Structural Changes Of Troponin Research Articles

Structural changes in troponin during activation of skeletal and heart muscle determined in situ by polarised fluorescence

AbstractCalcium binding to troponin triggers the contraction of skeletal and heart muscle through structural changes in the thin filaments that allow myosin motors from the thick filaments to bind to actin and drive filament sliding. Here, we review studies in which those changes were determined in demembranated fibres of skeletal and heart muscle using fluorescence for in situ structure (FISS), which determines domain orientations using polarised fluorescence from bifunctional rhodamine attached to cysteine pairs in the target domain. We describe the changes in the orientations of the N-terminal lobe of troponin C (TnCN) and the troponin IT arm in skeletal and cardiac muscle cells associated with contraction and compare the orientations with those determined in isolated cardiac thin filaments by cryo-electron microscopy. We show that the orientations of the IT arm determined by the two approaches are essentially the same and that this region acts as an almost rigid scaffold for regulatory changes in the more mobile regions of troponin. However, the TnCN orientations determined by the two methods are clearly distinct in both low- and high-calcium conditions. We discuss the implications of these results for the role of TnCN in mediating the multiple signalling pathways acting through troponin in heart muscle cells and the general advantages and limitations of FISS and cryo-EM for determining protein domain orientations in cells and multiprotein complexes.

Effects of cardiomyopathy-linked mutations K15N and R21H in tropomyosin on thin-filament regulation and pointed-end dynamics.

Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used Förster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca2+-dependent regulation of cardiac thin filaments. We found that the K15N mutation desensitizes thin filaments to Ca2+ and slows the kinetics of structural changes in troponin induced by Ca2+ dissociation from troponin, while the R21H mutation has almost no effect on these parameters. Expression of the K15N mutant in cardiomyocytes decreases leiomodin’s thin-filament pointed-end assembly but does not affect tropomodulin’s assembly at the pointed end. Our in vitro assays show that the R21H mutation causes a twofold decrease in tropomyosin’s affinity for F-actin and affects leiomodin’s function. We suggest that the K15N mutation causes DCM by altering Ca2+-dependent thin-filament regulation and that one of the possible HCM-causing mechanisms by the R21H mutation is through alteration of leiomodin’s function.

Distinct contributions of the thin and thick filaments to length-dependent activation in heart muscle

The Frank-Starling relation is a fundamental auto-regulatory property of the heart that ensures the volume of blood ejected in each heartbeat is matched to the extent of venous filling. At the cellular level, heart muscle cells generate higher force when stretched, but despite intense efforts the underlying molecular mechanism remains unknown. We applied a fluorescence-based method, which reports structural changes separately in the thick and thin filaments of rat cardiac muscle, to elucidate that mechanism. The distinct structural changes of troponin C in the thin filaments and myosin regulatory light chain in the thick filaments allowed us to identify two aspects of the Frank-Starling relation. Our results show that the enhanced force observed when heart muscle cells are maximally activated by calcium is due to a change in thick filament structure, but the increase in calcium sensitivity at lower calcium levels is due to a change in thin filament structure.

A FRET-Based Assay for Monitoring Actions of Calcium Sensitizers on the Thin Filament

