Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR

We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca(2+)-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca(2+) state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time tau=1.9ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca(2+) state showed that Cys133 existed on a stable segment (tau=4.8ns) held by TnC. Spectra of reconstituted thin filaments (-Ca(2+) state) revealed that slow mobility (tau=45ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca(2+) concentrations.     

Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C

The muscle thin filament protein troponin (Tn) regulates contraction of vertebrate striated muscle by conferring Ca2+ sensitivity to the interaction of actin and myosin. Troponin C (TnC), the Ca2+ binding subunit of Tn contains two homologous domains and four divalent cation binding sites. Two structural sites in the C-terminal domain of TnC bind either Ca2+ or Mg2+, and two regulatory sites in the N-terminal domain are specific for Ca2+. Interactions between TnC and the inhibitory Tn subunit troponin I (TnI) are of central importance to the Ca2+ regulation of muscle contraction and have been intensively studied. Much remains to be learned, however, due mainly to the lack of a three-dimensional structure for TnI. In particular, the role of amino acid residues near the C-terminus of TnI is not well understood. In this report, we prepared a mutant TnC which contains a single Trp-26 residue in the N-terminal, regulatory domain. We used fluorescence lifetime and quenching measurements to monitor Ca2+- and Mg2+-dependent changes in the environment of Trp-26 in isolated TnC, as well as in binary complexes of TnC with a Trp-free mutant of TnI or a truncated form of this mutant, TnI(1-159), which lacked the C-terminal 22 amino acid residues of TnI. We found that full-length TnI and TnI(1-159) affected Trp-26 similarly when all four binding sites of TnC were occupied by Ca2+. When the regulatory Ca2+-binding sites in the N-terminal domain of TnC were vacant and the structural sites in the C-terminal domain of were occupied by Mg2+, we found significant differences between full-length TnI and TnI(1-159) in their effect on Trp-26. Our results provide the first indica- tion that the C-terminus of TnI may play an important role in the regulation of vertebrate striated muscle through Ca2+-dependent interactions with the regula- tory domain of TnC.     

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.     

Time-resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: conformational analysis of intermediates and transition-state complexes.

Single tryptophan-containing mutants of low adenylylation state Escherichia coli glutamine synthetase have been studied by frequency-domain fluorescence spectroscopy in the presence of various substrates and inhibitors. At pH 6.5, the Mn-bound wild-type enzyme (wild type has two tryptophans/subunit) and the mutant enzymes exhibit heterogeneous fluorescence decay kinetics; the individual tryptophans are adequately described by a triple exponential decay scheme. The recovered lifetime values are 5.9 ns, 2.6 ns, and 0.4 ns for Trp-57 and 5.8 ns, 2.3 ns, and 0.4 ns for Trp-158. These values are nearly identical to the previously reported results at pH 7.5 (Atkins, W.M., Stayton, P.S., & Villafranca, J.J., 1991, Biochemistry 30, 3406-3416). In addition, Trp-57 and Trp-158 both exhibit an ATP-induced increase in the relative fraction of the long lifetime component, whereas only Trp-57 is affected by this ligand at pH 7.5. The transition-state analogue L-methionine-(R,S)-sulfoximine (MSOX) causes a dramatic increase in the fractional intensity of the long lifetime component of Trp-158. This ligand has no effect on the W158S mutant protein and causes a small increase in the fractional intensity of the long lifetime component of the W158F mutant protein. Addition of glutamate to the ATP complex, which affords the gamma-glutamylphosphate-ADP complex, results in the presence of new lifetime components at 7, 3.2, and 0.5 ns for Trp-158, but has no effect on Trp-57. Similar results were obtained when ATP was added to the MSOX complex; Trp-57 exhibits heterogeneous fluorescence decay with lifetimes of 7, 3.5, and 0.8 ns. Decay kinetics of Trp-158 are best fit to a nearly homogeneous decay with a lifetime of 5.5 ns in the MSOX-ATP inactivated complex. These results provide a model for the sequence of structural and dynamic changes that take place at the Trp-57 loop and the central loop (Trp-158) during several intermediate stages of catalysis.     

Effect of Ca2+ binding on troponin C. Changes in spin label mobility, extrinsic fluorescence, and sulfhydryl reactivity.

The Ca2+ binding component (TnC) of troponin has been selectively labeled with either a spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, or with a fluorescent probe, S-mercuric-N-dansyl cysteine, presumably at its single cysteine residue (Cys-98) in order to probe the interactions of TnC with divalent metals and with other subunits of troponin. The modified protein has the same Ca2+ binding properties as native TnC (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628), viz. two Ca2+ binding sites at which Mg2+ appears to compete (Ca2+-Mg2+ sites, KCa = 2 X 10(7) M-1) and two sites at which Mg2+ does not compete (Ca2+-specific sites, KCa = 2 X 10(5) M-1). Either Ca2+ or Mg2+ alters the ESR spectrum of spin-labeled TnC in a manner that indicates a decrease in the mobility of the label, Ca2+ having a slightly greater effect. In systems containing both Ca2+ and Mg2+ the mobility of the spin label is identical with that in systems containing Ca2+ alone. The binding constants for Ca2+ and Mg2+ deduced from ESR spectral changes are 10(7) and 10(3) M-1, respectively, and the apparent affinity for Ca2+ decreases by about an order of magnitude on adding 2 mM Mg2+. Thus, the ESR spectral change is associated with binding of Ca2+ to one or both of the Ca2+-Mg2+ sites. Addition of Ca2+ to the binary complexes of spin-labeled TnC with either troponin T (TnT) or troponin I (TnI) produces greater reduction in the mobility of the spin label than in the case of spin-labeled TnC alone, and in the case of the complex with TnI the affinity for Ca2+ is increased by an order of magnitude. The fluorescence of dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-labeled TnC is enhanced by Ca2+ binding to both high and low affinity sites with apparent binding constants of 2.6 X 10(7) M-1 and 2.9 X 10(5) M-1, respectively, calculated from the transition midpoints. The presence of 2 mM Mg2+, which produces no effect on dansyl fluorescence itself, in contrast to its effect on the spin label, shifts the high affinity constant to 2 X 10(6) M-1. Spectral changes produced by Ca2+ binding to the TnC-TnI complex furnish evidence that the affinity of TnC for Ca2+ is increased in the complex. The reactivity of Cys-98 to the labels and to 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is decreased by Ca2+ or Mg2+ both with native TnC and in 6 M urea. The reaction rate between Cys-98 and Nbs2 decreases to one-half the maximal value at a Ca2+ concentration that suggests binding to the Ca2+-Mg2+ sites. Formation of a binary complex between TnI and TnC reduces the rate of reaction, and there is a further reduction by Ca2+. The effect of Ca2+ takes place at concentrations that are 1 order of magnitude lower than in the case of TnC alone. These results suggest that the Ca2+ binding site adjacent to Cys-98 is one of the Ca2+-Mg2+ binding sites.     

Kinetics of Ca2+ release shows interactions between the two classes of sites of troponin-C

The kinetics of Ca2+-release from the two high affinity sites of troponin-C (TnC) was studied by the stopped flow technique following rapid mixing with either EDTA or excess TbCl3. The rate constants obtained by the two methods were 2.8 and 0.7 s-1, respectively. For the tryptic fragment of TnC that contains only the COOH-terminal half of the molecule, both methods generate rate constants of 2.2 s-1. These results are consistent with the interpretation that binding of Tb3+ to the Ca2+-specific sites reduces the rate of dissociation of Ca2+ from, and thereby enhances the affinity for, the Ca2+-Mg2+ sites; this, in turn, suggests interactions between the two halves of the TnC molecule.     

Ca(2+)-dependence of structural changes in troponin-C in demembranated fibers of rabbit psoas muscle.

The Ca(2+)-dependence of structural changes in troponin-C (TnC) has been detected by monitoring the fluorescence from TnC labeled at Methionine-25, in the NH2-terminal domain, with danzylaziridine (TnC-DANZ) and then exchanged for endogenous TnC in glycerinated single fibers. The fluorescence-pCa relation obtained from fibers stretched to a sarcomere length greater than 4.0 microns evidenced two transitions: a small one, attributable to the binding of Ca2+ to the high affinity, Ca(2+)-Mg(2+)-binding sites of TnC; and a large one, attributable to the binding of Ca2+ to the low affinity, Ca(2+)-specific binding sites of TnC. In the fluorescence-pCa relation determined with fibers set to a sarcomere length of 2.4 microns, hence obtained in the presence of cycling cross-bridges, the large transition had the same Ca(2+)-dependence as did the development of tension. These results indicate that the NH2-terminal globular domain of TnC is modified by the binding of Ca2+ to sites located in both globular domains and that the structural changes in TnC resulting from the binding of Ca2+ to the low-affinity sites, but not to the high-affinity sites, are directly associated with the triggering of contraction.     

Stimulation of sodium-calcium exchange by cholesterol incorporation into isolated cardiac sarcolemmal vesicles

Modification of the cholesterol content of highly purified cardiac sarcolemma from dog ventricles was accomplished by incubation with phosphatidylcholine liposomes containing various amounts of cholesterol. The degree of cholesterol enrichment could be varied by changing the liposomal cholesterol/phospholipid ratio or varying the liposome-membrane incubation time. Na+-Ca2+ exchange measured in cholesterol-enriched sarcolemmal vesicles was increased up to 48% over control values. The stimulation of Na+-Ca2+ exchange was associated with an increased affinity of the exchanger for Ca2+ (Km = 17 microM compared with Km = 22 microM for control preparations). Na+-Ca2+ exchange measured in cholesterol-depleted membrane preparations was decreased by 15%. This depressed activity was associated with a decreased affinity of the exchanger for Ca2+ (Km = 27 microM). These changes were not due to either a change in membrane permeability to Ca2+ or an increase in the amount of Ca2+ bound to sarcolemmal vesicles. The stimulating effect of cholesterol enrichment was specific to the Na+-Ca2+ exchange process since sarcolemmal Ca2+-Mg2+ ATPase activity was depressed 40% by cholesterol enrichment. Further, K+-p-nitrophenylphosphatase and Na+-K+ ATPase activities were depressed in both cholesterol-depleted and cholesterol-enriched sarcolemmal vesicles. In situ oxidation of membrane cholesterol completely eliminated Na+-Ca2+ exchange. These results suggest that cholesterol is intimately associated with Na+-Ca2+ exchange and may interact with the exchange protein and modulate its activity.     

Conformational changes in subdomain 2 of G-actin: fluorescence probing by dansyl ethylenediamine attached to Gln-41.

Gln-41 on G-actin was specifically labeled with a fluorescent probe, dansyl ethylenediamine (DED), via transglutaminase reaction to explore the conformational changes in subdomain 2 of actin. Replacement of Ca2+ with Mg2+ and ATP with ADP on G-actin produced large changes in the emission properties of DED. These substitutions resulted in blue shifts in the wavelength of maximum emission and increases in DED fluorescence. Excitation of labeled actin at 295 nm revealed energy transfer from tryptophans to DED. Structure considerations and Cu2+ quenching experiments suggested that Trp-79 and/or Trp-86 serves as energy donors to DED. Energy transfer from these residues to DED on Gln-41 increased with the replacement of Ca2+ with Mg2+ and ATP with ADP. Polymerization of Mg-G-actin with MgCl2 resulted in much smaller changes in DED fluorescence than divalent cation substitution. This suggests that the conformation of loop 38-52 on actin is primed for the polymerization reaction by the substitution of Ca2+ with Mg2+ on G-actin.     

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.     

Time-resolved fluorescence studies of genetically engineered Escherichia coli glutamine synthetase. Effects of ATP on the tryptophan-57 loop.

Single-tryptophan-containing mutants of low adenylation state Escherichia coli glutamine synthetase (wild type has two tryptophans at positions 57 and 158) have been constructed and studied by multifrequency phase/modulation fluorescence spectroscopy. The W57L mutant (retains tryptophan at residue 158) and the W158S mutant (retains tryptophan at residue 57) are both characterized by heterogeneous exponential decay kinetics. Global analysis indicates that for the Mn-bound form of the enzyme at pH 7.4 the fluorescence of both tryptophans is best described by a sum of three discrete expontials with recovered lifetimes of 4.77, 1.72, and 0.10 ns for Trp-57 and 5.04, 2.28, and 0.13 ns for Trp-158. The wild-type enzyme also exhibits decay kinetics described by a triple-exponential model with similar lifetime components. The individual tryptophans are distinguishable by the fractional intensities of the resolvable lifetimes. The wild-type and W158S enzymes are dominated by the 5-ns component which provides nearly 60% and 65%, respectively, of the fractional intensity at five wavelengths spanning the emission spectrum. In contrast, the W57L enzyme demonstrates a larger fraction of the 2-ns lifetime species (60%) and only 35% of the longer lifetime component. The substrate ATP induces a shift to approximately 90% of the 5-ns component for the wild-type and W158S enzymes, whereas the W57L protein is essentially unaffected by this ligand. Steady-state quenching studies with iodide indicate that addition of ATP results in a 3.0-3.5-fold decrease in the apparent Stern-Volmer quenching constants for the wild-type and W158S enzymes. Phase/modulation experiments at several iodide concentrations indicate that the median, 2 ns, lifetime component is selectively quenched compared to the 5-ns lifetime component. These results suggest a model where ATP binding results in a shift in the equilibrium distribution of microconformational states populated by Trp-57. ATP shifts this equilibrium nearly completely to the states exhibiting the long-lifetime component which, based on quenching studies, is less solvent-accessible than the conformational states associated with the other lifetime components.     

