Involvement of conserved, acidic residues in the N-terminal domain of troponin C in calcium-dependent regulation

We have mutated eight conserved, charged amino acid residues in the N-terminal, regulatory domain of troponin C (TnC) so we could investigate their role in troponin-linked Ca2+ regulation of muscle contraction. These residues surround a hydrophobic pocket in the N-terminal domain of TnC which, when Ca2+ binds to regulatory sites in this domain, is exposed and interacts with the inhibitory region of troponin I (TnI). We constructed three double mutants (E53A/E54A, E60A/E61A, and E85A/D86A) and two single mutants (R44A and R81A) of rabbit fast skeletal muscle troponin C (TnC) in which the charged residues were replaced with neutral alanines. All five of these mutants retained TnC's ability to bind TnI in a Ca2+-dependent manner, to neutralize TnI's inhibition of actomyosin S1 ATPase activity, and to form a ternary complex with TnI and troponin T (TnT). Ternary complexes formed with TnC(R44A) or TnC(R81A) regulated actomyosin S1 ATPase activity normally, with TnI-based inhibition in the absence of Ca2+ and TnT-based activation in the presence of Ca2+. TnC(E53A/E54A) and TnC(E85A/D86A) interacted weakly with TnT, as judged by native gel electrophoresis. Ternary complexes formed with these mutants inhibited actomyosin S1 ATPase activity in both the presence and absence of Ca2+, and did not undergo Ca2+-dependent structural changes in TnI which can be detected by limited chymotryptic digestion. TnC(E60A/E61A) interacted normally with TnT. Its ternary complex showed Ca2+-dependent structural changes in TnI, inhibited actomyosin S1 ATPase in the absence of Ca2+, but did not activate ATPase in the presence of Ca2+. This is the first demonstration that selective mutation of TnC can abolish the activating effect of troponin while its inhibitory function is retained. Our results suggest the existence of an elaborate network of protein-protein interactions formed by TnI, TnT, and the N-terminal domain of TnC, all of which are important in the Ca2+-dependent regulation of muscle contraction.     

Proximity relationships between residue 6 of troponin I and residues in troponin C: further evidence for extended conformation of troponin C in the troponin complex

Skeletal muscle troponin C (TnC) adopts an extended conformation when crystallized alone and a compact one when crystallized with an N-terminal troponin I (TnI) peptide, TnI(1-47) [Vassylyev et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4847-4852]. The N-terminal region of TnI (residues 1-40) was suggested to play a functional role of facilitating the movement of TnI's inhibitory region between TnC and actin [Tripet et al. (1997) J. Mol. Biol. 271, 728-750]. To test this hypothesis and to investigate the conformation of TnC in the intact troponin complex and in solution, we attached fluorescence and photo-cross-linking probes to a mutant TnI with a single cysteine at residue 6. Distances from this residue to residues of TnC were measured by the fluorescence resonance energy transfer technique, and the sites of photo-cross-linking in TnC were determined by microsequencing and mass spectrometry following enzymatic digestions. Our results show that in the troponin complex neither the distance between TnI residue 6 and TnC residue 89 nor the photo-cross-linking site in TnC, Ser133, changes with Ca(2+), in support of the notion that this region plays mainly a structural rather than a regulatory role. The distances to residues 12 and 41 in TnC's N-domain are both considerably longer than those predicted by the crystal structure of TnC.TnI(1-47), supporting an extended rather than a compact conformation of TnC. In the binary TnC.TnI complex and the presence of Ca(2+), Met43 in TnC's N-domain was identified as the photo-cross-linking site, and multiple distances between TnI residue 6 and TnC residue 41 were detected. This was taken to indicate increased flexibility in TnC's central helix and that TnC dynamically changes between a compact and an extended conformation when troponin T (TnT) is absent. Our results further emphasize the difference between the binary TnC.TnI and the ternary troponin complexes and the importance of using intact proteins in the study of structure-function relationships of troponin.     

