Interaction of aldolase with the troponin-tropomyosin complex of bovine muscle.
Ontology highlight
ABSTRACT:
SUBMITTER: Clarke FM
PROVIDER: S-EPMC1166344 | biostudies-other | 1974 Jun
REPOSITORIES: biostudies-other
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Project description:In an effort to elucidate the mechanism of calmodulin regulation of muscle contraction, we investigated the interaction between calmodulin and troponin components in the presence of Ca2+ or Sr2+ by the use of ultracentrifugation methods and polyacrylamide-gel electrophoresis. Skeletal-muscle troponin C bound to troponin I and dissociated it from the tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin T was absent, calmodulin bound to troponin I and dissociated it from the tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin T was present, calmodulin hardly bound to troponin I even in the presence of bivalent cations. Trifluoperazine, a calmodulin antagonist, inhibited the bivalent-cation-dependent interaction between calmodulin and troponin I. Calmodulin migrated more slowly in the presence of Sr2+ than it did in the presence of EGTA but faster than it did in the presence of Ca2+ on polyacrylamide-gel electrophoresis under non-denaturing conditions. It is concluded that troponin T is not required in the calmodulin regulation of muscle contraction because troponin T inhibits the bivalent-cation-dependent interaction between calmodulin and troponin I and because calmodulin binds to troponin I and dissociates it from the tropomyosin-actin complex in a bivalent-cation-dependent manner. Sr2+-induced exposure of the hydrophobic region enables calmodulin to bind to troponin I, as is the case with Ca2+.
| S-EPMC1147646 | biostudies-other
Project description:Bovine cardiac troponin is similar to rabbit skeletal troponin with respect to secondary structure, amino acid composition and molecular weight of the subunits, but differs slightly with respect to biological activity and surface charges of the subunits. Previous circular-dichroic studies of the subunits and recombination of subunits have indicated significant Ca2+-induced delocalized conformational changes. Present studies of the native troponin complex are not in accord with such changes. Furthermore the formation of the troponin-tropomyosin complex in vitro results in no delocalized conformational changes, nor does it sensitize troponin to Ca2+-induced changes. It is suggested that the troponin complex cannot be dissociated into subunits without significant and irreversible conformational perturbation.
| S-EPMC1186048 | biostudies-other
Project description: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.
| S-EPMC2483953 | biostudies-literature
Project description:1. Eight peptides were separated from the CNBr digest of troponin T from rabbit white skeletal muscle and characterized. 2. By study of the amino acid sequence of the methionine-containing peptides isolated after chymotryptic and tryptic digestion and of the N- and C-terminals of the CNBr peptides, six of the latter were shown to be arranged in the sequence CNB1-CNB2-CNB5-CNB6-CNB8-CNB7. The other two peptides, CNB1' and CNB3, have been shown to be partial digestion products. 3. The CNBr peptides CNB1' and CNB2 contained a common sequence and were the only peptides in CNBr digests of troponin T that formed a complex with tropomyosin as judged by viscometric and electrophoretic studies. 4. It is concluded that tropomyosin interacts with the N-terminal half of the troponin T molecule approximately in the region lying between residues 70 and 160. 5. Electrophoretic evidence indicates that tropomyosin and troponin C interact with troponin T. 6. None of the major CNBr peptides of troponin T isolated formed a complex with troponin C on electrophoresis at pH 8.6.