Calorimetric studies on calcium and magnesium binding by troponin C from bullfrog skeletal muscle

Microcalorimetic titrations were carried out to measure the thermodynamic functions of bullfrog skeletal muscle troponin C (TnC) in the interaction with Ca2+ and Mg2+, at 25 degrees C and at pH 7.0. Enthalpy titration curves with Ca2+ were composed of three stages both in the presence and in the absence of Mg2+. The first (0-2 mol Ca2+/mol TnC) and the third (greater than 3 mol Ca2+/mol TnC) stages were exothermic and the second stage (2-3 mol Ca2+/mol TnC) was endothermic. Mg2+ affected the first stage to decrease the amount of heat produced but not the second and third stages. The enthalpy titration with Mg2+, in the absence of Ca2+, was slightly exothermic initially and then became endothermic beyond 2-3 mol Mg2+/mol TnC. Absorption of heat was observed throughout the additions of Mg2+ in the presence of 1 mM Ca2+. The results indicate that bullfrog TnC has two high-affinity Ca2+-Mg2+ sites, two low-affinity Ca2(+)-specific sites, and two or around two Mg2(+)-specific sites. Based on the enthalpy and entropy changes, the Ca2+ binding reactions of TnC were classified into three types, indicating thermodynamic variety in the binding sites of the molecule.     

Static and kinetic studies on rabbit skeletal muscle troponin

Fluorescence titration curves of 2-[4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid-labeled troponin (IAANS-labeled Tn) and troponin-1-anilinonaphthalene-8-sulfonic acid (Tn-ANS) complex indicated that the fluorescent moiety, IAANS or ANS, detects conformational change of troponin I (TnI) or Tn due to the Ca2+ binding or removal reaction with the low affinity Ca2+-binding sites of troponin C (TnC) component. A fluorescence stopped-flow study showed that the kinetic behavior of IAANS-labeled Tn reflects a change in state of the TnI component induced by the Ca2+ binding or removal reaction with the low affinity Ca2+-binding sites of TnC component. The state change of TnI induced by the Ca2+ binding was complete within the instrumental dead time. On the other hand, that induced by the Ca2+ removal had a rate constant of around 13 s-1. ANS, which is noncovalently bound to Tn, reflects the kinetic properties of both the TnI component and the low affinity Ca2+-binding region of TnC component. The fluorescence intensity change of ANS induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC was complete within the instrumental dead time, while that induced by the Ca2+ removal from the same sites was biphasic. The rate constants of the biphasic process were found to be 62 +/- 7 s-1 and 16 +/- 4 s-1. The former value corresponds to the rate constant of the Ca2+ removal reaction from the low affinity Ca2+-binding sites of TnC component, and the latter value to the rate constant observed in the case of IAANS-labeled Tn. Based on these experimental results and on the discussion in our previous paper (Iio, T. & Kondo, H. (1981) J. Biochem. 90, 163-175), we have refined the two-way information-transfer mechanism which we previously proposed in order to explain the biological function of Tn.     

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

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

Co-operative activation of skeletal muscle thin filaments by rigor crossbridges. The effect of troponin C extraction