Characterization of zero-length cross-links between rabbit skeletal muscle troponin C and troponin I: evidence for direct interaction between the inhibitory region of troponin I and the NH2-terminal, regulatory domain of troponin C

Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca2(+)-dependent regulation of vertebrate striated muscle contraction. Previous attempts to elucidate the molecular details of TnC-TnI interactions, mainly involving chemically modified proteins or fragments thereof, have led to the widely accepted idea that the "inhibitory region" (residues 96-116) of TnI binds to an alpha-helical segment of TnC comprising residues 89-100 in the nonregulatory, COOH-terminal domain. In an attempt to identify other possible physiologically important interactions between these proteins, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) was used to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI. TnC was activated with EDC and N-hydroxysuccinimide (NHS) and then mixed with an equimolar amount of TnI [Grabarek, Z., & Gergely, J. (1988) Biophys. J. 53, 392a]. The resulting cross-linked TnCXI was cleaved with cyanogen bromide, trypsin, and Staphylococcus aureus V8 protease (SAP). Cross-linked peptides were purified by reverse-phase HPLC and characterized by sequence analysis. The results indicated that residues from the regulatory Ca2(+)-binding site II in the NH2-terminal domain of TnC (residues 46-78) formed cross-links with TnI segments spanning residues 92-167. The most highly cross-linked residues in TnI were Lys-105 and Lys-107, located in the inhibitory region. These results yield the first evidence for an interaction between the N-terminal domain of TnC and the inhibitory region of TnI.     

Site-directed spin labeling electron paramagnetic resonance study of the calcium-induced structural transition in the N-domain of human cardiac troponin C complexed with troponin I

Calcium-induced structural transition in the amino-terminal domain of troponin C (TnC) triggers skeletal and cardiac muscle contraction. The salient feature of this structural transition is the movement of the B and C helices, which is termed the "opening" of the N-domain. This movement exposes a hydrophobic region, allowing interaction with the regulatory domain of troponin I (TnI) as can be seen in the crystal structure of the troponin ternary complex [Takeda, S., Yamashita, A., Maeda, K., and Maeda, Y. (2003) Nature 424, 35-41]. In contrast to skeletal TnC, Ca(2+)-binding site I (an EF-hand motif that consists of an A helix-loop-B helix motif) is inactive in cardiac TnC. The question arising from comparisons with skeletal TnC is how both helices move according to Ca(2+) binding or interact with TnI in cardiac TnC. In this study, we examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a cardiac TnC monomer state and TnC-TnI binary complex by means of site-directed spin labeling electron paramagnetic resonance (EPR). Doubly spin-labeled TnC mutants were prepared, and the spin-spin distances were estimated by analyzing dipolar interactions with the Fourier deconvolution method. An interspin distance of 18.4 A was estimated for mutants spin labeled at G42C on the B helix and C84 on the D helix in a Mg(2+)-saturated monomer state. The interspin distance between Q58C on the C helix and C84 on the D helix was estimated to be 18.3 A under the same conditions. Distance changes were observed by the addition of Ca(2+) ions and the formation of a complex with TnI. Our data indicated that the C helix moved away from the D helix in a distinct Ca(2+)-dependent manner, while the B helix did not. A movement of the B helix by interaction with TnI was observed. Both Ca(2+) and TnI were also shown to be essential for the full opening of the N-domain in cardiac TnC.     

Calcium-induced flexibility changes in the troponin C-troponin I complex

The contraction of vertebrate striated muscle is modulated by Ca(2+) binding to the regulatory protein troponin C (TnC). Ca(2+) binding causes conformational changes in TnC which alter its interaction with the inhibitory protein troponin I (TnI), initiating the regulatory process. We have used the frequency domain method of fluorescence resonance energy transfer (FRET) to measure distances and distance distributions between specific sites in the TnC-TnI complex in the presence and absence of Ca(2+) or Mg(2+). Using sequences based on rabbit skeletal muscle proteins, we prepared functional, binary complexes of wild-type TnC and a TnI mutant which contains no Cys residues and a single Trp residue at position 106 within the TnI inhibitory region. We used TnI Trp-106 as the FRET donor, and we introduced energy acceptor groups into TnC by labeling at Met-25 with dansyl aziridine or at Cys-98 with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our distance distribution measurements indicate that the TnC-TnI complex is relatively rigid in the absence of Ca(2+), but becomes much more flexible when Ca(2+) binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, helping to release the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distributions between TnC and TnI in their binary complex.     

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.     