| S-EPMC1172328 | biostudies-other
Project description:Regulation of the crossbridge cycle that drives muscle contraction involves a reconfiguration of the troponin-tropomyosin complex on actin filaments. By comparing atomic models of troponin-tropomyosin fitted to cryo-EM structures of inhibited and Ca2+-activated thin filaments, we find that tropomyosin pivots rather than rolls or slides across actin as generally thought. We propose that pivoting can account for the Ca2+ activation that initiates muscle contraction and then relaxation influenced by troponin-I (TnI). Tropomyosin is well-known to occupy either of three meta-stable configurations on actin, regulating access of myosin motorheads to their actin-binding sites and thus the crossbridge cycle. At low Ca2+ concentrations, tropomyosin is trapped by TnI in an inhibitory B-state that sterically blocks myosin binding to actin, leading to muscle relaxation. Ca2+ binding to TnC draws TnI away from tropomyosin, while tropomyosin moves to a C-state location over actin. This partially relieves the steric inhibition and allows weak binding of myosin heads to actin, which then transition to strong actin-bound configurations, fully activating the thin filament. Nevertheless, the reconfiguration that accompanies the initial Ca2+-sensitive B-state/C-state shift in troponin-tropomyosin on actin remains uncertain and at best is described by moderate-resolution cryo-EM reconstructions. Our recent computational studies indicate that intermolecular residue-to-residue salt-bridge linkage between actin and tropomyosin is indistinguishable in B- and C-state thin filament configurations. We show here that tropomyosin can pivot about relatively fixed points on actin to accompany B-state/C-state structural transitions. We argue that at low Ca2+ concentrations C-terminal TnI domains attract tropomyosin, causing it to bend and then pivot toward the TnI, thus blocking myosin binding and contraction.
| S-EPMC10227645 | biostudies-literature
Project description:Tropomyosin and troponin regulate muscle contraction by participating in a macromolecular scale steric-mechanism to control myosin-crossbridge - actin interactions and consequently contraction. At low-Ca2+, the C-terminal 30% of troponin subunit-I (TnI) is proposed to trap tropomyosin in a position on thin filaments that sterically interferes with myosin-binding, thus causing muscle relaxation. In contrast, at high-Ca2+, inhibition is released after the C-terminal domains dissociate from F-actin-tropomyosin as its component switch-peptide domain binds to the N-lobe of troponin-C (TnC). Recent, paradigm-shifting, cryo-EM reconstructions by the Namba group have revealed density attributed to TnI along cardiac muscle thin filaments at both low- and high-Ca2+ concentration. Modeling the reconstructions showed expected high-Ca2+ hydrophobic interactions of the TnI switch-peptide and TnC. However, under low-Ca2+ conditions, sparse interactions of TnI and tropomyosin, and in particular juxtaposition of non-polar switch-peptide residues and charged tropomyosin amino acids in the published model seem difficult to reconcile with an expected steric-blocking conformation. This anomaly is likely due to inaccurate fitting of tropomyosin into the cryo-EM volume. In the current study, the low-Ca2+ cryo-EM volume was fitted with a more accurate tropomyosin model and representation of cardiac TnI. Our results show that at low-Ca2+ a cluster of hydrophobic residues at the TnI switch-peptide and adjacent H4 helix (Ala149, Ala151, Met 154, Leu159, Gly160, Ala161, Ala163, Leu167, Leu169, Ala171, Leu173) draw-in tropomyosin surface residues (Ile143, Ile146, Ala151, Ile154), presumably attracting the entire tropomyosin cable to its myosin-blocking position on actin. The modeling confirms that neighboring TnI "inhibitory domain" residues (Arg145, Arg148) bind to thin filaments at actin residue Asp25, as previously suggested. ClusPro docking of TnI residues 137-184 to actin-tropomyosin, including the TnI inhibitory-domain, switch-peptide and Helix H4, verified the modeled configuration. Our residue-to-residue contact-mapping of the TnI-tropomyosin association lends itself to experimental validation and functional localization of disease-bearing mutations.
