Changes In Troponin C Research Articles

Thermodynamics of Calcium Binding to Zebrafish Troponin C (TnC) Visualized through Isothermal Titration Calorimetry (ITC) and Molecular Dynamics (MD)

Interaction of calcium Ca2+ and troponin C (TnC) is a fundamental process governing regulation of cardiac contractility. Increased affinity of cTnC's regulatory site II for Ca2+ elevates sensitivity, leading to greater force production capabilities. Maintenance of cardiac function in the face of varying temperature is an important property of the heart and sequence based changes in TnC may alter the ability to modulate force with changing temperature. Two distinct genes express TnC in the teleost heart: cardiac (cTnC) and fish-specific slow skeletal (ssTnC). 18 of 161 amino acids differ between zebrafish paralogs.qPCR experiments in temperature-acclimated zebrafish at 28°C showed ssTnC mRNA levels to be two-fold greater than cTnC mRNA levels while at 18°C, cTnC expression was 1.5-fold higher in the atrium.Isothermal Titration Calorimetry (ITC) was used to study the heat change upon titration of Ca2+ with site II in the N-terminal domain of zebrafish TnC paralogs. The interaction of Ca2+ with TnC was an endothermic, entropically driven reaction. Our work suggests that ssTnC may have a higher Ca2+ affinity (Kd ∼11 and 14 vs. 13 and 17 μM in cTnC at 28 and 18 °C, respectively) at both temperatures indicating a more favorable Gibbs free energy of interaction.The effect of sequence variations between paralogs have been modeled through equilibrium Molecular Dynamics (MD) simulations. The resultant structures were similar, however a measurable difference was seen in the free energy of Ca2+ binding. ΔG of binding for the ssTnC is lower at 18°C and higher at 28°C than the cardiac paralog. Taken together, these results suggest that the increase in expression of the ssTnC at higher temperatures compensates for the decrease in the ability of the cTnC to bind calcium.

HD exchange and PLIMSTEX determine the affinities and order of binding of Ca2+ with troponin C.

Troponin C (TnC), present in all striated muscle, is the Ca(2+)-activated trigger that initiates myocyte contraction. The binding of Ca(2+) to TnC initiates a cascade of conformational changes involving the constituent proteins of the thin filament. The functional properties of TnC and its ability to bind Ca(2+) have significant regulatory influence on the contractile reaction of muscle. Changes in TnC may also correlate with cardiac and various other muscle-related diseases. We report here the implementation of the PLIMSTEX strategy (protein ligand interaction by mass spectrometry, titration, and H/D exchange) to elucidate the binding affinity of TnC with Ca(2+) and, more importantly, to determine the order of Ca(2+) binding of the four EF hands of the protein. The four equilibrium constants, K(1) = (5 ± 5) × 10(7) M(-1), K(2) = (1.8 ± 0.8) × 10(7) M(-1), K(3) = (4.2 ± 0.9) × 10(6) M(-1), and K(4) = (1.6 ± 0.6) × 10(6) M(-1), agree well with determinations by other methods and serve to increase our confidence in the PLIMSTEX approach. We determined the order of binding to the four EF hands to be III, IV, II, and I by extracting from the H/DX results the deuterium patterns for each EF hand for each state of the protein (apo through fully Ca(2+) bound). This approach, demonstrated for the first time, may be general for determining binding orders of metal ions and other ligands to proteins.

Mechano-chemical effects of Ca 2+ in cross-linked troponin-C films

Changes in troponin C (TnC) conformation upon Ca 2+ binding forms the basis for regulatory and structural functions of TnC molecules. In the present study, Ca 2+-induced conformational changes in TnC were observed by mechanical measurements. TnC films were prepared by drying or electrospraying TnC solutions, cross-linked with glutaraldehyde, and isometric tension and stiffness measured as a function of pCa. An increase in Ca 2+ from a pCa of 9 to 4 induced large-scale mechanical changes in the TnC films causing several percent shrinkage of the unloaded films. This shrinkage could be partially assigned to Ca 2+ binding to the Ca 2+/Mg 2+ sites of TnC.

