we investigated two classes of model TnT molecules: (1) NH2-terminal truncated cardiac TnT and (2) chimera proteins consisting of an acidic or basic skeletal muscle TnT NH2-terminus spliced to the cardiac TnT core. Deletion of the TnT hypervariable NH2-terminus preserved binding to troponin I and tropomyosin and sustained cardiac muscle contraction in the heart of transgenic mice. Further deletion of the conserved central region diminished binding to tropomyosin. The reintroduction of differently charged NH2-terminal domains in the chimeric molecules produced long-range conformational changes in the central and COOH-terminal regions to alter troponin I and tropomyosin binding. Similar NH2-terminal charge effects are demonstrated in naturally occurring cardiac TnT isoforms, indicating a physiological significance. These results suggest that the hypervariable NH2-terminal region modulates the conformation and function of the TnT core structure to fine-tune muscle contractility.     

A modulatory role for the troponin T tail domain in thin filament regulation

In striated muscle the force generating acto-myosin interaction is sterically regulated by the thin filament proteins tropomyosin and troponin (Tn), with the position of tropomyosin modulated by calcium binding to troponin. Troponin itself consists of three subunits, TnI, TnC, and TnT, widely characterized as being responsible for separate aspects of the regulatory process. TnI, the inhibitory unit is released from actin upon calcium binding to TnC, while TnT performs a structural role forming a globular head region with the regulatory TnI- TnC complex with a tail anchoring it within the thin filament. We have examined the properties of TnT and the TnT(1) tail fragment (residues 1-158) upon reconstituted actin-tropomyosin filaments. Their regulatory effects have been characterized in both myosin S1 ATPase and S1 kinetic and equilibrium binding experiments. We show that both inhibit the actin-tropomyosin-activated S1 ATPase with TnT(1) producing a greater inhibitory effect. The S1 binding data show that this inhibition is not caused by the formation of the blocked B-state but by significant stabilization of the closed C-state with a 10-fold reduction in the C- to M-state equilibrium, K(T), for TnT(1). This suggests TnT has a modulatory as well as structural role, providing an explanation for its large number of alternative isoforms.     

Cardiac Muscle Activation Blunted by a Mutation to the Regulatory Component,Troponin T

The striated muscle thin filament comprises actin, tropomyosin, and troponin. The Tn complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnT may serve as a bridge between the Ca²⁺ sensor (TnC) and the actin filament. In the short helix preceding the IT-arm region, H1(T2), there are known dilated cardiomyopathy-linked mutations (among them R205L). Thus we hypothesized that there is an element in this short helix that plays an important role in regulating the muscle contraction, especially in Ca²⁺ activation. We mutated Arg-205 and several other amino acid residues within and near the H1(T2) helix. Utilizing an alanine replacement method to compare the effects of the mutations, the biochemical and mechanical impact on the actomyosin interaction was assessed by solution ATPase activity assay, an in vitro motility assay, and Ca²⁺ binding measurements. Ca²⁺ activation was markedly impaired by a point mutation of the highly conserved basic residue R205A, residing in the short helix H1(T2) of cTnT, whereas the mutations to nearby residues exhibited little effect on function. Interestingly, rigor activation was unchanged between the wild type and R205A TnT. In addition to the reduction in Ca²⁺ sensitivity observed in Ca²⁺ binding to the thin filament, myosin S1-ADP binding to the thin filament was significantly affected by the same mutation, which was also supported by a series of S1 concentration-dependent ATPase assays. These suggest that the R205A mutation alters function through reduction in the nature of cooperative binding of S1.     

Binding of troponin I and the troponin I-troponin C complex to actin-tropomyosin. Dissociation by myosin subfragment 1

Troponin I (TnI) is the component of the troponin complex, TnI, TnC, TnT, that is responsible for inhibition of actomyosin ATPase activity. Using the fluorescence of pyrene-labeled tropomyosin (Tm), we probed the interaction of TnI and TnIC with Tm on the reconstituted muscle thin filament. The results indicate that TnI and TnIC(-Ca(2+)) bind specifically and strongly to actin-Tm with a stoichiometry of 1 TnI or 1 TnIC/1 Tm/7 actin, in agreement with previous results. The binding of myosin heads (S1) to actin-Tm at low levels of saturation caused TnI and TnIC to dissociate from actin-Tm. These results are interpreted in terms of the S1-binding state allosteric-cooperative model of the actin-Tm thin filament, closed/open. Thus, TnI and TnIC(-Ca(2+)) bind to the closed state of actin-Tm and their binding is greatly weakened in the S1-induced open state, indicating that they act as allosteric inhibitors. The fluorescence change and the stoichiometry indicate that the TnI-binding site is composed of regions from both actin and Tm probably in the vicinity of Cys 190.     