Cardiac troponin (cTn) is composed of troponins C, I, and T. cTnC is the Ca2+-dependent switch for cardiac muscle contraction. A decrease in calcium activation, and a subsequent decrease in cardiac contractility, has been linked to cardiac diseases such as familial hypertrophic cardiomyopathy. Calcium sensitizers targeting cTn are a promising pharmacological approach to stimulating contractility through increased sensitization or stabilization of Ca2+-induced changes. We developed an in vitro assay to measure the actions of Ca2+ sensitizers on cTn within reconstituted regulated actin (rAc) (cTn, tropomyosin, f-actin). The assay utilizes fluorescence resonance energy transfer (FRET) to monitor structural changes of troponin as a function of pCa. A donor and acceptor fluorophore were covalently attached to the mobile domain of cTnI and the C-lobe of cTnC, respectively. The fluorophore positions were selected due to the large observed Ca2+-induced distance change (1.8 nm), allowing for an assay highly sensitive to pCa fluctuations and Ca2+-sensitizing effects. Steady-state calcium titrations were performed to monitor fluctuations in Ca2+ affinity in the presence of two Ca2+ sensitizers known to bind to cTnC: Levosimendan and bepridil. Bepridil was shown to increase Ca2+ sensitivity, while Levosimendan was shown to have little effect on Ca2+ sensitivity. Additionally, bepridil appeared to prevent troponin from closing under Ca2+-saturating conditions, a structural effect that may give insight into the mechanism of Ca2+ sensitization of cTn. This assay is a fast approach to monitor the effects of Ca2+ sensitizers on cTn within the thin filament.

Structural dynamics of troponin during activation of skeletal muscle

Time-resolved changes in the conformation of troponin in the thin filaments of skeletal muscle were followed during activation in situ by photolysis of caged calcium using bifunctional fluorescent probes in the regulatory and the coiled-coil (IT arm) domains of troponin. Three sequential steps in the activation mechanism were identified. The fastest step (1,100 s(-1)) matches the rate of Ca(2+) binding to the regulatory domain but also dominates the motion of the IT arm. The second step (120 s(-1)) coincides with the azimuthal motion of tropomyosin around the thin filament. The third step (15 s(-1)) was shown by three independent approaches to track myosin head binding to the thin filament, but is absent in the regulatory head. The results lead to a four-state structural kinetic model that describes the molecular mechanism of muscle activation in the thin filament-myosin head complex under physiological conditions.

Cardiac Thin Filament Structural Modulation by Sarcomere Length

Myofilament Length Dependent Activation (LDA) forms the cellular basis of the Frank-Starling law observed on the heart. LDA has been studied intensively and appears to be modulated through various mechanisms, such as contractile proteins composition and their phosphorylation status. However, the cellular molecular mechanisms that underlie this phenomenon are still not well characterized.The aim of our study is to determine whether LDA is regulated through cTnC structural changes upon stretch. Accordingly, we used a single attached skinned cardiac myocyte in combination with confocal fluorescent measurement. Using this technique, we found that stretch of a relaxed cell, in the absence of Ca2+, resulted in marked alterations of cTnC structure as reported by cTnC-T53C-IAF confocal fluorescence. Moreover, titin mutant cells show a drastic alteration of both passive tension and myofilament sensitivity to calcium (pCa50) upon stretch. Consistent with this finding, by employing time-resolved x-ray diffraction of intact, electrically stimulated rat myocardium, we found marked changes in troponin and myosin structure upon stretch in the diastolic phase (i.e. when cross-bridges are not active). Moreover, we repeated these x-ray experiments using rat myocardium that expresses an unusually long titin molecule and found that, when compared to WT, diastolic stretch in these muscles did not induce the structural changes in troponin and myosin observed in WT. These results strongly suggest a direct effect of sarcomere length on thin and thick filament and implicate titin to be the molecule underling the length signal transduction for LDA.

The Ca2+-Induced Structural Changes in Troponin In-Situ and In-Vitro: A FRET Study in Permeabilized Cardiac Muscle Fibers and Reconstituted Thin Filaments