Cross-linking of residue 57 in the regulatory domain of a mutant rabbit skeletal muscle troponin C to the inhibitory region of troponin I

Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca(2+)-dependent regulation of vertebrate striated muscle contraction. In the present study, we investigated the sites of interaction between the N-terminal regulatory domain of TnC and the inhibitory region (residues 96-116) of TnI, using a mutant rabbit skeletal TnC (designated as TnC57) that contains a single Cys at residue 57 in the C-helix. TnC57 was modified with the photoreactive cross-linker 4-maleimidobenzophenone (BP-Mal), and, after formation of a binary complex with TnI, cross-linking between the proteins was induced by photolysis. The resulting product was cleaved with CNBr and several proteases, and peptides containing cross-links were purified and subjected to amino acid sequencing. The results show that Cys-57 of TnC57 is cross-linked to the segment of TnI spanning residues 113-121. Previously, we showed that Cys-98 of TnC can be cross-linked via BP-Mal to TnI residues 103-110 (Leszyk, J., Collins, J.H., Leavis, P.C., and Tao, T. (1987) Biochemistry 26, 7042-7047). Taken together, these results demonstrate that both the C- and the N-terminal domains of TnC interact with the inhibitory region of TnI and are consistent with the hypothesis that, in a complex with TnI, TnC adopts a more compact conformation than in the crystal structure.     

Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity.

A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities.     

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)     

Photocrosslinking of benzophenone-labeled single cysteine troponin I mutants to other thin filament proteins

The interaction sites of rabbit skeletal troponin I (TnI) with troponin C (TnC), troponin T (TnT), tropomyosin (Tm) and actin were mapped systematically using nine single cysteine residue TnI mutants with mutation sites at positions 6, 48, 64, 89, 104, 121, 133, 155 or 179 (TnI6, TnI48 etc.). Each mutant was labeled with the heterobifunctional photocrosslinker 4-maleimidobenzophenone (BP-Mal), and incorporated into the TnI.TnC binary complex, the TnI.TnC.TnT ternary troponin (Tn) complex, and the Tn.Tm.F-actin synthetic thin filament. Photocrosslinking reactions carried out in the presence and absence of Ca(2+) yielded the following results: (1) BP-TnI6 photocrosslinked primarily to TnC with a small degree of Ca(2+)-dependence in all the complex forms. (2) BP-TnI48, TnI64 and TnI89 photocrosslinked to TnT with no Ca(2+)-dependence. Photocrosslinking to TnC was reduced in the ternary versus the binary complex. BP-TnI89 also photocrosslinked to actin with higher yields in the absence of Ca(2+) than in its presence. (3) BP-TnI104 and TnI133 photocrosslinked to actin with much higher yields in the absence than in the presence of Ca(2+). (4) BP-TnI121 photocrosslinked to TnC with a small degree of Ca(2+)-dependence, and did not photocrosslink to actin. (5) BP-TnI155 and TnI179 photocrosslinked to TnC, TnT and actin, but all with low yields. All the labeled mutants photocrosslinked to TnC with varying degrees of Ca(2+)-dependence, and none to Tm. These results, along with those published allowed us to construct a structural and functional model of TnI in the Tn complex: in the presence of Ca(2+), residues 1-33 of TnI interact with the C-terminal domain hydrophobic cleft of TnC, approximately 48-89 with TnT, approximately 90-113 with TnC's central helix, approximately 114-125 with TnC's N-terminal domain hydrophobic cleft, and approximately 130-150 with TnC's A-helix. In the absence of Ca(2+), residues approximately 114-125 move out of TnC's N-terminal domain hydrophobic cleft and trigger the movements of residues approximately 89-113 and approximately 130-150 away from TnC and towards actin.     

Kinetic analysis of the interactions between troponin C (TnC) and troponin I (TnI) binding peptides: evidence for separate binding sites for the 'structural' N-terminus and the 'regulatory' C-terminus of TnI on TnC