When Ca2+ binds to troponin C (TnC), all 26 troponin-tropomyosin (Tn-Tm) complexes of a regulatory strand change in concert from the inactive to the active configuration. To see if the complexes respond similarly when they are activated by rigor crossbridges in the absence of Ca2+, we determined the slope (ns) of the bell-shaped pS/tension (pS = -log [MgATP], where S = MgATP2-) relationship between pS 5, where the tension is maximal, and pS 2.3, where fibers are fully relaxed. In control skinned rabbit psoas fibers the ns value is greater than 4; it progressively decreases with TnC extraction. This decrease in ns with TnC extraction is analogous to the decrease in the slope (Hill coefficient) of the pCa/tension (pCa = -log [Ca2+]) relationship with extraction. Complete TnC extraction reduces the maximum substrate-induced tension by only 25%; in contrast, it reduces the maximum Ca2+ induced tension to zero. The effects of TnC extraction on the slope of the pS/tension curve are explained by the assumptions that (1) extracted Tn-Tm complexes no longer change in concert with their neighbors but change independently of them, and (2) co-operative signals cannot cross extracted Tn-Tm complexes. The ns value, therefore, like the nH, is a direct function of the number of contiguous, intact, Tn-Tm complexes in a stretch of a regulatory strand. To describe qualitatively the bi-phasic pS/tension relationship, the mono-phasic pCa/tension relationship, and the effects of TnC extraction on them, we introduce a version of the concerted-transition formalism which includes two activating ligands, Ca2+ and rigor crossbridges.     

Salt-induced conformational changes in skeletal myosin light chains, troponin-C, and parvalbumin

Evidence for salt-induced changes in myosin light chains [dissociated by treatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)], troponin-C (TnC), and parvalbumin was obtained from chymotryptic digestion, circular dichroism, fluorescence, and difference absorption studies. High salt (0.6 M NaCl) protects the DTNB light chain from proteolysis, increases its alpha-helical content, and quenches the tryptophan fluorescence. These effects are similar to the changes induced by Ca2+ but smaller in magnitude. TnC is affected by monovalent cations in a similar manner. Changes in the alpha-helical content resemble the effect of Ca2+. The enhancement of tyrosine fluorescence reflects conformational changes in the Ca2+-Mg2+ binding sites. The increase in the fluorescence of dansylaziridine-labeled TnC suggests perturbation of Ca2+-specific sites by salt. Cancellation of this effect by Mg2+ binding to the high-affinity sites is indicative of site-site interactions. In Whiting parvalbumin, salt-induced a perturbation of tryptophan absorption similar in nature to the Ca2+ effect.     

Kinetics of the conformational change of skeletal troponin C induced by Ca2+-Mg2+ exchange at the high-affinity Ca2+-binding sites

The rate constant of the conformational change of skeletal troponin C (TnC) induced by the Ca2+ binding reaction with the high-affinity Ca2+-binding sites was determined in the presence of Mg2+ by the fluorescence stopped-flow method in 0.1 M KCl, 50 mM Na-cacodylate-HCl pH 7.0 at 20 degrees C. The [MgCl2] dependence of the rate constants of the observed biphasic conformational change leveled off at the high [MgCl2] region: the rate constants were 60 +/- 9 s-1 and 8 +/- 2 s-1, respectively. These values are larger than the rate constants of the biphasic fluorescence intensity change of TnC induced by Mg2+ removal reaction at the high-affinity Ca2+-binding sites (37 +/- 7 s-1 and 3.0 +/- 0.6 s-1) under the same experimental conditions. These results suggest that the Ca2+-Mg2+ exchange reaction at the high-affinity Ca2+-binding sites is faster than the resultant conformational change accompanying the fluorescence intensity change. Based on these results, we also reexamine the molecular kinetic mechanism of the conformational change of the protein induced by the Mg2+ binding or removal reaction with the high affinity Ca2+-binding sites of skeletal TnC.     

Rapid charge translocation by the cardiac Na(+)-Ca2+ exchanger after a Ca2+ concentration jump.

The kinetics of Na(+)-Ca2+ exchange current after a cytoplasmic Ca2+ concentration jump (achieved by photolysis of DM-nitrophen) was measured in excised giant membrane patches from guinea pig or rat heart. Increasing the cytoplasmic Ca2+ concentration from 0.5 microM in the presence of 100 mM extracellular Na+ elicits an inward current that rises with a time constant tau 1 < 50 microseconds and decays to a plateau with a time constant tau 2 = 0.65 +/- 0.18 ms (n = 101) at 21 degrees C. These current signals are suppressed by Ni2+ and dichlorobenzamil. No stationary current, but a transient inward current that rises with tau 1 < 50 microseconds and decays with tau 2 = 0.28 +/- 0.06 ms (n = 53, T = 21 degrees C) is observed if the Ca2+ concentration jump is performed under conditions that promote Ca(2+)-Ca2+ exchange (i.e., no extracellular Na+, 5 mM extracellular Ca2+). The transient and stationary inward current is not observed in the absence of extracellular Ca2+ and Na+. The application of alpha-chymotrypsin reveals the influence of the cytoplasmic regulatory Ca2+ binding site on Ca(2+)-Ca2+ and forward Na(+)-Ca2+ exchange and shows that this site regulates both the transient and stationary current. The temperature dependence of the stationary current exhibits an activation energy of 70 kj/mol for temperatures between 21 degrees C and 38 degrees C, and 138 kj/mol between 10 degrees C and 21 degrees C. For the decay time constant an activation energy of 70 kj/mol is observed in the Na(+)-Ca2+ and the Ca(2+)-Ca2+ exchange mode between 13 degrees C and 35 degrees C. The data indicate that partial reactions of the Na(+)-Ca2+ exchanger associated with Ca2+ binding and translocation are very fast at 35 degrees C, with relaxation time constants of about 6700 s-1 in the forward Na(+)-Ca2+ exchange and about 12,500 s-1 in the Ca(2+)-Ca2+ exchange mode and that net negative charge is moved during Ca2+ translocation. According to model calculations, the turnover number, however, has to be at least 2-4 times smaller than the decay rate of the transient current, and Na+ inward translocation appears to be slower than Ca2+ outward movement.     

Calcium binding to the low affinity sites in troponin C induces conformational changes in the high affinity domain. A possible route of information transfer in activation of muscle contraction

Residues 89-100 of troponin C (C89-100) and 96-116 of troponin I (I96-116) interact with each other in the troponin complex (Dalgarno, D.C., Grand, R.J.A., Levine, B.A. Moir, A., J.G., Scott, G.M.M., and Perry, S.V. (1982) FEBS Lett. 150, 54-58) and are necessary for the Ca2+ sensitivity of actomyosin ATPase (Syska, H., Wilkinson, J.M., Grand, R.J.A., and Perry, S.V. (1976) Biochem. J. 153, 375-387 and Grabarek, Z., Drabikowski, W., Leavis, P.C., Rosenfeld, S.S., and Gergely, J. (1981) J. Biol. Chem. 256, 13121-13127). We have studied Ca2+-induced changes in the region C89-100 by monitoring the fluorescence of troponin C (TnC) labeled at Cys-98 with 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid. Equilibrium titration of the labeled TnC with Ca2+ indicates that the probe is sensitive to binding to both classes of sites in free TnC as well as in its complex with TnI. When Mg2 X TnC is mixed with Ca2+ in a stopped flow apparatus, there is a rapid fluorescence increase related to Ca2+ binding to the unoccupied sites I and II followed by a slower increase (k = 9.9 s-1) that represents Mg2+-Ca2+ exchange at sites III and IV. In the TnC X TnI complex, the fast phase is much larger and the Mg2+-Ca2+ exchange at sites III and IV results in a small decrease rather than an increase in the fluorescence of the probe. The possibility is discussed that the fast change in the environment of Cys-98 upon Ca2+ binding to sites I and II may be instrumental in triggering activation of the thin filament by facilitating a contact between C89-100 and I96-116.     

Quantification of the calcium-induced secondary structural changes in the regulatory domain of troponin-C.

The backbone resonance assignments have been completed for the apo (1H and 15N) and calcium-loaded (1H, 15N, and 13C) regulatory N-domain of chicken skeletal troponin-C (1-90), using multidimensional homonuclear and heteronuclear NMR spectroscopy. The chemical-shift information, along with detailed NOE analysis and 3JHNH alpha coupling constants, permitted the determination and quantification of the Ca(2+)-induced secondary structural change in the N-domain of TnC. For both structures, 5 helices and 2 short beta-strands were found, as was observed in the apo N-domain of the crystal structure of whole TnC (Herzberg O, James MNG, 1988, J Mol Biol 203:761-779). The NMR solution structure of the apo form is indistinguishable from the crystal structure, whereas some structural differences are evident when comparing the 2Ca2+ state solution structure with the apo one. The major conformational change observed is the straightening of helix-B upon Ca2+ binding. The possible importance and role of this conformational change is explored. Previous CD studies on the regulatory domain of TnC showed a significant Ca(2+)-induced increase in negative ellipticity, suggesting a significant increase in helical content upon Ca2+ binding. The present study shows that there is virtually no change in alpha-helical content associated with the transition from apo to the 2Ca2+ state of the N-domain of TnC. Therefore, the Ca(2+)-induced increase in ellipticity observed by CD does not relate to a change in helical content, but more likely to changes in spatial orientation of helices.