Ca2+ binding to N-domain of cardiac troponin C (N-cTnC) initiates a cascade of structural changes within the thin filament. Among these changes, Ca2+-induced structural changes in troponin play a critical role in the allosteric regulation of the force-generating acto-myosin interactions. Although previous in-vitro studies have provided insight into these structural changes, how they are modulated by geometric and mechanical constraints conferred by the sarcomeric lattice structure in muscle tissue remains unknown. Recently we have developed a technique to quantitatively measure these structural changes in chemically-skinned muscle fibers using time-resolved FRET. In this study, we use this technique to monitor the Ca2+-induced N-cTnC opening in skinned muscle fibers and compare the obtained results to in-vitro studies. FRET donor (AEDANS) and acceptor (DDPM) modified double-cysteine mutant cTnC(13C/51C)AEDANS-DDPM was reconstituted into skinned muscle fibers to examine the N-cTnC opening. To study the effects of crossbridge and sarcomere-length based activation on this structural change, simultaneous measurements of fluorescence intensity decays and isometric tension were performed. Our results show that at steady-state, the Ca2+-induced N-cTnC opening is proportional to muscle force development, but the level of opening varies depending on the presence or absence of strong crossbridges (XB). For instance, in the presence of sodium vanadate (inhibitor of XB) no Ca2+-induced force response was observed, but Ca2+-induced opening of N-cTnC in the fibers was evident but with a smaller magnitude. Furthermore, when compared to in-vitro measurements, results from muscle fibers suggest that N-cTnC may adopt a more compact structure in muscle fibers due to the constraints imposed by the myofilament lattice structure. The effects of the myofilament lattice structure on structural dynamics of N-cTnC are also discussed in terms of FRET distance distributions.

Cardiac Thin Filament Activation Modulation by Stretch

Myofilament Length Dependent Activation (LDA) forms the cellular basis of the Frank-Starling law observed on the heart. LDA has been studied intensively and appears to be modulated through various mechanisms, such as the composition of the contractile proteins and their phosphorylation status. However, the cellular molecular mechanisms that underlie this phenomenon are still not well characterized.The aim of our study is to determine whether LDA is regulated through cTnC structural changes upon stretch. Accordingly, we used a single attached skinned cardiac myocyte in combination with confocal fluorescent measurement. using this technique, we found that stretch of a relaxed cell, in the absence of Ca2+, resulted in marked alterations of cTnC structure as reported by cTnC-T53C-IAF confocal fluorescence. Moreover, titin mutant cells show a drastic alteration of both passive tension and myofilament sensitivity to calcium (pCa50) upon stretch. Consistent with this finding, by employing time-resolved x-ray diffraction of intact, electrically stimulated rat myocardium, we found marked changes in troponin and myosin structure upon stretch in the diastolic phase (i.e. when cross-bridges are not active). Moreover, we repeated these x-ray experiments using rat myocardium that expresses an unusually long titin molecule and found that, when compared to WT, diastolic stretch in these muscles did not cause structural changes in troponin and myosin structures. These results strongly implicate titin to be the molecule that transmits the length signal for LDA.

Monitoring the Structural Behavior of Troponin and Myoplasmic Free Ca 2+ Concentration during Twitch of Frog Skeletal Muscle

The interaction of troponin molecules on the thin filament with Ca 2+ plays a key role in regulating muscle contraction. To characterize the structural changes of troponin caused by Ca 2+ and crossbridge formation, we recorded the small-angle x-ray intensity and the myoplasmic free Ca 2+ concentration using fluo-3 AM in the same frog skeletal muscle during twitch elicited by a single electrical pulse at 16°C. In an overstretched muscle, the intensity of the meridional reflection from troponin at 1/38.5 nm −1 began to change at 4 ms after the stimulus, reached a peak at 10 ms, and returned to the resting level with a halftime of 25 ms. The concentration of troponin-bound Ca 2+ began to increase at 1–2 ms after the stimulus, reached a peak at 5 ms, and returned to the resting level with a halftime of 40 ms, indicating that troponin begins to change conformation only after a sizable amount of Ca 2+ has bound to it, and returns to the resting structure even when there is still some bound Ca 2+. In a muscle with a filament overlap, crossbridge formation appears to slow down Ca 2+ release from troponin and have a large effect on its conformation.

