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

The interaction of troponin molecules on the thin filament with Ca²⁺ plays a key role in regulating muscle contraction. To characterize the structural changes of troponin caused by Ca²⁺ and crossbridge formation, we recorded the small-angle x-ray intensity and the myoplasmic free Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ has bound to it, and returns to the resting structure even when there is still some bound Ca²⁺. In a muscle with a filament overlap, crossbridge formation appears to slow down Ca²⁺ release from troponin and have a large effect on its conformation.     

Structural Basis for the Activation of Muscle Contraction by Troponin and Tropomyosin

The molecular regulation of striated muscle contraction couples the binding and dissociation of Ca²⁺ on troponin (Tn) to the movement of tropomyosin on actin filaments. In turn, this process exposes or blocks myosin binding sites on actin, thereby controlling myosin crossbridge dynamics and consequently muscle contraction. Using 3D electron microscopy, we recently provided structural evidence that a C-terminal extension of TnI is anchored on actin at low Ca²⁺ and competes with tropomyosin for a common site to drive tropomyosin to the B-state location, a constrained, relaxing position on actin that inhibits myosin-crossbridge association. Here, we show that release of this constraint at high Ca²⁺ allows a second segment of troponin, probably representing parts of TnT or the troponin core domain, to promote tropomyosin movement on actin to the Ca²⁺-induced C-state location. With tropomyosin stabilized in this position, myosin binding interactions can begin. Tropomyosin appears to oscillate to a higher degree between respective B- and C-state positions on troponin-free filaments than on fully regulated filaments, suggesting that tropomyosin positioning in both states is troponin-dependent. By biasing tropomyosin to either of these two positions, troponin appears to have two distinct structural functions; in relaxed muscles at low Ca²⁺, troponin operates as an inhibitor, while in activated muscles at high Ca²⁺, it acts as a promoter to initiate contraction.     

Troponin Regulatory Function and Dynamics Revealed by H/D Exchange-Mass Spectrometry

Muscle contraction is tightly regulated by Ca²⁺ binding to the thin filament protein troponin. The mechanism of this regulation was investigated by detailed mapping of the dynamic properties of cardiac troponin using amide hydrogen exchange-mass spectrometry. Results were obtained in the presence of either saturation or non-saturation of the regulatory Ca²⁺ binding site in the NH₂ domain of subunit TnC. Troponin was found to be highly dynamic, with 60% of amides exchanging H for D within seconds of exposure to D₂O. In contrast, portions of the TnT-TnI coiled-coil exhibited high protection from exchange, despite 6 h in D₂O. The data indicate that the most stable portion of the trimeric troponin complex is the coiled-coil. Regulatory site Ca²⁺ binding altered dynamic properties (i.e. H/D exchange protection) locally, near the binding site and in the TnI switch helix that attaches to the Ca²⁺-saturated TnC NH₂ domain. More notably, Ca²⁺ also altered the dynamic properties of other parts of troponin: the TnI inhibitory peptide region that binds to actin, the TnT-TnI coiled-coil, and the TnC COOH domain that contains the regulatory Ca²⁺ sites in many invertebrate as opposed to vertebrate troponins. Mapping of these affected regions onto the troponin highly extended structure suggests that cardiac troponin switches between alternative sets of intramolecular interactions, similar to previous intermediate resolution x-ray data of skeletal muscle troponin.     

Kinetic Mechanism of Ca2+-controlled Changes of Skeletal Troponin I in Psoas Myofibrils

Conformational changes in the skeletal troponin complex (sTn) induced by rapidly increasing or decreasing the [Ca²⁺] were probed by 5-iodoacetamidofluorescein covalently bound to Cys-133 of skeletal troponin I (sTnI). Kinetics of conformational changes was determined for the isolated complex and after incorporating the complex into rabbit psoas myofibrils. Isolated and incorporated sTn exhibited biphasic Ca²⁺-activation kinetics. Whereas the fast phase (k_obs∼1000 s⁻¹) is only observed in this study, where kinetics were induced by Ca²⁺, the slower phase resembles the monophasic kinetics of sTnI switching observed in another study (Brenner and Chalovich. 1999. Biophys. J. 77:2692–2708) that investigated the sTnI switching induced by releasing the feedback of force-generating cross-bridges on thin filament activation. Therefore, the slower conformational change likely reflects the sTnI switch that regulates force development. Modeling reveals that the fast conformational change can occur after the first Ca²⁺ ion binds to skeletal troponin C (sTnC), whereas the slower change requires Ca²⁺ binding to both regulatory sites of sTnC. Incorporating sTn into myofibrils increased the off-rate and lowered the Ca²⁺ sensitivity of sTnI switching. Comparison of switch-off kinetics with myofibril force relaxation kinetics measured in a mechanical setup indicates that sTnI switching might limit the rate of fast skeletal muscle relaxation.     