Deletion of the first 45 NH2-terminal residues of rabbit skeletal troponin T strengthens binding of troponin to immobilized tropomyosin.

The binding of the NH2-terminal region of troponin T (TnT) to the COOH-terminal region of tropomyosin (Tm) and the head-to-tail overlap between Tm molecules is thought to provide a pivotal link between troponin (Tn) and Tm (White, S.P., Cohen, C., and Phillips, G.N., Jr. (1987) Nature 325, 826-828). To further explore the structure-function relationship of the NH2-terminal region of TnT, we studied the binding of a 26,000-dalton TnT fragment (26K-TnT, Ohtsuki, I., Shiraishi, F., Suenaga, N., Miyata, T., and Tanokura, M.J. (1984) J. Biochem. (Tokyo) 95, 1337-1342) which corresponds to residues 46-259 of TnT2f, the major isoform of TnT in rabbit fast twitch muscle, to immobilized alpha-Tm. Both 26K-TnT and TnT2f were retained by the alpha-Tm affinity column in the presence of 150 mM NaCl. However, upon increasing the NaCl concentration 26K-TnT was eluted from the column at a higher ionic strength than was TnT. When applied alone, the binary complex of TnI and TnC (TnC.TnI) was not retained by the alpha-Tm affinity column. When applied subsequently to prebound TnT2f or 26K-TnT, TnI.TnC was retained by the alpha-Tm affinity column and eluted together with TnT2f or 26K-TnT as ternary troponin complexes. Whether Ca2+ was present or not, Tn containing 26K-TnT was eluted at a higher ionic strength than was Tn containing TnT2f, indicating that removal of the first 45 residues of TnT2f strengthens the binding of Tn to Tm. In the presence of Tm, reconstituted Tn containing 26K-TnT conferred Ca2+ sensitivity on actomyosin-S1 MgATPase, and the steepness of the pCa-ATPase relation was unchanged with respect to the actoS1 ATPase regulated by TnT2f. It is concluded that the first 45 residues of TnT2f are not essential for anchoring the troponin complex to the thin filament and do not play a crucial role in the cooperative response of regulated actoS1 ATPase to Ca2+.     

Effect of different troponin T-tropomyosin combinations on thin filament activation

The response of permeabilized rabbit fast skeletal muscle fibers to calcium is determined by the troponin T (TnT) and tropomyosin (Tm) isoforms they express. Fibers expressing primarily TnT2f and alpha 2 Tm exhibit steeper pCa/tension relations than those in which either TnT1f or TnT3f and alpha beta Tm predominate. Troponin C extraction studies show that lower slopes do not result from a less concerted transition on the thin filament: the Tn-Tm regulatory strand activates as a unit in all fast fibers. Because the TnT variants differ in their N-terminal segments, and this region overlaps adjacent Tms on the regulatory strand, we propose that both the end-to-end overlap of Tm and the effect of TnT on that interaction are the basis of the concerted transition of the regulatory strand to the active state that occurs in the presence of calcium. Moreover, the effect of different Tn-Tm combinations on the ratio of the affinities of TnC for calcium in the relaxed and active states appears to be a significant determinant of the contractile properties of fast fibers in vivo.     

Cardiac troponin T isoforms affect the Ca2+ sensitivity and inhibition of force development. Insights into the role of troponin T isoforms in the heart

At least four isoforms of troponin T (TnT) exist in the human heart, and they are expressed in a developmentally regulated manner. To determine whether the different N-terminal isoforms are functionally distinct with respect to structure, Ca(2+) sensitivity, and inhibition of force development, the four known human cardiac troponin T isoforms, TnT1 (all exons present), TnT2 (missing exon 4), TnT3 (missing exon 5), and TnT4 (missing exons 4 and 5), were expressed, purified, and utilized in skinned fiber studies and in reconstituted actomyosin ATPase assays. TnT3, the adult isoform, had a slightly higher alpha-helical content than the other three isoforms. The variable region in the N terminus of cardiac TnT was found to contribute to the determination of the Ca(2+) sensitivity of force development in a charge-dependent manner; the greater the charge the higher the Ca(2+) sensitivity, and this was primarily because of exon 5. These studies also demonstrated that removal of either exon 4 or exon 5 from TnT increased the cooperativity of the pCa force relationship. Troponin complexes reconstituted with the four TnT isoforms all yielded the same maximal actin-tropomyosin-activated myosin ATPase activity. However, troponin complexes containing either TnT1 or TnT2 (both containing exon 5) had a reduced ability to inhibit this ATPase activity when compared with wild type troponin (which contains TnT3). Interestingly, fibers containing these isoforms also showed less relaxation suggesting that exon 5 of cardiac TnT affects the ability of Tn to inhibit force development and ATPase activity. These results suggest that the different N-terminal TnT isoforms would produce different functional properties in the heart that would directly affect myocardial contraction.     

Ca(2+)- and S1-induced movement of troponin T on reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy

Troponin T (TnT) is an essential component of troponin (Tn) for the Ca(2+)-regulation of vertebrate striated muscle contraction. TnT consists of an extended NH(2)-terminal domain that interacts with tropomyosin (Tm) and a globular COOH-terminal domain that interacts with Tm, troponin I (TnI), and troponin C (TnC). We have generated two mutants of a rabbit skeletal beta-TnT 25-kDa fragment (59-266) that have a unique cysteine at position 60 (N-terminal region) or 250 (C-terminal region). To understand the spatial rearrangement of TnT on the thin filament in response to Ca(2+) binding to TnC, we measured distances from Cys-60 and Cys-250 of TnT to Gln-41 and Cys-374 of F-actin on the reconstituted thin filament by using fluorescence resonance energy transfer (FRET). The distances from Cys-60 and Cys-250 of TnT to Gln-41 of F-actin were 39.5 and 30.0 A, respectively in the absence of Ca(2+), and increased by 2.6 and 5.8 A, respectively upon binding of Ca(2+) to TnC. The rigor binding of myosin subfragment 1 (S1) further increased these distances by 4 and 5 A respectively, when the thin filaments were fully decorated with S1. This indicates that not only the C-terminal but also the N-terminal region of TnT showed the Ca(2+)- and S1-induced movement, and the C-terminal region moved more than N-terminal region. In the absence of Ca(2+), the rigor S1 binding also increased the distances to the same extent as the presence of Ca(2+) when the thin filaments were fully decorated with S1. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding both in the presence and absence of Ca(2+). However, plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed hyperbolic curve in the presence of Ca(2+) but sigmoidal curve in the absence of Ca(2+). FRET measurement of the distances from Cys-60 and Cys-250 of TnT to Cys-374 of actin showed almost the same results as the case of Gln-41 of actin. The present FRET measurements demonstrated that not only TnI but also TnT change their positions on the thin filament corresponding to three states of thin filaments (relaxed, Ca(2+)-induced or closed, and S1-induced or open states).     

Exon skipping in cardiac troponin T of turkeys with inherited dilated cardiomyopathy

Troponin T is a central component of the thin filament-associated troponin-tropomyosin system and plays an essential role in the Ca(2+) regulation of striated muscle contraction. The importance of the structure and function of troponin T is evident in the regulated isoform expression during development and the point mutations resulting in familial hypertrophic and dilated cardiomyopathies. We report here that turkeys with inherited dilated cardiomyopathy and heart failure express an unusual low molecular weight cardiac troponin T missing 11 amino acids due to the splice out of the normally conserved exon 8-encoded segment. The deletion of a 9-bp segment from intron 7 of the turkey cardiac troponin T gene may be responsible for the weakened splicing of the downstream exon 8 during mRNA processing. The exclusion of the exon 8-encoded segment results in conformational changes in cardiac troponin T, an altered binding affinity for troponin I and tropomyosin, and an increased calcium sensitivity of the actomyosin ATPase. Expression of the exon 8-deleted cardiac troponin T prior to the development of cardiomyopathy in turkeys indicates a novel RNA splicing disease and provides evidence for the role of troponin T structure-function variation in myocardial pathogenesis and heart failure.     

Effects of the amino-terminal regions of tropomyosin and troponin T on thin filament assembly.

Bacterially expressed alpha-tropomyosin lacks the amino-terminal acetylation present in muscle tropomyosin and binds poorly to actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Using a linear lattice model, we determined the affinity (Ko) of unacetylated tropomyosin or troponin-unacetylated tropomyosin for an isolated site on the actin filament and the fold increase in affinity (y) when binding is to an adjacent site. The absence of tropomyosin acetylation decreased Ko 2 orders of magnitude in the absence of troponin. Tropomyosin acetylation also enhanced troponin-tropomyosin binding to actin, not by increasing cooperativity (y), but rather by increasing Ko. These results suggest that the amino-terminal region of tropomyosin is a crucial actin binding site. Troponin promoted unacetylated tropomyosin binding to actin, increasing Ko more than 1,000-fold. Troponin70-259, which lacks the troponin T peptide (1-69) spanning the overlap between adjacent tropomyosins, behaved similarly to intact troponin. Cooperative interactions between adjacent troponin-tropomyosin complexes remained strong despite the use of a nonpolymerizable tropomyosin and a troponin unable to bridge neighboring tropomyosins physically. The Ko for troponin70-259-unacetylated tropomyosin was 500-fold greater than for troponin159-259-unacetylated tropomyosin, indicating that troponin T residues 70-158 are critical for anchoring troponin-tropomyosin to F-actin. The mechanism of cooperative thin filament assembly is discussed.     