The Ca(2+)/Mg(2+)-dependent interactions between TnC and TnI play a critical role in regulating the 'on' and 'off' states of muscle contraction as well as maintaining the structural integrity of the troponin complex in the off state. In the present study, we have investigated the binding interactions between the N-terminus of TnI (residues 1-40 of skeletal TnI) and skeletal TnC in the presence of Ca(2+) ions, Mg(2+) ions and in the presence of the C-terminal regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). Our results show the N-terminus of TnI can bind to TnC with high affinity in the presence of Ca(2+) or Mg(2+) ions with apparent equilibrium dissociation constants of K(d(Ca(2+) ) ) = 48 nM and K(d(Mg(2+) ) ) = 29 nM. The apparent association and dissociation rate constants for the interactions were, k(on) = 4.8 x 10(5) M (-1) s(-1), 3.4 x 10(5) M (-1) s(-1) and k(off) = 2.3 x 10(-2) s(-1), 1.0 x 10(-2) s(-1) for TnC(Ca(2+)) and TnC(Mg(2+)) states, respectively. Competition studies between each of the TnI regions and TnC showed that both TnI regions can bind simultaneously to TnC while native gel electrophoresis and SEC confirmed the formation of stable ternary complexes between TnI(96-139) (or TnI(96-131)) and TnC-TnI(1-40). Further analysis of the binding interactions in the ternary complex showed the binding of the TnI regulatory region to TnC was critically dependent upon the presence of both TnC binding sites (i.e. TnI(96-115) and TnI(116-131)) and the presence of Ca(2+). Furthermore, the presence of TnI(1-40) slightly weakened the affinity of the regulatory peptides for TnC. Taken together, these results support the model for TnI-TnC interaction where the N-terminus of TnI remains bound to the C-domain of TnC in the presence of high and low Ca(2+) levels while the TnI regulatory region (residues 96-139) switches in its binding interactions between the actin-tropomyosin thin filament and its own sites on the N- and C-domain of TnC at high Ca(2+) levels, thus regulating muscle contraction.     

Solution structure of the chicken skeletal muscle troponin complex via small-angle neutron and X-ray scattering

Troponin is a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle by participating in a series of conformational events within the actin-based thin filament. Troponin is a heterotrimeric complex consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT). Ternary troponin complexes have been produced by assembling recombinant chicken skeletal muscle TnC, TnI and the C-terminal portion of TnT known as TnT2. A full set of small-angle neutron scattering data has been collected from TnC-TnI-TnT2 ternary complexes, in which all possible combinations of the subunits have been deuterated, in both the +Ca2+ and -Ca2+ states. Small-angle X-ray scattering data were also collected from the same troponin TnC-TnI-TnT2 complex. Guinier analysis shows that the complex is monomeric in solution and that there is a large change in the radius of gyration of TnI when it goes from the +Ca2+ to the -Ca2+ state. Starting with a model based on the human cardiac troponin crystal structure, a rigid-body Monte Carlo optimization procedure was used to yield models of chicken skeletal muscle troponin, in solution, in the presence and in the absence of regulatory calcium. The optimization was carried out simultaneously against all of the scattering data sets. The optimized models show significant differences when compared to the cardiac troponin crystal structure in the +Ca2+ state and provide a structural model for the switch between +Ca2+ and -Ca2+ states. A key feature is that TnC adopts a dumbbell conformation in both the +Ca2+ and -Ca2+ states. More importantly, the data for the -Ca2+ state suggest a long extension of the troponin IT arm, consisting mainly of TnI. Thus, the troponin complex undergoes a large structural change triggered by Ca2+ binding.     

Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin.

Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.     

Differential pH effect on calcium-induced conformational changes of cardiac troponin C complexed with cardiac and fast skeletal isoforms of troponin I and troponin T

The aim of this study is to investigate the molecular events associated with the deleterious effects of acidosis on the contractile properties of cardiac muscle as in the ischemia of heart failure. We have conducted a study of the effects of increasing acidity on the Ca(2+) induced conformational changes of pyrene labelled cardiac troponin C (PIA-cTnC) in isolation and in complex with porcine cardiac or chicken pectoral skeletal muscle TnI and/or TnT. The pyrene label has been shown to serve as a useful fluorescence reporter group for conformational and interaction events of the N-terminal regulatory domain of TnC with only minimal fluorescence changes associated with C-terminal domain. Results obtained show that the significant decreases at pH 6.0 of site II Ca(2+) affinity of PIA-cTnC when complexed as a binary complex with either cTnI or cTnT are significantly reduced when cTnI is replaced with sTnI or cTnT with sTnT. However, this effect is appreciably diminished when the cTnI and cTnT in the ternary complex are replaced by sTnI and sTnT. The smaller effects in the ternary complex of replacing both cTnI and cTnT by their skeletal counterparts on depressing the Ca(2+) affinity from pH 7.0 to 6.0 arise from TnI replacement. Thus, changes in TnC conformation resulting from isoform-specific interactions with TnI and TnT could be an integral part of the effect of pH on myofilament Ca(2+)sensitivity.     

Biologically important interactions between synthetic peptides of the N-terminal region of troponin I and troponin C.