The solution structure of the Mg2+ form of soybean calmodulin isoform 4 reveals unique features of plant calmodulins in resting cells

Soybean calmodulin isoform 4 (sCaM4) is a plant calcium‐binding protein, regulating cellular responses to the second messenger Ca²⁺. We have found that the metal ion free (apo‐) form of sCaM4 possesses a half unfolded structure, with the N‐terminal domain unfolded and the C‐terminal domain folded. This result was unexpected as the apo‐forms of both soybean calmodulin isoform 1 (sCaM1) and mammalian CaM (mCaM) are fully folded. Because of the fact that free Mg²⁺ ions are always present at high concentrations in cells (0.5–2 mM), we suggest that Mg²⁺ should be bound to sCaM4 in nonactivated cells. CD studies revealed that in the presence of Mg²⁺ the initially unfolded N‐terminal domain of sCaM4 folds into an α‐helix‐rich structure, similar to the Ca²⁺ form. We have used the NMR backbone residual dipolar coupling restraints ¹D_NH, ¹D_CαHα, and ¹D_C′Cα to determine the solution structure of the N‐terminal domain of Mg²⁺‐sCaM4 (Mg²⁺‐sCaM4‐NT). Compared with the known structure of Ca²⁺‐sCaM4, the structure of the Mg²⁺‐sCaM4‐NT does not fully open the hydrophobic pocket, which was further confirmed by the use of the fluorescent probe ANS. Tryptophan fluorescence experiments were used to study the interactions between Mg²⁺‐sCaM4 and CaM‐binding peptides derived from smooth muscle myosin light chain kinase and plant glutamate decarboxylase. These results suggest that Mg²⁺‐sCaM4 does not bind to Ca²⁺‐CaM target peptides and therefore is functionally similar to apo‐mCaM. The Mg²⁺‐ and apo‐structures of the sCaM4‐NT provide unique insights into the structure and function of some plant calmodulins in resting cells.     

Terbium binding to troponin C: Binding stoichiometry and structural changes induced in the protein

Terbium (Tb³⁺) binding to skeletal muscle troponin C was studied by fluorescence spectroscopy and circular dichroism. Titrations indicate that Tb³⁺, like Ca²⁺, preferentially binds to the two high affinity Ca²⁺-Mg²⁺ sites (III and IV) inducing structural changes similar to those induced by Ca²⁺. Tb³⁺ readily displaces Ca²⁺ from these sites suggesting a K_(Tb³⁺) ≥ 10⁹ M^?1 In 6 M urea, both Ca²⁺ and Tb³⁺ bind preferentially to a single site on troponin C. The spectral changes suggest this to be site III.     

Ca-Dependent Photocrosslinking of Tropomyosin Residue 146 to Residues 157-163 in the C-Terminal Domain of Troponin I in Reconstituted Skeletal Muscle Thin Filaments

The Ca²⁺-dependent interaction of troponin I (TnI) with actin·tropomyosin (Tm) in muscle thin filaments is a critical step in the regulation of muscle contraction. Previous studies have suggested that, in the absence of Ca²⁺, TnI interacts with Tm and actin in reconstituted muscle thin filaments, maintaining Tm at the outer domain of actin and blocking myosin-actin interaction. To obtain direct evidence for this Tm-TnI interaction, we performed photochemical crosslinking studies using Tm labeled with 4-maleimidobenzophenone at position 146 or 174 (Tm*146 or Tm*174, respectively), reconstituted with actin and troponin [composed of TnI, troponin T (TnT), and troponin C] or with actin and TnI. After near-UV irradiation, SDS gels of the Tm*146-containing thin filament showed three new high-molecular-weight bands determined to be crosslinked products Tm*146-TnI, Tm*146-troponin C, and Tm*146-TnT using fluorescence-labeled TnI, mass spectrometry, and Western blot analysis. While Tm*146-TnI was produced only in the absence of Ca²⁺, the production of other crosslinked species did not show Ca²⁺ dependence. Tm*174 mainly crosslinked to TnT. In the absence of actin, a similar crosslinking pattern was obtained with a much lower yield. A tryptic peptide from Tm*146-TnI with a molecular mass of 2601.2 Da that was not present in the tryptic peptides of Tm*146 or TnI was identified using HPLC and matrix-assisted laser desorption/ionization time-of-flight. This was shown, using absorption and fluorescence spectroscopy, to be the 4-maleimidobenzophenone-labeled peptide from Tm crosslinked to TnI peptide 157-163. These data, which show that a region in the C-terminal domain of TnI interacts with Tm in the absence of Ca²⁺, support the hypothesis that a TnI-Tm interaction maintains Tm at the outer domain of actin and will help efforts to localize troponin in actin·Tm muscle thin filaments.     