Structural changes in troponin in response to Ca 2+ and myosin binding to thin filaments during activation of skeletal muscle

Contraction of skeletal and cardiac muscle is regulated by Ca2+ -dependent structural changes in troponin that control the interaction between myosin and actin. We measured the orientations of troponin domains in skeletal muscle fibers using polarized fluorescence from bifunctional rhodamine probes on the C and E helices of troponin C. The C helix, in the regulatory head domain, tilts by approximately 30 degrees when muscle is activated in physiological conditions, with a Ca2+ -sensitivity similar to that of active force. Complete inhibition of active force did not affect C-helix orientation, and binding of rigor myosin heads did not affect its orientation at saturating [Ca2+]. The E helix, in the IT arm of troponin, tilted by approximately 10 degrees on activation, and this was reduced to only 3 degrees when active force was inhibited. Binding of rigor myosin heads produced a larger tilt of the E helix. Thus, in situ, the regulatory head acts as a pure Ca2+ -sensor, whereas the IT arm is primarily sensitive to myosin head binding. The polarized fluorescence data from active muscle are consistent with an in vitro structure of the troponin core complex in which the D and E helices of troponin C are collinear. The present data were used to orient this structure in the fiber and suggest that the IT arm is at approximately 30 degrees to the filament axis in active muscle. In relaxed muscle, the IT arm tilts to approximately 40 degrees but the D/E helix linker melts, allowing the regulatory head to tilt through a larger angle.

時分割X線回折法を用いた筋収縮過程におけるトロポニンの構造変化の解析

時分割X線回折法を用いた筋収縮過程におけるトロポニンの構造変化の解析

1P102ESRを用いたトロポニンCのカルシウム依存的構造変化の解析

1P102ESRを用いたトロポニンCのカルシウム依存的構造変化の解析

The changes in heat capacity and entropy of troponin C induced by calcium binding.

We have used the method of enthalpy titration to analyze the structural changes of troponin C caused by calcium binding. Successive additions of calcium to metal-free troponin C in a microcalorimeter cell result in at least three distinguishable transitions. Analysis of the results has shown that troponin C has at least three classes of calcium binding sites; one site of highest affinity (binding constant, 10(8)-10(10) M-1), one site of next highest affinity (binding constant, 10(6)-10(7) M-1) and two low affinity sites (binding constant, 10(5)-10(6) M-1). Titrations of troponin C with calcium at various temperatures have shown that calcium binding causes large changes in the heat capacity of troponin C. Following the method of Sturtevant (1977), the magnitudes of the hydrophobic and intramolecular vibrational contributions to the heat capacity and entropy changes of troponin C on calcium binding have been estimated. In Mg-free solutions, calcium binding to the 1st site of highest affinity gives rise to a strong hydrophobic effect and to a tightening of the molecular structure. In contrast, calcium binding to the 2nd site of next highest affinity gives rise to a strong hydrophobic effect in the reverse direction and to a "softening" of the structure. Calcium binding to the two low affinity sites has a moderately strong hydrophobic effect and also causes a moderate tightening of the structure. These results are in many respects similar to those obtained with proton magnetic resonance spectroscopy by Levine et al. (1977). These studies are mutually complementary. When 1 mM magnesium is present the changes caused by calcium binding to the two high affinity sites are greatly altered, whereas those involving the two low affinity sites are not much affected. The moderate tightening of the structure which is caused by calcium binding to the two low affinity sites, and which is seen both in the absence and presence of magnesium, is most likely to be involved in the regulation of contraction.

A laser Raman spectroscopic study of Ca2+ binding to troponin C

Laser Raman spectroscopy has been used detect structural changes in troponin C induced by Ca2+ binding. Addition of Ca2+ - Mg2+ sites produces perturbations in the amide III region of the spectrum indicative of increased alpha-helical content, and in regions of the spectrum corresponding to carboxylate, thiol, and phenol side chains. However, Ca2+ binding to the low affinity Ca2+ - specific sites is not detected by laser Raman spectral changes.

Calcium binding of troponin C. I. A potentiometric titration study.

Structural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2 mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222 nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites.