Acid-induced dimerization of skeletal troponin C

We have investigated pH-dependent changes of the properties of troponin C from rabbit skeletal muscle. At pH 7.5 this protein is a monomer and at pH 5.2 it is a dimer. In contrast, bovine cardiac troponin C remains essentially monomeric at pH 5.2. Bovine brain calmodulin is not a dimer, but significantly aggregated at the same acidic pH. The dimerization of skeletal troponin C was demonstrated by low-speed (16,000 rpm) sedimentation equilibrium measurements carried out at 20 degrees C and by polyacrylamide gel electrophoresis under nondenaturing conditions. Dimer formation was significantly inhibited in the ultracentrifuge at rotor speeds of 30,000 and 40,000 rpm at 20 degrees C, and was completely prevented at a rotor speed of 40,000 rpm and 4 degrees C. This temperature and pressure dependence of dimerization strongly suggests that hydrophobic bonding is a major factor in promoting skeletal troponin C association at pH 5.2. The intramolecular distance between Met-25 and Cys-98 of rabbit skeletal troponin C deduced from fluorescence resonance energy transfer measurements increased by a factor of two upon lowering the pH from 7.5 to 5.2, indicating a pH-dependent transition in which the protein changed from a relatively compact conformation to an elongated conformation. The proton-induced increase in the energy transfer distance is related to the acid-induced dimerization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-dependent structural transition in rabbit skeletal troponin C

Although the crystal structure of troponin C is known (Herzberg, O., and James, M. N. G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S. T., Roychowdhury, P., Greaser, M., and Wang, B. C. (1985) Science 227, 945-948), its structure in solution, particularly under physiological conditions, has not been established. We examined the conformation of troponin C under a variety of conditions by measuring the distance between sites located in the N- and C-terminal domains using the technique of resonance energy transfer. The donor was the luminescent lanthanide ion Tb3+ bound at the low affinity metal sites in the N-terminal domain. The acceptor was 4-dimethylaminophenylazophenyl-4'-maleimide attached at Cys-98 in the C-terminal domain. The distance between these sites was found to be greater than 5.2 nm at pH 5.0, 2.7 nm at pH 6.8 for uncomplexed troponin C, and 4.1 nm for troponin C complexed with troponin I at pH 6.8. These findings suggest that uncomplexed troponin C undergoes a pH-dependent transition from an elongated conformation, compatible with the crystal structure at acidic pH, to a more compact conformation at neutral pH. When complexed with troponin I, troponin C adopts a conformation of intermediate length compared to the uncomplexed molecule at pH 6.8 and 5.0.     

An interdomain distance in cardiac troponin C determined by fluorescence spectroscopy

The distance between Ca2+-binding site III in the C-terminal domain and Cys35 in the N-terminal domain in cardiac muscle troponin C (cTnC) was determined with a single-tryptophan mutant using bound Tb3+ as the energy donor and iodoacetamidotetramethylrhodamine linked to the cysteine residue as energy acceptor. The luminescence of bound Tb3+ was generated through sensitization by the tryptophan located in the 12-residue binding loop of site III upon irradiation at 295 nm, and this sensitized luminescence was the donor signal transferred to the acceptor. In the absence of bound cation at site II, the mean interdomain distance was found to be 48-49 A regardless of whether the cTnC was unbound or bound to cardiac troponin I, or reconstituted into cardiac troponin. These results suggest that cTnC retains its overall length in the presence of bound target proteins. The distribution of the distances was wide (half-width >9 A) and suggests considerable interdomain flexibility in isolated cTnC, but the distributions became narrower for cTnC in the complexes with the other subunits. In the presence of bound cation at the regulatory site II, the interdomain distance was shortened by 6 A for cTnC, but without an effect on the half-width. The decrease in the mean distance was much smaller or negligible when cTnC was complexed with cTnI or cTnI and cTnT under the same conditions. Although free cTnC has considerable interdomain flexibility, this dynamics is slightly reduced in troponin. These results indicate that the transition from the relaxed state to an activated state in cardiac muscle is not accompanied by a gross alteration of the cTnC conformation in cardiac troponin.     