Myosin and actomyosin from human skeletal muscle

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (±2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37°C. Activation of the Mg-ATPase activity of Ca²⁺-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 μM Ca²⁺ concentration (CaEGTA binding constant = 4.4 · 10⁵ at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6–9 range, the Ca²⁺-ATPase activity of the subfragment 1 was 1.8-and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6–10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca²⁺-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca²⁺ is similar to that of rabbit fast or slow muscle     

Intrinsic fluorescence studies on troponin C.

Evidence for a proton transfer mechanism in the Ca²⁺-induced enhancement of the Tyr fluorescence of troponin C was obtained by studying the effects, in D₂O and H₂O, of Ca²⁺, Mg²⁺, and H⁺ on the fluorescence of both the protein and a model system containing L-Tyr in the presence of citrate. In the model system, it is shown that citrate quenches the fluorescence of L-Tyr, that there is a large deuterium isotope effect on the quenching, and that binding of Ca², Mg²⁺, or H⁺ by citrate results in a fluorescence enhancement. These results can be explained by the transfer of the phenolic proton of the excited Tyr to the carboxylates of citrate. Similar effects on the fluorescence of troponin C suggest that, in the apoprotein, the fluorescence of Tyr is quenched by a similar mechanism. Thus, the Ca²⁺, Mg²⁺, and H⁺-induced Tyr fluorescence enhancement in troponin C is due to “dequenching” resulting from coordination or protonation of vicinal carboxylates. Studies of troponin C fluorescence and fluorescence depolarization as a function of urea concentration enabled an estimate of the separate fluorescence contributions of its two Tyr residues (Nos. 10 and 109) in its various conformational states. Further evidence was also obtained to support the earlier proposal that the Ca²⁺ enhancement is primarily due to the direct loss of quenching by nearby carboxylates, by showing that the pH-induced fluorescence enhancement did not occur in parallel with the ellipticity increase at 222 nm.     

Cardiac Muscle Activation Blunted by a Mutation to the Regulatory Component,Troponin T

The striated muscle thin filament comprises actin, tropomyosin, and troponin. The Tn complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnT may serve as a bridge between the Ca²⁺ sensor (TnC) and the actin filament. In the short helix preceding the IT-arm region, H1(T2), there are known dilated cardiomyopathy-linked mutations (among them R205L). Thus we hypothesized that there is an element in this short helix that plays an important role in regulating the muscle contraction, especially in Ca²⁺ activation. We mutated Arg-205 and several other amino acid residues within and near the H1(T2) helix. Utilizing an alanine replacement method to compare the effects of the mutations, the biochemical and mechanical impact on the actomyosin interaction was assessed by solution ATPase activity assay, an in vitro motility assay, and Ca²⁺ binding measurements. Ca²⁺ activation was markedly impaired by a point mutation of the highly conserved basic residue R205A, residing in the short helix H1(T2) of cTnT, whereas the mutations to nearby residues exhibited little effect on function. Interestingly, rigor activation was unchanged between the wild type and R205A TnT. In addition to the reduction in Ca²⁺ sensitivity observed in Ca²⁺ binding to the thin filament, myosin S1-ADP binding to the thin filament was significantly affected by the same mutation, which was also supported by a series of S1 concentration-dependent ATPase assays. These suggest that the R205A mutation alters function through reduction in the nature of cooperative binding of S1.     

