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.     

Functional importance of Ca2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

Ca(2+)-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca(2+) due to substitutions of the amino acid residues responsible for Ca(2+) liganding. To evaluate the functional importance of the Ca(2+)-deficient N-terminal lobe in the Ca(2+)-regulatory function of molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using (45)Ca(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca(2+)/mol and 1 mol Ca(2+)/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca(2+) binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6M urea, if Ca(2+) was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca(2+)-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca(2+) sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca(2+) sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca(2+).     

Aluminum fluoride interactions with troponin C.

The increasing interest in the metal ion aluminum fluoride and beryllium fluoride complexes as phosphate analogs in the myosin ATPase reaction and in muscle fiber studies prompted the examination of their interactions with the regulatory system of troponin and tropomyosin. In this work, the effects of these metal ion analogs on the spectral properties of the Ca(2+)-binding subunit of troponin, troponin C (TnC), were examined. In contrast to beryllium fluoride which did not change the spectral properties of TnC, aluminum fluoride binding induced an increase in both the alpha-helicity and the tyrosine fluorescence of TnC and exposed a hydrophobic region on this protein for fluorescent probe binding. Aluminum fluoride also reduced the Ca2+ and/or Mg(2+)-induced changes on TnC. These results indicate a direct interaction of aluminum fluoride with TnC and merit consideration in designing muscle fiber experiments with this phosphate analog.     

Engineered troponin C constructs correct disease-related cardiac myofilament calcium sensitivity

Aberrant myofilament Ca(2+) sensitivity is commonly observed with multiple cardiac diseases, especially familial cardiomyopathies. Although the etiology of the cardiomyopathies remains unclear, improving cardiac muscle Ca(2+) sensitivity through either pharmacological or genetic approaches shows promise of alleviating the disease-related symptoms. Due to its central role as the Ca(2+) sensor for cardiac muscle contraction, troponin C (TnC) stands out as an obvious and versatile target to reset disease-associated myofilament Ca(2+) sensitivity back to normal. To test the hypothesis that aberrant myofilament Ca(2+) sensitivity and its related function can be corrected through rationally engineered TnC constructs, three thin filament protein modifications representing different proteins (troponin I or troponin T), modifications (missense mutation, deletion, or truncation), and disease subtypes (familial or acquired) were studied. A fluorescent TnC was utilized to measure Ca(2+) binding to TnC in the physiologically relevant biochemical model system of reconstituted thin filaments. Consistent with the pathophysiology, the restrictive cardiomyopathy mutation, troponin I R192H, and ischemia-induced truncation of troponin I (residues 1-192) increased the Ca(2+) sensitivity of TnC on the thin filament, whereas the dilated cardiomyopathy mutation, troponin T ΔK210, decreased the Ca(2+) sensitivity of TnC on the thin filament. Rationally engineered TnC constructs corrected the abnormal Ca(2+) sensitivities of the thin filament, reconstituted actomyosin ATPase activity, and force generation in skinned trabeculae. Thus, the present study provides a novel and versatile therapeutic strategy to restore diseased cardiac muscle Ca(2+) sensitivity.     

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

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

Binding properties of the calcium-activated F2 isoform of Lethocerus troponin C

While in most muscles contraction is triggered by calcium effluxes, insect flight muscles are also activated by mechanical stretch. We are interested in understanding the role that the troponin C protein, usually the calcium sensor, plays in stretch activation. In the flight muscles of Lethocerus, a giant water bug often used as a model system, there are two isoforms of TnC, F1 and F2, present in an approximately 10:1 ratio. F1 TnC is responsible for activating the muscle following a stretch, whereas F2 TnC produces a sustained contraction, the magnitude of which depends on the concentration of Ca(2+) in the fiber. We have previously shown that F1 TnC binds only one Ca(2+) ion in its C-terminal domain and that interaction with troponin H, the insect ortholog of troponin I, is insensitive to Ca(2+). Here, we have studied the effect of Ca(2+) and Mg(2+) on the affinities of the interaction of F2 TnC with troponin H peptides. We show that the presence of two Ca(2+) ions, one in each of the globular domains, increases the affinity for TnH by at least 1 order of magnitude. The N lobe has a lower affinity for Ca(2+), but it is also sensitive to Mg(2+). The C lobe is insensitive to Mg(2+) as previously demonstrated by mutations of the individual EF-hands. The interaction with TnH seems also to have significant structural differences from that observed for the F1 TnC isoform. We discuss how our findings could account for stretch activation.     