Ca2+ dependence of the distance between Cys-98 of troponin C and Cys-133 of troponin I in the ternary troponin complex. Resonance energy transfer measurements

We have used resonance energy transfer to study the spatial relationship between Cys-98 of rabbit skeletal troponin C and Cys-133 of rabbit skeletal troponin I in the reconstituted ternary troponin complex. The donor was introduced by labeling either troponin C or troponin I with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, while the acceptor was introduced by labeling either protein with N-[4-(dimethylamino)phenyl-4'-azophenyl]maleimide. The extent of energy transfer was determined by measuring the quenching of the donor fluorescence decay. The results indicate first that the distance between these two sites is not fixed, suggesting that the protein regions involved possess considerable segmental flexibility. Second, the mean distance between the two sites is dependent on the metal-binding state of troponin C, being 39.1 A when none of the metal-binding sites are occupied, 41.0 A when Mg2+ ions bind at the high-affinity sites, and 35.5 A when Ca2+ ions bind to the low-affinity sites. Neither the magnitude of the distances nor the trend of change with metal ions differs greatly when the locations of the probes are switched or when steady-state fluorometry was used to determine the transfer efficiency. Since the low-affinity sites have been implicated as the physiological triggering sites, our findings suggest that one of the key events in Ca2+ activation of skeletal muscle contraction is a approximately 5-A decrease in the distance between the Cys-98 region of troponin C and the Cys-133 region of troponin I.     

A model for the Ca2+-induced conformational transition of troponin C. A trigger for muscle contraction

The initial contractile event in muscle is the binding of Ca2+ ions to troponin C of the troponin complex, leading to a series of conformational changes in the members of the thin and thick filaments. Knowledge of the crystal structure of turkey skeletal muscle troponin C has provided a structural basis for the modeling of the first stage of this process in atomic detail. This crystal structure probably represents the molecule in the relaxed state of muscle, with two of the maximum of 4 Ca2+ ions bound. The basis for the model presented here is that upon binding of the additional two Ca2+ ions, the regulatory domain of the molecule undergoes a conformational transition to become closely similar in structure to the domain which always binds Ca2+ or Mg2+ under physiological conditions. The root mean square discrepancy in atomic coordinates between the apo and the modeled Ca2+-bound states of the regulatory domain is 4.8 A, with some shifts as large as 10-15 A in the region near the linker between the two Ca2+ binding sites. It is demonstrated that this Ca2+-bound conformation of the regulatory domain conforms to accepted protein structure rules and that the change in conformation can be accomplished without encountering any barriers too high to be surmounted on the physiological time scale.     

Nanosecond study of fluorescently labeled troponin C.

The time-resolved extrinsic fluorescence of rabbit skeletal troponin C was studied with the protein labeled at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. Both the intensity and anisotropy decays followed a biexponential decay law, regardless of the ionic condition, pH, viscosity or temperature. The lifetimes and their fractional amplitudes were insensitive to Mg2+, and the lifetimes were also insensitive to Ca2+. In response to Ca2+ binding to all four sites, the fractional amplitude (alpha 1) associated with the short lifetime (tau 1) decreased by a factor of two, thus increasing the ratio of the two amplitudes alpha 2/alpha 1 from 1.6 to 4.3. These amplitude changes suggest the existence of two conformational states of TnC-IAEDANS, with the conformation associated with the long-decay component (tau 2) being promoted by saturation of the two Ca(2+)-specific sites. At pH 5.2 the ratio alpha 2/alpha 1 for the apo-protein was 3.5 indicating different relative populations of the two decay components when compared with pH 7.2. In the presence of Ca2+ at the lower pH, alpha 2/alpha 1 decreased to 2.1, suggesting a shift of the conformations in favor of the short-decay component. Thus Ca2+ elicited different conformational changes in TnC at the two pH values. The recovered anisotropies suggest that there were fast molecular motions that were not resolved in the present experiments, and some of these motions were sensitive to Ca2+ binding to the specific sites. These results support the notion of communication between the N-domain and the C-terminal end of the central helix of troponin C.     