Troponin Revealed: Uncovering the Structure of the Thin Filament On-Off Switch in Striated Muscle

Recently, our understanding of the structural basis of troponin-tropomyosin’s Ca²⁺-triggered regulation of striated muscle contraction has advanced greatly, particularly via cryo-electron microscopy data. Compelling atomic models of troponin-tropomyosin-actin were published for both apo- and Ca²⁺-saturated states of the cardiac thin filament. Subsequent electron microscopy and computational analyses have supported and further elaborated the findings. Per cryo-electron microscopy, each troponin is highly extended and contacts both tropomyosin strands, which lie on opposite sides of the actin filament. In the apo-state characteristic of relaxed muscle, troponin and tropomyosin hinder strong myosin-actin binding in several different ways, apparently barricading the actin more substantially than does tropomyosin alone. The troponin core domain, the C-terminal third of TnI, and tropomyosin under the influence of a 64-residue helix of TnT located at the overlap of adjacent tropomyosins are all in positions that would hinder strong myosin binding to actin. In the Ca²⁺-saturated state, the TnI C-terminus dissociates from actin and binds in part to TnC; the core domain pivots significantly; the N-lobe of TnC binds specifically to actin and tropomyosin; and tropomyosin rotates partially away from myosin’s binding site on actin. At the overlap domain, Ca²⁺ causes much less tropomyosin movement, so a more inhibitory orientation persists. In the myosin-saturated state of the thin filament, there is a large additional shift in tropomyosin, with molecular interactions now identified between tropomyosin and both actin and myosin. A new era has arrived for investigation of the thin filament and for functional understandings that increasingly accommodate the recent structural results.     

Model of Sarcomeric Ca2+ Movements,Including ATP Ca2+ Binding and Diffusion,during Activation of Frog Skeletal Muscle

Cannell and Allen (1984. Biophys. J. 45:913–925) introduced the use of a multi-compartment model to estimate the time course of spread of calcium ions (Ca²⁺) within a half sarcomere of a frog skeletal muscle fiber activated by an action potential. Under the assumption that the sites of sarcoplasmic reticulum (SR) Ca²⁺ release are located radially around each myofibril at the Z line, their model calculated the spread of released Ca²⁺ both along and into the half sarcomere. During diffusion, Ca²⁺ was assumed to react with metal-binding sites on parvalbumin (a diffusible Ca²⁺- and Mg²⁺-binding protein) as well as with fixed sites on troponin. We have developed a similar model, but with several modifications that reflect current knowledge of the myoplasmic environment and SR Ca²⁺ release. We use a myoplasmic diffusion constant for free Ca²⁺ that is twofold smaller and an SR Ca²⁺ release function in response to an action potential that is threefold briefer than used previously. Additionally, our model includes the effects of Ca²⁺ and Mg²⁺ binding by adenosine 5′-triphosphate (ATP) and the diffusion of Ca²⁺-bound ATP (CaATP). Under the assumption that the total myoplasmic concentration of ATP is 8 mM and that the amplitude of SR Ca²⁺ release is sufficient to drive the peak change in free [Ca²⁺] (Δ[Ca²⁺]) to 18 μM (the approximate spatially averaged value that is observed experimentally), our model calculates that (a) the spatially averaged peak increase in [CaATP] is 64 μM; (b) the peak saturation of troponin with Ca²⁺ is high along the entire thin filament; and (c) the half-width of Δ[Ca²⁺] is consistent with that observed experimentally. Without ATP, the calculated half-width of spatially averaged Δ[Ca²⁺] is abnormally brief, and troponin saturation away from the release sites is markedly reduced. We conclude that Ca²⁺ binding by ATP and diffusion of CaATP make important contributions to the determination of the amplitude and the time course of Δ[Ca²⁺].     

Significance of Troponin Dynamics for Ca2+-mediated Regulation of Contraction and Inherited Cardiomyopathy

Ca²⁺ dissociation from troponin causes cessation of muscle contraction by incompletely understood structural mechanisms. To investigate this process, regulatory site Ca²⁺ binding in the NH₂-lobe of subunit troponin C (TnC) was abolished by mutagenesis, and effects on cardiac troponin dynamics were mapped by hydrogen-deuterium exchange (HDX)-MS. The findings demonstrate the interrelationships among troponin''s detailed dynamics, troponin''s regulatory actions, and the pathogenesis of cardiomyopathy linked to troponin mutations. Ca²⁺ slowed HDX up to 2 orders of magnitude within the NH₂-lobe and the NH₂-lobe-associated TnI switch helix, implying that Ca²⁺ greatly stabilizes this troponin regulatory region. HDX of the TnI COOH terminus indicated that its known role in regulation involves a partially folded rather than unfolded structure in the absence of Ca²⁺ and actin. Ca²⁺-triggered stabilization extended beyond the known direct regulatory regions: to the start of the nearby TnI helix 1 and to the COOH terminus of the TnT-TnI coiled-coil. Ca²⁺ destabilized rather than stabilized specific TnI segments within the coiled-coil and destabilized a region not previously implicated in Ca²⁺-mediated regulation: the coiled-coil''s NH₂-terminal base plus the preceding TnI loop with which the base interacts. Cardiomyopathy-linked mutations clustered almost entirely within influentially dynamic regions of troponin, and many sites were Ca²⁺-sensitive. Overall, the findings demonstrate highly selective effects of regulatory site Ca²⁺, including opposite changes in protein dynamics at opposite ends of the troponin core domain. Ca²⁺ release triggers an intramolecular switching mechanism that propagates extensively within the extended troponin structure, suggests specific movements of the TnI inhibitory regions, and prominently involves troponin''s dynamic features.     

Ca2+-dependent Muscle Dysfunction Caused by Mutation of the Caenorhabditis elegans Troponin T-1 Gene

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca²⁺]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061–1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca²⁺-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca²⁺ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca²⁺ signaling support that CeTnT-1 phenotypes are dependent on Ca²⁺ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca²⁺] does not require TnT.     

Nuclear magnetic resonance structure of calcium-binding protein 1 in a Ca(2+) -bound closed state: Implications for target recognition

Calcium‐binding protein 1 (CaBP1), a neuron‐specific member of the calmodulin (CaM) superfamily, regulates the Ca²⁺‐dependent activity of inositol 1,4,5‐triphosphate receptors (InsP3Rs) and various voltage‐gated Ca²⁺ channels. Here, we present the NMR structure of full‐length CaBP1 with Ca²⁺ bound at the first, third, and fourth EF‐hands. A total of 1250 nuclear Overhauser effect distance measurements and 70 residual dipolar coupling restraints define the overall main chain structure with a root‐mean‐squared deviation of 0.54 Å (N‐domain) and 0.48 Å (C‐domain). The first 18 residues from the N‐terminus in CaBP1 (located upstream of the first EF‐hand) are structurally disordered and solvent exposed. The Ca²⁺‐saturated CaBP1 structure contains two independent domains separated by a flexible central linker similar to that in calmodulin and troponin C. The N‐domain structure of CaBP1 contains two EF‐hands (EF1 and EF2), both in a closed conformation [interhelical angles = 129° (EF1) and 142° (EF2)]. The C‐domain contains EF3 and EF4 in the familiar Ca²⁺‐bound open conformation [interhelical angles = 105° (EF3) and 91° (EF4)]. Surprisingly, the N‐domain adopts the same closed conformation in the presence or absence of Ca²⁺ bound at EF1. The Ca²⁺‐bound closed conformation of EF1 is reminiscent of Ca²⁺‐bound EF‐hands in a closed conformation found in cardiac troponin C and calpain. We propose that the Ca²⁺‐bound closed conformation of EF1 in CaBP1 might undergo an induced‐fit opening only in the presence of a specific target protein, and thus may help explain the highly specialized target binding by CaBP1.     

Infrared study of synthetic peptide analogues of the calcium‐binding site III of troponin C: The role of helix F of an EF‐hand motif

The EF‐hand motif (helix–loop–helix) is a Ca²⁺‐binding domain that is common among many intracellular Ca²⁺‐binding proteins. We applied Fourier‐transform infrared spectroscopy to study the synthetic peptide analogues of site III of rabbit skeletal muscle troponin C (helix E–loop–helix F). The 17‐residue peptides corresponding to loop–helix F (DRDADGYIDAEELAEIF), where one residue is substituted by the D ‐type amino acid, were investigated to disturb the α‐helical conformation of helix F systematically. These D ‐type‐substituted peptides showed no band at about 1555 cm^?1 even in the Ca²⁺‐loaded state although the native peptide (L ‐type only) showed a band at about 1555 cm^?1 in the Ca²⁺‐loaded state, which is assigned to the side‐chain COO^? group of Glu at the 12th position, serving as the ligand for Ca²⁺ in the bidentate coordination mode. Therefore, helix F is vital to the interaction between the Ca²⁺ and the side‐chain COO^? group of Glu at the 12th position. Implications of the COO^? antisymmetric stretch and the amide‐I′ of the synthetic peptide analogues of the Ca²⁺‐binding sites are discussed. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 342–347, 2013.     