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.     

Evolutionary and physiological variation in cardiac troponin C in relation to thermal strategies of fish

Striated muscle contraction is initiated when troponin C (TnC) binds Ca(2+), which activates actinomyosin ATPase. We investigated (i) the variation between cardiac TnC (cTnC) primary structure within teleost fish and (ii) the pattern of TnC expression in response to temperature acclimation. There were few differences between rainbow trout (Oncorhynchus mykiss), yellowfin tuna (Thunnus albacares), yellow perch (Perca flavescens), goldfish (Carassius auratus), white sucker (Catostomus commersoni), and icefish (Chaenocephalus aceratus) in cTnC amino acid sequence. No variation existed in the regulatory Ca(2+)-binding site (site 2). The site 3 and 4 substitutions were limited to residues not directly involved in Ca(2+) coordination. Fish cTnC primary structure was highly conserved between species (93%-98%) and collectively divergent from the highly conserved sequence seen in birds and mammals. Northern blots and polymerase chain reaction showed that thermal acclimation of trout (3 degrees, 18 degrees C) did not alter the TnC isoform pattern. While cardiac and white muscle had the expected isoforms-cTnC and fast troponin C (fTnC), respectively-red muscle unexpectedly expressed primarily ftnC. Cold acclimation did not alter myofibrillar ATPase Ca(2+) sensitivity, but maximal velocity increased by 60%. We found no evidence that TnC variants, arising between species or in response to thermal acclimation, play a major role in mitigating the effects of temperature on contractility of the adult fish heart.     

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.     

Computational detection and analysis of sequences with duplex-derived interstrand G-quadruplex forming potential

Troponin is well known as a Ca(2+)-dependent regulator of striated muscle contraction and it has been generally accepted that troponin functions as an inhibitor of muscle contraction or actin-myosin interaction at low Ca(2+) concentrations, and Ca(2+) at higher concentrations removes the inhibitory action of troponin. Recently, however, troponin became detectable in non-striated muscles of several invertebrates and in addition, unique troponin that functions as a Ca(2+)-dependent activator of muscle contraction has been detected in protochordate animals, although troponin in vertebrate striated muscle is known as an inhibitor of the contraction in the absence of a Ca(2+). Further studies on troponin in invertebrate muscle, especially in non-striated muscle, would provide new insight into the evolution of regulatory systems for muscle contraction and diverse function of troponin and related proteins. The methodology used for preparation and characterization of functional properties of protochordate striated and smooth muscles will be helpful for further studies of troponin in other invertebrate animals.     

The Rates of Ca2+ Dissociation and Cross-bridge Detachment from Ventricular Myofibrils as Reported by a Fluorescent Cardiac Troponin C

The rate-limiting step of cardiac muscle relaxation has been proposed to reside in the myofilament. Both the rates of cross-bridge detachment and Ca(2+) dissociation from troponin C (TnC) have been hypothesized to rate-limit myofilament inactivation. In this study we used a fluorescent TnC to measure both the rate of Ca(2+) dissociation from TnC and the rate of cross-bridge detachment from several different species of ventricular myofibrils. The fluorescently labeled TnC was sensitive to both Ca(2+) dissociation and cross-bridge detachment at low Ca(2+) (presence of EGTA), allowing for a direct comparison between the two proposed rates of myofilament inactivation. Unlike Ca(2+) dissociation from TnC, cross-bridge detachment varied in myofibrils from different species and was rate-limited by ADP release. At subphysiological temperatures (<20 °C), the rate of Ca(2+) dissociation from TnC was faster than the rate of cross-bridge detachment in the presence of ADP. These results support the hypothesis that cross-bridge detachment rate-limits relaxation. However, Ca(2+) dissociation from TnC was not as temperature-sensitive as cross-bridge detachment. At a near physiological temperature (35 °C) and ADP, the rate of cross-bridge detachment may actually be faster than the rate of Ca(2+) dissociation. This provides evidence that there may not be a simple, single rate-limiting step of myofilament inactivation.     

Microtiter plate monoclonal antibody epitope analysis of Ca2+- and Mg2+-induced conformational changes in troponin C

Spectroscopic methods such as circular dichroism and F?rster resonance energy transfer are current approaches for monitoring protein conformational changes. Those analyses require special equipment and expertise. The need for fluorescence labeling of the protein may interfere with the native structure. We have developed a microtiter plate-based monoclonal antibody (mAb) epitope analysis to detect protein conformational changes in a high throughput manner. This method is based on the concept that the affinity of the antigen-binding site of an antibody for the specific antigenic epitope will change when the 3-D structure of the epitope changes. The effectiveness of this approach was demonstrated in the present study on troponin C (TnC), an allosteric protein in the Ca(2+) regulatory system of striated muscle. Using TnC purified by a highly effective rapid procedure and mAbs developed against epitopes in the N- and C-domains of TnC enzyme-linked immunosorbant assay (ELISA) clearly detected Ca(2+)-induced conformational changes in both the N-terminal regulatory domain and the C-terminal structural domain of TnC. On the other hand, Mg(2+)-binding to the C-domain of TnC resulted in a long-range effect on the N-domain conformation, indicating a functional significance of Ca(2+)-Mg(2+) exchange at the C-domain metal ion-binding sites. In addition to further understanding of the structure-function relationship of TnC, the data demonstrate that the mAb epitope analysis provides a simple high throughput method for monitoring 3-D structural changes in native proteins under physiological condition and has broad applications in protein structure-function relationship studies.     

NMR and mutagenesis studies on the phosphorylation region of human cardiac troponin I

Phosphorylation of the cardiac troponin complex by PKA at S22 and S23 of troponin I (TnI) accelerates Ca(2+) release from troponin C (TnC). The region of TnI around the bisphosphorylation site binds to, and stabilizes, the Ca(2+) bound N-terminal domain of TnC. Phosphorylation interferes with this interaction between TnI and TnC resulting in weaker Ca(2+) binding. In this study, we used (1)H-(15)N-HSQC NMR to investigate at the atomic level the interaction between an N-terminal fragment of TnI consisting of residues 1-64 of TnI (I1-64) and TnC. We produced several mutants of I1-64, TnI, and TnC to test the contribution of certain residues to the transmission of the phosphorylation signal in both NMR experiments and functional assays. We also investigated how phosphorylation of the PKC sites in I1-64 (S41 and S43) affects the interaction of I1-64 with TnC. We found that phosphorylation of S22 and S23 produced only localized effects in the structure of I1-64 between residues 24 and 34. Residues 1-17 of I1-64 did not bind to TnC, and residues 38-64 bound tightly to the C-terminal domain of TnC regardless of phosphorylation. Residues 22-34 bound weakly to TnC in a phosphorylation sensitive manner. Bisphosphorylation prevented this phosphorylation switch region from interacting with TnC. Systematic mutation of residues in the phosphorylation switch did not prevent PKA phosphorylation from accelerating Ca(2+) release from troponin. We conclude that the phosphorylation switch binds to TnC via an extended interaction site spanning residues R19 to A34.     

Biological function and site II Ca2+-induced opening of the regulatory domain of skeletal troponin C are impaired by invariant site I or II Glu mutations

To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control. Circular dichroism measurements of wild-type N domain as a function of pCa (= -log[Ca(2+)]) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca(2+) binding. With E41A, E77A, and cardiac TnC N domains this [theta](222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+). Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.     

Ca2+-dependent interaction of the inhibitory region of troponin I with acidic residues in the N-terminal domain of troponin C

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96-116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95-116, 95-119 or 95-124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117-124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.     

Characterization of troponin T dilated cardiomyopathy mutations in the fetal troponin isoform