Solution structure of a polypeptide dimer comprising the fourth Ca(2+)-binding site of troponin C by nuclear magnetic resonance spectroscopy

The structure of a 39 amino acid proteolytic fragment of rabbit skeletal troponin C containing the fourth Ca(2+)-binding site has been determined by an approach involving nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Hydrodynamic and NMR evidence establishes unambiguously that the fragment forms a stable dimer in solution in the presence of excess Ca2+. The calculation of the dimeric structure is based on a total of 1056 experimental restraints comprising 422 interproton distances, 35 phi, 28 psi, and 28 chi 1 torsion angle restraints within each subunit, 30 intermonomer distance restraints, and 6 Ca2+ restraints per subunit. A total of 48 final structures were calculated having an rms deviation about the mean atomic backbone coordinate positions of 1.0 A for residues Asp128-Glu156. The solution structure consists of a dimer of helix-loop-helix motifs related by a 2-fold axis of symmetry. The overall architecture of the dimer is very similar to the C-terminal domain in the crystal structure of chicken skeletal troponin C.     

Calcium binding to troponin C and troponin: effects of Mg2+, ionic strength and pH

Calcium binding to troponin C and troponin was examined by a metallochromic indicator method under various conditions to obtain a further understanding of the regulatory roles of these proteins in muscle contraction. Troponin C has four Ca binding sites, of which 2 sites have a high affinity of 4.5 X 10(6) M-1 for Ca2+ and the other 2 sites have a low affinity of 6.4 X 10(4) M-1 in a reaction medium consisting of 100 mM KCl, 20 mM MOPS-KOH pH 6.80 and 0.13 mM tetramethylmurexide at 20 degrees C. Magnesium also binds competitively to both the high and low affinity sites: the apparent binding constants are 1,000 M-1 and 520 M-1, respectively. Contrary to the claim by Potter and Gergely (J. Biol. Chem. 250, 4628-4633, 1975), the low affinity sites are not specific only for Ca2+. The high and low affinity sites of troponin C showed different dependence on the ionic strength: the high affinity sites were similar to GEDTA, while the low affinity sites were similar to calmodulin, which has a steeper ionic strength dependence than GEDTA. Ca binding to troponin C was not affected by change of pH between 6.5 and 7.2. Troponin I enhanced the apparent affinity of troponin C for Ca2+ to a value similar to that for troponin. Trifluoperazine also increased Ca binding to troponin C. Troponin has four Ca binding sites as does troponin C, but the affinities are so high that the precise analysis was difficult by this method. The apparent binding constants for Ca2+ and Mg2+ were determined to be 3.5 X 10(6) M-1 and 440 M-1, respectively, for low affinity sites under the same conditions as for troponin C, being independent of change in pH between 6.5 and 7.2. The competitive binding of Mg2+ to the low affinity sites of troponin is consistent with the results of Kohama (J. Biochem. 88, 591-599, 1980). The estimate for the high affinity sites is compatible with the reported results.     

Energetics of the binding of calcium and troponin I to troponin C from rabbit skeletal muscle.

We determined the free energy of interaction between rabbit skeletal troponin I (TNI) and troponin C (TNC) at 10 degrees and 20 degrees C with fluorescently labeled proteins. The sulfhydryl probe 5-iodoacetamidoeosin (IAE) was attached to cysteine (Cys)-98 of TNC and to Cys-133 of TNI, and each of the labeled proteins was titrated with the other unlabeled protein. The association constant for formation of the complex between labeled TNC (TNC*) and TNI was 6.67 X 10(5) M-1 in 0.3 M KCl, and pH 7.5 at 20 degrees C. In the presence of bound Mg2+, the binding constant increased to 4.58 X 10(7) M-1 and in the presence of excess of Ca2+, the association constant was 5.58 X 10(9) M-1. Very similar association constants were obtained when labeled TNI was titrated with unlabeled TNC. The energetics of Ca2+ binding to TNC* and the complex TNI X TNC* were also determined at 20 degrees C. The two sets of results were used to separately determine the coupling free energy for binding TNI and Mg2+, or Ca2+ to TNC. The results yielded a total coupling free energy of -5.4 kcal. This free energy appeared evenly partitioned into the two species: TNI X TNC(Mg)2 or TNI X TNC(Ca)2, and TNI X TNC(Ca)4. The first two species were each stabilized by -2.6 kcal, with respect to the Ca2+ free TNI X TNC complex, and TNI X TNC(Ca)4 was stabilized by -2.8 kcal, respect to TNI X TNC(Ca)2 or TNI X TNC(Mg)2. The coupling free energy was shown to produce cooperatively complexes formed between TNI and TNC in which the high affinity sites were initially saturated as a function of free Ca2+ to yield TNI X TNC(Ca)4. This saturation occurred in the free Ca2+ concentration range 10(-7) to 10(-5) M. The cooperative strengthening of the linkage between TNI and TNC induced by Ca2+ binding to the Ca2+-specific sites of TNC may have a direct relationship to activation of actomyosin ATPase. The nature of the forces involved in the Ca2+-induced strengthening of the complex is discussed.     

Structural change of troponin C molecule and its domains upon Ca2+ binding in the presence of Mg2+ ions measured by a solution X-ray scattering technique

A small-angle X-ray scattering study on troponin C showed that two domains of the molecule move closer to each other and the molecule shrinks along its long axis upon Ca2+ binding in the absence of Mg2+ ions (Fujisawa, T., Ueki, T., & Iida S. (1988) J. Biochem. 105, 377-383). When Mg2+ ions bind to troponin-C, the radius of gyration changes from 27.8 to 24.3 A and the average radius of gyration of the two domains is estimated to be 15.1 A. These radii indicate that the distance between the centers of the two domains is 38.1 A. Such a change is analogous to the previous result for troponin C with two Ca2+ ions bound at the high-affinity sites. Thus, the structural behavior of troponin C molecule is essentially the same when Ca2+/Mg2+ ions bind to its high-affinity sites. On the other hand, the effect of Ca2+ binding to the low-affinity sites in the presence of Mg2+ ions is quite different from the previous result. The binding of Ca2+ ions causes a dimerization of troponin C molecules with an apparent constant of 511 M-1. Such a characteristic behavior, implying the occurrence of a surface property change, may be related to the physiological role of troponin C molecule in the muscle. The scattering experiments on the tryptic fragments of troponin C also had interesting and important results: the C-domain shrinks, with the radius of gyration changing from 17.0 to 14.9 A while the N-domain swells from 13.9 to 15.0 A upon Ca2+ binding. Such an opposite change is consistent with the results of circular dichroism and spectroscopic studies of the domains.     

Small-angle x-ray scattering investigation of the solution structure of troponin C

X-ray crystallographic studies of troponin C (Herzberg, O., and James, M.N.G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S.T., and Roychowdhury, P. (1985a) Science 227, 945-948) have revealed a novel protein structure consisting of two globular domains, each containing two Ca2+-binding sites, connected via a nine-turn alpha-helix, three turns of which are fully exposed to solvent. Since the crystals were grown at pH approximately 5, it is of interest to determine whether this structure is applicable to the protein in solution under physiological conditions. We have used small-angle x-ray scattering to examine the solution structure of troponin C at pH 6.8 and the effect of Ca2+ on the structure. The scattering data are consistent with an elongated structure in solution with a radius of gyration of approximately 23.0 A, which is quite comparable to that computed for the crystal structure. The experimental scattering profile and the scattering profile computed from the crystal structure coordinates do, however, exhibit differences at the 40-A level. A weak Ca2+-facilitated dimerization of troponin C was observed. The data rule out large Ca2+-induced structural changes, indicating rather that the molecule with Ca2+ bound is only slightly more compact than the Ca2+-free molecule.     