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

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

The effect of regulatory Ca2+ on the in situ structures of troponin C and troponin I: a neutron scattering study

The effects of regulatory amounts of Ca2+ on the in situ structures of troponin C (TnC) and troponin I (TnI) in whole troponin have been investigated by neutron scattering. In separate difference experiments, 97% deuterated TnC and TnI within whole troponin were studied ±Ca2+ in 41.6% 2H2O buffers in which protonated subunits were rendered “invisible”. We found that the radius of gyration (Rg) of TnI decreased by ≈10% upon addition of regulatory Ca2+ indicating that it was significantly more compact in the presence of Ca2+. The apparent cross-sectional radius of gyration (Rc) of TnI increased by about 9% when regulatory Ca2+ was bound to TnC. Modeling studies showed that the high-Q scattering patterns of TnI could be fit by a TnI which consisted of two subdomains: one, a highly oblate ellipsoid of revolution containing about 65% of the mass and the other, a highly prolate ellipsoid of revolution consisting of about 35% of the mass. No other fits could be found with this class of models. Best fits were achieved when the axes of revolution of these ellipsoids were steeply inclined with respect to each other. Ca2+ addition decreased the center of mass separation by about 1.5 nm. The Rg of TnI, its high-Q scattering pattern, and the resultant structure were different from previous results on neutron scattering by TnI in the (+Ca2+) TnC·TnI complex.The Rg of TnC indicated that it was elongate in situ. The Rg of TnC was not sensitive to the Ca2+ occupancy of its regulatory sites. However, Rc increased upon Ca2+ addition in concert with expectations from NMR and crystallography of isolated TnC. The present observations indicate that TnI acts like a molecular switch which is controlled by smaller Ca2+-induced changes in TnC.

Troponin T and Ca2+ dependence of the distance between Cys48 and Cys133 of troponin I in the ternary troponin complex and reconstituted thin filaments.

Contraction of vertebrate striated muscle is regulated by the interaction of Ca2+ with the heterotrimeric protein troponin (Tn), composed of troponin-C (TnC), troponin-I (TnI), and troponin-T (TnT). Although much is known about the Ca2+-induced conformational changes in TnC, the Ca2+-binding subunit of Tn, little is known about how TnI, the inhibitory subunit, responds to the binding of Ca2+ to TnC. In this work, we used resonance energy transfer to measure the distance between probes attached at Cys48 and Cys133 in the N- and C-terminal domains, respectively, of TnI. A mutant rabbit skeletal TnI, TnI48/133 (C64S), was constructed by converting Cys64 into Ser. The remaining two thiols at Cys48 and Cys133 were labeled with the fluorescent donor 1,5-IAEDANS, and the nonfluorescent acceptor, DAB-Mal. We found an interprobe distance of approximately 41 A for both uncomplexed TnI and TnI in the binary complex with TnC. This distance increased to 51 A in the ternary Tn complex with TnT. These distances did not change significantly on binding of Ca2+ to TnC. In the reconstituted thin filament, this distance remained to be 50 A in the presence of saturating Ca2+, but increased to approximately 66 A on removing Ca2+ with EGTA in the presence of Mg2+. Our results indicate firstly that while TnC has only small effects on the global conformation of TnI, the presence of TnT in the ternary Tn complex gives rise to an apparent elongation of TnI. Secondly, whereas there is no detectable Ca2+-dependent change in the global conformation of TnI in the Tn complex free in solution, the removal of Ca2+ caused a substantial separation of the N- and C-terminal TnI regions in the reconstituted thin filament, owing to the interaction between the C-terminal region of TnI and actin in the relaxed state.

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.

Heat capacity and entropy changes of troponin C from bullfrog skeletal muscle induced by calcium binding

In order to elucidate the structural changes of bullfrog skeletal muscle troponin C (TnC) caused by Ca2+ binding, microcalorimetric titrations were performed in both the absence and presence of Mg2+, at pH 7.0, and at 5 degrees, 15 degrees, and 25 degrees C. The results indicate that, in the absence of Mg2+, Ca2+ binding to sites 1 and 2 gives rise to a large hydrophobic effect, a sequestering of nonpolar groups on the surface of molecule to the interior, and a tightening of the molecular structure as a whole. In contrast, hydrophobic groups were exposed from the interior to the surface of molecule and the molecular mobility was increased, upon Ca2+ binding to site 3. Ca2+ binding to site 4 induced much less change in the conformation of TnC molecule than that to the other sites. The presence of 5 mM Mg2+ dramatically reduced the magnitude of the conformational changes in TnC on Ca2+ binding to sites 1 and 2. On the other hand, the conformational changes by Ca2+ binding to sites 3 and 4 were not affected by Mg2+. Ca2+ binding to site 3 of TnC, thus, causes the conformational changes distinctly different from those upon Ca2+ binding to the other sites. The conformational changes upon Ca2+ binding to site 3 of TnC are also clearly distinct from the Ca2(+)-induced conformational changes of parvalbumins, but are similar to those of calmodulin. Site 3 of TnC may, therefore, be the regulatory site in muscle contraction, and the exposure or generation of hydrophobic groups on the surface of molecule as well as the increase in molecular mobility may be the common characteristics of the regulation by Ca2(+)-binding proteins.

Reciprocal coupling between troponin C and myosin crossbridge attachment.

The attachment of cycling myosin crossbridges to actin and the resultant muscle contraction are regulated in skeletal muscle by the binding of Ca2+ to the amino-terminal, regulatory sites of the troponin C (TnC) subunit of the thin filament protein troponin. Conversely, the attachment of crossbridges to actin has been shown to alter the affinity of TnC for Ca2+. In this study, fluorescently labeled TnC incorporated into reconstituted thin filaments was used to investigate the relationship between crossbridge attachment to actin and structural changes in the amino-terminal region of TnC. Fluorescence intensity changes were measured under the following conditions: saturating [Ca2+] in the absence of crossbridges, rigor crossbridge attachment in the presence and absence of Ca2+, and cycling crossbridge attachment. The percent of heavy meromyosin crossbridges associated with the thin filaments under these conditions was also determined. The results show that, in addition to the binding of Ca2+ to TnC, the attachment of both rigor and cycling crossbridges to actin alters the structure of TnC near the regulatory, Ca2+-specific sites of the molecule. A differential coupling between weakly versus strongly bound crossbridge states and TnC structure was detected, suggesting a possible differential regulation of these states by conformational changes in TnC. These findings illustrate a reciprocal coupling, via thin filament protein interactions, between structural changes in TnC and the attachment of myosin crossbridges to actin, such that each can influence the other, and indicate that TnC is not simply an on-off switch but may exist in a number of different conformations.

Troponin I enhances acidic pH-induced depression of Ca2+ binding to the regulatory sites in skeletal troponin C.

Inhibition of muscle force development by acidic pH is a well known phenomenon, yet the exact mechanism by which a decrease in pH inhibits the Ca2+-activated force in striated myofilaments remains poorly understood. Whether or not the deactivation by acidic pH involves direct competition between Ca2+ and protons for regulatory binding sites on fast skeletal troponin C (TnC) or whether other proteins in thin filament regulation are important remains unclear. We measured the effects of acidic pH on Ca2+-dependent fluorescent changes in TnC labeled with the probe danzylaziridine (Danz), which reports Ca2+ binding to the regulatory (Ca2+-specific) sites. Measurements were also made with TnCDanz complexed with the inhibitory Tn unit, TnI, and in the whole Tn complex. Our results show that a drop in pH from 7.0 to 6.5 is associated with a 1.6-fold increase in the midpoint for the relation between free Ca2+ and Ca2+ binding to the regulatory sites on TnCDanz. However, when TnCDanz was present in its complex with either TnI alone or with TnI-TnT, the increase in midpoint free Ca2+ was increased by 3.5-fold. We tested whether this potentiation in the effect of acidic pH on Ca2+ binding to TnC is due to a pH-induced alteration in the binding of TnI to TnC. A decrease in pH from 7.0 to 6.5 was associated with a halving of the affinity of TnI for TnC. We also probed the effect of acidic pH on TnI. This was done (i) by measuring the intrinsic fluorescence of tryptophan residues in TnI alone and (ii) by measuring fluorescence of TnI (in the Tn complex) labeled at Cys-133 with 5-iodoacetamidofluorescein. A drop in pH from 7.0 to 6.5 was associated with a 15% decrease in intrinsic fluorescence and with a 30% decrease in the fluorescence of the 5-iodoacetamidofluorescein probe. We conclude, therefore, that while protons and Ca2+ may directly affect Ca2+ binding to regulatory sites on fast skeletal TnC, the effect of acidic pH on TnC Ca2+ binding is amplified in the TnI-TnC and Tn complexes by a pH-related effect on the affinity of TnI for TnC.