Structural basis for the regulation of muscle contraction by troponin and tropomyosin

The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca²⁺ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosin-crossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca²⁺ binding to TnC (the Ca²⁺ sensor of troponin that relieves inhibition) presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI (the “inhibitory” subunit of troponin) with tropomyosin and actin that cause tropomyosin movement are unknown, and thus, the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI, and then, 3D electron microscopy was used to resolve where troponin is anchored on actin-tropomyosin. Electron microscopy reconstruction showed how TnI binding to both actin and tropomyosin at low Ca²⁺ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus, the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated steric interference of actin-myosin interaction that regulates muscle contraction.     

Three-Dimensional Organization of Troponin on Cardiac Muscle Thin Filaments in the Relaxed State

Muscle contraction is regulated by troponin-tropomyosin, which blocks and unblocks myosin binding sites on actin. To elucidate this regulatory mechanism, the three-dimensional organization of troponin and tropomyosin on the thin filament must be determined. Although tropomyosin is well defined in electron microscopy helical reconstructions of thin filaments, troponin density is mostly lost. Here, we determined troponin organization on native relaxed cardiac muscle thin filaments by applying single particle reconstruction procedures to negatively stained specimens. Multiple reference models led to the same final structure, indicating absence of model bias in the procedure. The new reconstructions clearly showed F-actin, tropomyosin, and troponin densities. At the 25 Å resolution achieved, troponin was considerably better defined than in previous reconstructions. The troponin density closely resembled the shape of troponin crystallographic structures, facilitating detailed interpretation of the electron microscopy density map. The orientation of troponin-T and the troponin core domain established troponin polarity. Density attributable to the troponin-I mobile regulatory domain was positioned where it could hold tropomyosin in its blocking position on actin, thus suggesting the underlying structural basis of thin filament regulation. Our previous understanding of thin filament regulation had been limited to known movements of tropomyosin that sterically block and unblock myosin binding sites on actin. We now show how troponin, the Ca²⁺ sensor, may control these movements, ultimately determining whether muscle contracts or relaxes.     

Three-Dimensional Organization of Troponin on Cardiac Muscle Thin Filaments in the Relaxed State

Muscle contraction is regulated by troponin-tropomyosin, which blocks and unblocks myosin binding sites on actin. To elucidate this regulatory mechanism, the three-dimensional organization of troponin and tropomyosin on the thin filament must be determined. Although tropomyosin is well defined in electron microscopy helical reconstructions of thin filaments, troponin density is mostly lost. Here, we determined troponin organization on native relaxed cardiac muscle thin filaments by applying single particle reconstruction procedures to negatively stained specimens. Multiple reference models led to the same final structure, indicating absence of model bias in the procedure. The new reconstructions clearly showed F-actin, tropomyosin, and troponin densities. At the 25 Å resolution achieved, troponin was considerably better defined than in previous reconstructions. The troponin density closely resembled the shape of troponin crystallographic structures, facilitating detailed interpretation of the electron microscopy density map. The orientation of troponin-T and the troponin core domain established troponin polarity. Density attributable to the troponin-I mobile regulatory domain was positioned where it could hold tropomyosin in its blocking position on actin, thus suggesting the underlying structural basis of thin filament regulation. Our previous understanding of thin filament regulation had been limited to known movements of tropomyosin that sterically block and unblock myosin binding sites on actin. We now show how troponin, the Ca²⁺ sensor, may control these movements, ultimately determining whether muscle contracts or relaxes.     

Calcium binding to cardiac troponin C

The binding of Ca²⁺ to cardiac troponin C was studied by determining changes in the fluorescence and circular dichroism of the protein and by following changes in the free Ca²⁺ concentration by means of a Ca²⁺-specific electrode. Cardiac troponin C contains three Ca²⁺-binding sites which fall into two classes —two sites with a higher affinity and one with a lower affinity. The higher-affinity sites also bind Mg²⁺ which competes with the Ca²⁺.