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.     

Mg2+-paracrystal formation of tropomyosin as a condensation phenomenon. Effects of pH, salt, temperature, and troponin binding.

Tropomyosin (Tm) paracrystal formation induced by Mg2+ was studied by monitoring increases in light scattering. Paracrystals formed above a critical Tm concentration with lag phases in the time courses at pH 7.5 and 6.0, indicating that condensation polymerization processes are involved. The kinetic data at pH 7.5 reasonably fit a model in which nucleation and elongation are taken into account. The rate and extent of light scattering increased at low [Mg2+] and decreased at high [Mg2+] with a maximum at [Mg2+] = 15 mM, indicating different effects of Mg2+ in the two [Mg2+] ranges. The paracrystals were destabilized by increasing the salt concentration and decreasing the temperature. Mg2+ produces paracrystals at pH 6.0 and pH 7.5 by different kinetic mechanisms. Different Tm intermolecular interactions at the two pH values were indicated by studies of the excimer fluorescence of pyrene-labeled Tm and by effects of salt and temperature on the kinetics. At pH 6.0 Tm more readily formed paracrystals with decreased electrostatic effects. Effects of troponin on Mg2+-paracrystal formation of Tm at the two pH values correlated with the known differences in paracrystal structure when troponin is bound to Tm.     

Isolation and characterization of a highly phosphorylated troponin from bovine heart

A modified procedure for isolation of troponin from bovine heart is described, which results in a stable and highly phosphorylated protein. 31P-NMR spectra show up to four phosphoserine signals indicating that at least four serine residues of cardiac troponin are phosphorylated in the intact organ. The hydrodynamic parameters of phosphotroponin are almost identical to those previously published. Characteristically cardiac troponin shows a strong tendency to associate that is dependent on protein concentration. Mg2+ may specifically induce an aggregation, which can be observed during sedimentation. This phenomenon seems to be analogous to the Mg2+-induced dimerization of cardiac troponin C [Jaquet, K. and Heilmeyer, L. M. G., Jr (1987) Biochem. Biophys. Res. Commun. 145, 1390-1396]. Upon Mg2+ saturation a shift of one of the four 31P-NMR signals is observed. The affinity of troponin to Ca2+ is reduced when the protein concentration is enhanced only in the presence of Mg2+. This effect of Mg2+ suggests a model for the regulation of the Ca2+-binding affinity of cardiac troponin.     

Ca2+-induced conformational changes in cardiac troponin C as measured by N-(1-pyrene)maleimide fluorescence

Bovine cardiac troponin C was modified by N-(1-pyrene)maleimide at Cys-35 and Cys-84; the Ca2+-induced conformational changes were followed by measuring pyrene fluorescence. In isolated troponin C, the saturation of Ca2+, Mg2+-sites leads to a simultaneous increase in the pyrene monomer as well as to a decrease in the pyrene excimer fluorescence, whereas the saturation of Ca2+-specific sites results in a slight decrease in the fluorescence of pyrene monomer. Troponin T does not influence the dependence of pyrene-troponin C fluorescence on Ca2+ concentration. Within the equimolar complex of troponin C and troponin I, the saturation of Ca2+, Mg2+-sites has no effect on pyrene fluorescence, whereas the saturation of Ca2+-specific sites leads to a simultaneous decrease of both pyrene monomer and pyrene excimer fluorescence. It is supposed that troponin I diminishes the conformational changes in troponin C that are induced by the saturation of Ca2+, Mg2+-sites and enhances the conformational changes induced by the saturation of Ca2+-specific sites of troponin C.     