The interaction between troponin I and troponin C plays a critical role in the regulation of muscle contraction. In this study the interaction between troponin C (TnC) and the N-terminal region of TnI was investigated by the synthesis of three TnI peptides (residues 1-40/Rp, 10-40, and 20-40). The regulatory peptide (Rp) on binding to TnC prevents the ability of TnC to release the inhibition of the acto-S1-tropomyosin ATPase activity caused by TnI or the TnI inhibitory peptide (Ip), residues 104-115. A stable complex between TnC and Rp in the presence of Ca2+ was demonstrated by polyacrylamide gel electrophoresis in the presence of 6 M urea. Rp was able to displace TnI from a preformed TnI.TnC complex. In the absence of Ca2+, Rp was unable to maintain a complex with TnC in benign conditions of polyacrylamide gel electrophoresis which demonstrates the Ca(2+)-dependent nature of this interaction. Size-exclusion chromatography demonstrated that the TnC.Rp complex consisted of a 1:1 complex. The results of these studies have shown that the N-terminal region of TnI (1-40) plays a critical role in modulating the Ca(2+)-sensitive release of TnI inhibition by TnC.     

Mapping subdomains in the C-terminal region of troponin I involved in its binding to troponin C and to thin filament.

Troponin I (TnI) is the inhibitory component of troponin, the ternary complex that regulates skeletal and cardiac muscle contraction. Previous work showed that the C-terminal region of TnI, when linked to the "inhibitory region" (residues 98-116), possesses the major regulatory functions of the molecule (Farah, C. S., Miyamoto, C. A., Ramos, C. H. I., Silva, A. C. R., Quaggio, R. B., Fujimori, K., Smillie, L. B., and Reinach, F. C. (1994) J. Biol. Chem. 269, 5230-5240). To investigate these functions in more detail, serial deletion mutants of the C-terminal region of TnI were constructed. These experiments showed that longer C-terminal deletions result in lower inhibition of the actomyosin ATPase activity and weaken the interaction with the N-terminal domain of troponin C (TnC), consistent with the antiparallel model for the interaction between these two proteins. The conclusion is that the whole C-terminal region of TnI is necessary for its full regulatory activity. The region between residues 137 and 144, which was shown to have homology with residues 108-115 in the inhibitory region (Farah, C. S., and Reinach, F. C. (1995) FASEB J. 9, 755-767), is involved in the binding to TnC. The region between residues 98 and 129 is involved in modulating the affinity of TnC for calcium. The C-terminal residues 166-182 are involved in the binding of TnI to thin filament. A model for the function of TnI is discussed.     

Fast and slow isoforms of troponin I and troponin C. Distribution in normal rabbit muscles and effects of chronic stimulation

Polyclonal antibodies were raised against troponin I (TnI) and troponin C (TnC) purified from fast-twitch and slow-twitch rabbit muscles. These antibodies were used to elucidate the distribution of fast and slow isoforms of TnI and TnC in normal and chronically stimulated rabbit hind limb muscles by immunoblots of one-dimensional and two-dimensional electrophoreses. In contrast to the multiplicity of fast and slow troponin T (TnT) isoforms, TnI and TnC were present as unique fast and slow isoforms. Whereas no charge variants were detected for slow TnI, fast TnI was present in at least three charge variants. As judged from the results of alkaline phosphatase digestion, these charge variants represent differently phosphorylated forms. Fast and slow TnC both exist as two charge variants which, however, were unaffected by alkaline phosphatase treatment. Chronic low-frequency stimulation of fast-twitch muscles induced progressive increases in the slow isoforms of TnC and TnI at the expense of their fast isoforms. The extent of the fast-to-slow transition was more pronounced in the case of TnC than in that of TnI. Long-term stimulated muscles with a complete fast-to-slow transition, at the level of the TnT isoforms, still contained fast and slow isoforms of both TnI and TnC. The coexistence of fast and slow isoforms of the three troponin subunits in the transforming muscle was interpreted as indicating the presence of hybrid troponin molecules composed of fast and slow isoforms. Studies at the mRNA level showed changes similar to those at the protein level. However, in long-term stimulated muscles, the fast-to-slow transition of TnI was more pronounced at the mRNA level than at the protein level.     