Bovine cardiac troponin T: amino acid sequences of the two isoforms

Troponin T (TnT) is the tropomyosin-binding subunit of troponin, the thin filament regulatory complex that confers calcium sensitivity to striated muscle contraction and actomyosin ATPase activity. Bovine cardiac muscle contains two isoforms (TnT-1 and TnT-2) of TnT that differ in sequence near their amino termini. Thin filaments containing TnT-2 require less calcium to activate the MgATPase rate of myosin than do thin filaments containing TnT-1. Using whole troponin T purified from adult bovine cardiac muscle, we have determined the complete amino acid sequence of the larger, more abundant isoform TnT-1. We confirmed that sequence differences between TnT-1 and TnT-2 are confined to the amino-terminal regions and found that TnT-1 makes up approximately 75% of the total troponin T isolated. Partial sequencing of the separated isoforms showed that the difference between them is due solely to residues 15-19 (Glu-Ala-Ala-Glu-Glu) of TnT-1 being absent from TnT-2. The deleted segment may correspond to the product of exon 4 of the chicken cardiac TnT gene [Cooper, T.A., & Ordahl, C.P. (1985) J. Biol. Chem. 260, 11140-11148]. Exon 5, which is developmentally regulated in the chicken, is not expressed in either TnT-1 or TnT-2. TnT-1 contains 284 amino acid residues and has a Mr of 33,808, while TnT-2 contains 279 amino acid residues and has a Mr of 33,279. Bovine cardiac TnT contains the only known thiol group in any isolated TnT (Cys-39 of TnT-1, Cys-34 of TnT-2). Comparison of bovine, rabbit, and chicken cardiac TnT sequences shows near identity of the amino-terminal 13 amino acid residues (exons 2 and 3 of the chicken cardiac gene), many differences in the following 60 residues (exons 4-8), and great similarity in the C-terminal 230 residues (exons 9-18).     

Conformational modulation of slow skeletal muscle troponin T by an NH(2)-terminal metal-binding extension

TroponinT (TnT) is an essential element in the thin filamentCa²⁺-regulatory system controlling striated musclecontraction. Alternative RNA splicing generates developmental andmuscle type-specific TnT isoforms differing in the hypervariableNH₂-terminal region. Using avian fast skeletal muscle TnTcontaining a metal-binding segment, we have demonstrated a role of theNH₂-terminal domain in modulating the conformation of TnT(Wang J and Jin JP. Biochemistry 37: 14519-14528,1998). To further investigate the structure-function relationship ofTnT, the present study constructed and characterized a recombinantprotein in which the metal-binding peptide present in avian fastskeletal muscle TnT was fused to the NH₂ terminus of mouseslow skeletal muscle TnT. Metal ion or monoclonal antibody binding tothe NH₂-terminal extension induced conformational changes in other domains of the model TnT molecule. This was shown by thealtered affinity to a monoclonal antibody against the COOH-terminal region and a polyclonal antiserum recognizing multiple epitopes. Protein binding assays showed that metal binding to theNH₂-terminal extension had effects on the interaction ofTnT with troponin I, troponin C, and most significantly, tropomyosin.The data indicate that the NH₂-terminal Tx [4-7repeats of a sequence motif His-(Glu/Ala)-Glu-Ala-His] extension confers a specific conformational modulation in the slowskeletal muscle TnT.

     

Role of the head-to-tail overlap region in smooth and skeletal muscle beta-tropomyosin

Tropomyosin (Tm) is an alpha-helical, parallel, two-chain coiled coil which binds along the length of actin filaments in both muscle and non-muscle cells. Smooth and skeletal muscle Tms differ extensively at the C-terminus encoded by exon 9. Replacement of the striated muscle specific exon 9a-encoded C-terminus with that encoded by exon 9d expressed in smooth muscle and non-muscle cells increases the affinity of unacetylated alpha-SkTm for actin [Cho, Y. J., and Hitchcock-Degregori, S. E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10153-10157]. Here we show that swapping 10 amino acids at the C-terminus of beta-SkTm with the corresponding 10 amino acids of beta-SmTm had little effect on the regulation of S1 binding to actin, but Tm viscosity, Tm binding to actin, and troponin T1 binding to Tm all become like smooth rather than SkTm. beta-SkTm point mutations show that these properties are largely defined by the amino acids at two positions, 277 and 279. The N279L mutation reduces the viscosity of beta-SkTm to close to beta-SmTm values, while both residues contribute to the binding of TnT1. We also show that removing the first 11 N-terminal amino acids of beta-SmTm to make the mutant DeltaN-betaSmTm results in a 10-fold weakening in actin affinity compared to that of beta-SmTm. CD studies show no difference in thermal unfolding between beta-SmTm and DeltaN-betaSmTm; however, the viscosity of DeltaN-betaSmTm is much lower than that of the control. The results suggest that DeltaN-betaSmTm was unable to form filaments in solution but can form filaments on actin.