The major goal of this study was to elucidate how troponin T (TnT) dilated cardiomyopathy (DCM) mutations in fetal TnT and fetal troponin affect the functional properties of the fetal heart that lead to infantile cardiomyopathy. The DCM mutations R141W and DeltaK210 were created in the TnT1 isoform, the primary isoform of cardiac TnT in the embryonic heart. In addition to a different TnT isoform, a different troponin I (TnI) isoform, slow skeletal TnI (ssTnI), is the dominant isoform in the embryonic heart. In skinned fiber studies, TnT1-wild-type (WT)-treated fibers reconstituted with cardiac TnI.troponin C (TnC) or ssTnI.TnC significantly increased Ca(2+) sensitivity of force development when compared with TnT3-WT-treated fibers at both pH 7.0 and pH 6.5. Porcine cardiac fibers treated with TnT1 that contained the DCM mutations (R141W and DeltaK210), when reconstituted with either cardiac TnI.TnC or ssTnI.TnC, significantly decreased Ca(2+) sensitivity of force development compared with TnT1-WT at both pH values. The R141W mutation, which showed no significant change in the Ca(2+) sensitivity of force development in the TnT3 isoform, caused a significant decrease in the TnT1 isoform. The DeltaK210 mutation caused a greater decrease in Ca(2+) sensitivity and maximal isometric force development compared with the R141W mutation in both the fetal and adult TnT isoforms. When complexed with cardiac TnI.TnC or ssTnI.TnC, both TnT1 DCM mutations strongly decreased maximal actomyosin ATPase activity as compared with TnT1-WT. Our results suggest that a decrease in maximal actomyosin ATPase activity in conjunction with decreased Ca(2+) sensitivity of force development may cause a severe DCM phenotype in infants with the mutations.     

Effect of hydrophobic residue substitutions with glutamine on Ca(2+) binding and exchange with the N-domain of troponin C

Troponin C (TnC) is an EF-hand Ca(2+) binding protein that regulates skeletal muscle contraction. The mechanisms that control the Ca(2+) binding properties of TnC and other EF-hand proteins are not completely understood. We individually substituted 27 Phe, Ile, Leu, Val, and Met residues with polar Gln to examine the role of hydrophobic residues in Ca(2+) binding and exchange with the N-domain of a fluorescent TnC(F29W). The global N-terminal Ca(2+) affinities of the TnC(F29W) mutants varied approximately 2340-fold, while Ca(2+) association and dissociation rates varied less than 70-fold and more than 45-fold, respectively. Greater than 2-fold increases in Ca(2+) affinities were obtained primarily by slowing of Ca(2+) dissociation rates, while greater than 2-fold decreases in Ca(2+) affinities were obtained by slowing of Ca(2+) association rates and speeding of Ca(2+) dissociation rates. No correlation was found between the Ca(2+) binding properties of the TnC(F29W) mutants and the solvent accessibility of the hydrophobic amino acids in the apo state, Ca(2+) bound state, or the difference between the two states. However, the effects of these hydrophobic mutations on Ca(2+) binding were contextual possibly because of side chain interactions within the apo and Ca(2+) bound states of the N-domain. These results demonstrate that a single hydrophobic residue, which does not directly ligate Ca(2+), can play a crucial role in controlling Ca(2+) binding and exchange within a coupled and functional EF-hand system.     

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

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

Hybrid myosin light chains containing a calcium-specific site from troponin C.

Recombinant DNA approaches have allowed us to probe the mechanisms by which the regulatory light chains (RLCs) regulate myosin function by identifying the functional importance of specific regions of the RLC molecule. For example, we have demonstrated that the presence of high-affinity Ca2+/Mg(2+)-binding site in the N-terminal domain of the RLC is essential for the regulation of myosin-actin interaction [Reinach, F. C., Nagai, K. & Kendrick-Jones, J. (1986) Nature 322, 80-83]. To explore further the role of this metal-binding site in the RLC and generate an RLC with a Ca(2+)-specific site, we constructed four chicken skeletal muscle myosin regulatory light chain hybrid 'genes'. In these, the first domain containing the high-affinity Ca2+/Mg(2+)-binding site in the RLC was replaced with that containing the lower-affinity, Ca(2+)-specific, regulatory site from troponin C (TnC). In two of these hybrids, we replaced only the Ca(2+)-binding EF hand, while in the other two the EF hand and the N-terminal helix of TnC were transplanted. These hybrids were expressed in Escherichia coli in high yields and the purified proteins were used in calcium-binding experiments to assay the affinity and specificity of the sites and incorporated into scallop myosin to assay their regulatory behaviour. The results obtained show that the calcium-binding site from TnC, when transplanted into the RLC backbone, had a low affinity although most of its specificity appeared to be retained. As a result, although the TnC/RLC hybrids bound to scallop myosin and were able to activate the MgATPase activity of scallop acto-myosin, they were unable to regulate it. These results are in agreement with our previous findings that occupancy of the Ca2+/Mg2+ site in the RLC is essential for regulation. Our results suggest that the specificity and affinity of the calcium-binding site in troponin C is dependent on both intra- and inter-domain interactions within troponin C and that these latter interactions appear to be missing when this binding site is transplanted into the light chain backbone.