Cross-linking of rabbit skeletal muscle troponin subunits: labeling of cysteine-98 of troponin C with 4-maleimidobenzophenone and analysis of products formed in the binary complex with troponin T and the ternary complex with troponins I and T

The sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) was used to study the interaction of rabbit skeletal muscle troponin subunits TnC, TnT, and TnI. TnC was labeled at Cys-98 by the maleimide moiety of BPMal and then mixed with either TnT alone or TnI plus TnT, in the presence of Ca2+. Upon photolysis, TnI and/or TnT formed covalent cross-links with TnC. The cross-linked TnC-TnT heterodimer obtained from the binary complex was digested into progressively smaller cross-linked peptides that were purified by HPLC and then characterized by amino acid analysis and sequencing. An initial cross-linked CNBr fraction contained the expected peptide CB9 (residues 84-135) of TnC, plus CNBr peptides spanning residues 152-230 of TnT. Results from a peptic digest of the CNBr cross-linked fraction permitted the identification of residues 159-197 as the most highly cross-linked region in TnT. A final subtilisin digest yielded a heterogeneous cross-linked fraction, which suggested that an especially high degree of cross-links was formed in the vicinity of residues 175-178 (Met-Lys-Lys-Lys) of TnT. Although this region of TnT had previously been implicated in binding, we show here for the first time that it is close to Cys-98 of TnC. In an analogous study on the binary complex of TnC and TnI [Leszyk, J., Collins, J. H., Leavis, P. C., & Tao, T. (1987) Biochemistry 26, 7042-7047], we previously showed that Cys-98 of TnC was cross-linked mainly to CN4, the "inhibitory region", of TnI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence spectroscopic analysis of the proximity changes between the central helix of troponin C and the C-terminus of troponin T from chicken skeletal muscle

Recent structural studies of the troponin (Tn) core complex have shown that the regulatory head containing the N-lobe of TnC is connected to the IT arm by a flexible linker of TnC. The IT arm is a long coiled-coil formed by alpha-helices of TnI and TnT, plus the C-lobe of TnC. The TnT is thought to play a pivotal role in the linking of Ca(2+) -triggered conformational changes in thin filament regulatory proteins to the activation of cross-bridge cycling. However, a functional domain at the C-terminus of TnT is missing from the Tn core complex. In this study, we intended to determine the proximity relationship between the central helix of TnC and the TnT C-terminus in the binary and the ternary complex with and without Ca2+ by using pyrene excimer fluorescence spectroscopy and fluorescence resonance energy transfer. Chicken fast skeletal TnC contains a Cys102 at the E helix, while TnT has a Cys264 at its C-terminus. These two cysteines were specifically labeled with sulfhydryl-reactive fluorescence probes. The measured distance in the binary complex was about 19 Angstroms and slightly increased when they formed the ternary complex with TnI (20 Angstroms). Upon Ca2+ binding the distance was not affected in the binary complex but increased by approximately 4 Angstroms in the ternary complex. These results suggest that TnI plays an essential role in the Ca(2+) -mediated change in the spatial relationship between the C-lobe of TnC and the C-terminus of TnT.     

Cross-linking of rabbit skeletal muscle troponin with the photoactive reagent 4-maleimidobenzophenone: identification of residues in troponin I that are close to cysteine-98 of troponin C

We have used the sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) to study the interaction of rabbit skeletal muscle troponin C (TnC) and troponin I (TnI). TnC was specifically labeled at Cys-98 by the maleimide moiety of BPMal, and a binary complex was formed with TnI in the presence of Ca2+. Upon photolysis, covalent cross-links were formed between TnC and TnI [Tao, T., Scheiner, C.J., & Lamkin, M. (1986) Biochemistry 25, 7633-7639]. The cross-linked heterodimer was digested with cyanogen bromide, pepsin, and chymotrypsin into progressively smaller cross-linked peptides, which were purified by HPLC and then characterized by amino acid analysis and sequencing. We obtained a fraction from the initial CNBr digest that contained the expected peptide CB9 (residues 84-135) of TnC, cross-linked mainly to CN4 (residues 96-116), the "inhibitory region" of TnI. The peptides CN1 and CN3 of TnI were also detected in this fraction, but their molar ratios (compared to CB9) were only about 0.15 each, compared to 0.60 for CN4. Sequence analyses of fractions obtained after peptic and chymotryptic digests of the cross-linked CNBr fraction confirmed that CB9 and CN4 were the major cross-linked species. Quantitative analysis of sequencer results indicated that the residues in TnI that appeared to be most highly cross-linked to Cys-98 of TnC were Arg-108 and Pro-110, and to a lesser extent Arg-103 and Lys-107. These findings are consistent with previous studies on interactions between TnI and TnC and provide, for the first time, direct information on the identities of proximate amino acids in the two proteins.