Calmodulin and troponin C structures studied by Fourier transform infrared spectroscopy: effects of Ca2+ and Mg2+ binding

Fourier transform infrared (FTIR) spectroscopy has been used to examine the conformationally sensitive amide I' bands of calmodulin and troponin C. These are observed to undergo a sequence of spectroscopic changes which reflect conformational rearrangements that take place when Ca2+ is bound. Calmodulin and troponin C show similar though not identical changes on Ca2+ binding, and the effect of Mg2+ on troponin C is quite different from that of Ca2+. Both proteins show absorption maxima in the amide I' region at 1644 cm-1 which is significantly lower in frequency than has been generally observed for proteins that contain a high percentage of alpha-helix. It is proposed that an unusually high proportion of the helices in the structures of these proteins are distorted from the normal alpha-helical configuration such that the carbonyl stretching frequencies are lowered. It is further proposed that the shift to lower frequency is due to backbone carbonyl groups in the distorted helices that form strong hydrogen bonds with solvent molecules. A decrease in intensity at 1654 cm-1, the normal frequency assignment for alpha-helical structure, is observed as Ca2+ binds to calmodulin and troponin C. This suggests that Ca2+ binding results in a net decrease in "normal" alpha-helix conformation. There is a corresponding increase in intensity of the band at 1644 cm-1, possibly due to an increase in distorted helix content, allowing for a net increase in helix consistent with circular dichroism estimates of the Ca2+-dependent changes in helix content in calmodulin.     

Ca(2+) induces an extended conformation of the inhibitory region of troponin I in cardiac muscle troponin.

The inhibitory region of troponin I (TnI) plays a central regulatory role in the contraction and relaxation cycle of skeletal and cardiac muscle through its Ca(2+)-dependent interaction with actin. Detailed structural information on the interface between TnC and this region of TnI has been long in dispute. We have used fluorescence resonance energy transfer (FRET) to investigate the global conformation of the inhibitory region of a full-length TnI mutant from cardiac muscle (cTnI) in the unbound state and in reconstituted complexes with the other cardiac troponin subunits. The mutant contained a single tryptophan residue at the position 129 which was used as an energy transfer donor, and a single cysteine residue at the position 152 labeled with IAEDANS as energy acceptor. The sequence between Trp129 and Cys152 in cTnI brackets the inhibitory region (residues 130-149), and the distance between the two sites was found to be 19.4 A in free cTnI. This distance was insensitive to reconstitution of cTnI with cardiac troponin T (cTnT), cTnC, or cTnC and cTnT in the absence of bound regulatory Ca(2+) in cTnC. An increase of 9 A in the Trp129-Cys152 separation was observed upon saturation of the Ca(2+) regulatory site of cTnC in the complexes. This large increase suggests an extended conformation of the inhibitory region in the interface between cTnC and cTnI in holo cardiac troponin. This extended conformation is different from a recent model of the Ca(2+)-saturated skeletal TnI-TnC complex in which the inhibitory region is modeled as a beta-turn. The observed Ca(2+)-induced conformational change may be a switch mechanism by which movement of the regulatory region of cTnI to the exposed hydrophobic patch of the open regulatory N-domain of cTnC pulls the inhibitory region away from actin upon Ca(2+) activation in cardiac muscle.     

Introduction of negative charge mimicking protein kinase C phosphorylation of cardiac troponin I. Effects on cardiac troponin C

Protein kinase C phosphorylation of cardiac troponin, the Ca(2+)-sensing switch in muscle contraction, is capable of modulating the response of cardiac muscle to a Ca(2+) ion concentration. The N-domain of cardiac troponin I contains two protein kinase C phosphorylation sites. Although the physiological consequences of phosphorylation at Ser(43)/Ser(45) are known, the molecular mechanisms responsible for these functional changes have yet to be established. In this work, NMR was used to identify conformational and dynamic changes in cardiac troponin C upon binding a phosphomimetic troponin I, having Ser(43)/Ser(45) mutated to Asp. Chemical shift perturbation mapping indicated that residues in helix G were most affected. Smaller chemical shift changes were observed in residues located in the Ca(2+)/Mg(2+)-binding loops. Amide hydrogen/deuterium exchange rates in the C-lobe of troponin C were compared in complexes containing either the wild-type or phosphomimetic N-domain of troponin I. In the presence of a phosphomimetic domain, exchange rates in helix G increased, whereas a decrease in exchange rates for residues mapping to Ca(2+)/Mg(2+)-binding loops III and IV was observed. Increased exchange rates are consistent with destabilization of the Thr(129)-Asp(132) helix capping box previously characterized in helix G. The perturbation of helix G and metal binding loops III and IV suggests that phosphorylation alters metal ion affinity and inter-subunit interactions. Our studies support a novel mechanism for protein kinase C signal transduction, emphasizing the importance of C-lobe Ca(2+)/Mg(2+)-dependent troponin interactions.     

Ca2+-induced conformational transition in the inhibitory and regulatory regions of cardiac troponin I

Cardiac muscle activation is initiated by the binding of Ca(2+) to the single N-domain regulatory site of cardiac muscle troponin C (cTnC). Ca(2+) binding causes structural changes between cTnC and two critical regions of cardiac muscle troponin I (cTnI): the regulatory region (cTnI-R, residues 150-165) and the inhibitory region (cTnI-I, residues130-149). These changes are associated with a decreased cTnI affinity for actin and a heightened affinity for cTnC. Using F?rster resonance energy transfer, we have measured three intra-cTnI distances in the deactivated (Mg(2+)-saturated) and Ca(2+)-activated (Ca(2+)-saturated) states in reconstituted binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT) troponin complexes. Distance A (spanning cTnI-R) was unaltered by Ca(2+). Distances B (spanning both cTnI-R and cTnI-I) and C (from a residue flanking cTnI-I to a residue in the center of cTnI-R) exhibited Ca(2+)-induced increases of >8 A. These results compliment our previous determination of the distance between residues flanking cTnI-I alone. Together, the data suggest that Ca(2+) activation causes residues within cTnI-I to switch from a beta-turn/coil to an extended quasi-alpha-helical conformation as the actin-contacts are broken, whereas cTnI-R remains alpha-helical in both Mg(2+)- and Ca(2+)-saturated states. We have used the data to construct a structural model of the cTnI inhibitory and regulatory regions in the Mg(2+)- and Ca(2+)-saturated states.     

pH-dependent conformational changes of wheat germ calmodulin

Fluorescence energy transfer measurements were carried out between landmarks on wheat germ calmodulin to measure the interdomain distance. Tb3+ ions bound at the four Ca2+-binding sites were used as energy donors, and an organic chromophore, [4-(dimethylamino)-phenyl-4'-azophenyl]maleimide, attached to the single cysteine residue at position 27, was used as the acceptor. At pH's near neutrality all bound Tb3+ ions emit luminescence with shortened lifetimes as a result of energy transfer to the acceptor; at pH 5, however, part of the metal emission becomes unquenched. When the protein is subjected to limited digestion with trypsin in the presence of Ca2+, resulting in the formation of two fragments, each corresponding to half of the molecule, the decay of Tb3+ emission is no longer pH sensitive. These results suggest that, like rabbit skeletal troponin C [Wang, C.-L. A., Zhan, Q., Tao, T., & Gergely, J. (1987) J. Biol. Chem. 257, 8372-8375], wheat germ calmodulin exists in a relatively compact conformation at neutral pH's, but becomes more elongated at pH 5.