| S-EPMC8026701 | biostudies-literature
Project description:The cardiac muscle comprises dynamically interacting components that use allosteric/cooperative mechanisms to yield unique heart-specific properties. An essential protein in this allosteric/cooperative mechanism is cardiac muscle troponin T (cTnT), the central region (CR) and the T2 region of which differ significantly from those of fast skeletal muscle troponin T (fsTnT). To understand the biological significance of such sequence heterogeneity, we replaced the T1 or T2 domain of rat cTnT (RcT1 or RcT2) with its counterpart from rat fsTnT (RfsT1or RfsT2) to generate RfsT1-RcT2 and RcT1-RfsT2 recombinant proteins. In addition to contractile function measurements, dynamic features of RfsT1-RcT2- and RcT1-RfsT2-reconstituted rat cardiac muscle fibers were captured by fitting the recruitment-distortion model to the force response of small-amplitude (0.5%) muscle length changes. RfsT1-RcT2 fibers showed a 40% decrease in tension and a 44% decrease in ATPase activity, but RcT1-RfsT2 fibers were unaffected. The magnitude of length-mediated increase in crossbridge (XB) recruitment (E0) decreased by ~33% and the speed of XB recruitment (b) increased by ~100% in RfsT1-RcT2 fibers. Our data suggest the following: (1) the CR of cTnT modulates XB recruitment dynamics; (2) the N-terminal end region of cTnT has a synergistic effect on the ability of the CR to modulate XB recruitment dynamics; (3) the T2 region is important for tuning the Ca(2+) regulation of cardiac thin filaments. The combined effects of CR-tropomyosin interactions and the modulating effect of the N-terminal end of cTnT on CR-tropomyosin interactions may lead to the emergence of a unique property that tunes contractile dynamics to heart rates.
| S-EPMC3633627 | biostudies-literature
Project description:Skeletal-muscle troponin I and troponin T were found to be rapidly phosphorylated by cardiac phospholipid-sensitive Ca2+-dependent protein kinase, with Km values of 6.66 and 0.13 microM respectively. Stoichiometric phosphorylation of skeletal troponin I (endogenous phosphate content 0.7 mol/mol) indicated that the Ca2+-dependent enzyme and cyclic AMP-dependent protein kinase incorporated 0.9 and 0.8 mol/mol respectively. The same experiments with skeletal troponin T (endogenous phosphate content 1.9 mol/mol) revealed a maximal phosphorylation of 2 mol/mol by the Ca2+-dependent enzyme, whereas the cyclic AMP-dependent enzyme was unable to phosphorylate troponin T. The Ca2+-dependent enzyme phosphorylated both serine and threonine residues in skeletal and cardiac troponin I or troponin T; the cyclic AMP-dependent enzyme, in comparison, phosphorylated only serine in skeletal and cardiac troponin I. Although an equimolar amount of skeletal or cardiac troponin C markedly inhibited (80-90%) phosphorylation of skeletal and cardiac troponin I by the Ca2+-dependent enzyme, these troponin C preparations inhibited only phosphorylation of skeletal troponin I, but not that of cardiac troponin I, by the cyclic AMP-dependent enzyme. Calmodulin and Ca2+-binding protein S-100a could mimic the inhibitory effect of troponin C. A tissue specificity appeared to exist for the skeletal troponin T-skeletal troponin C interaction. Inhibition of troponin T phosphorylation by an equimolar amount of troponin C was lower than that of troponin I phosphorylation; these findings might explain in part why troponin T was the major substrate for the Ca2+-dependent enzyme in the troponin complex. The present studies indicate that skeletal and cardiac troponin I and troponin T were effective substrates for phospholipid-sensitive Ca2+-dependent protein kinase, suggesting a potential involvement of this Ca2+-effector enzyme in the regulation of myofibrillar activity.
| S-EPMC1153349 | biostudies-other
Project description:Stretch activation is important in the mechanical properties of vertebrate cardiac muscle and essential to the flight muscles of most insects. Despite decades of investigation, the underlying molecular mechanism of stretch activation is unknown. We investigated the role of recently observed connections between myosin and troponin, called "troponin bridges," by analyzing real-time X-ray diffraction "movies" from sinusoidally stretch-activated Lethocerus muscles. Observed changes in X-ray reflections arising from myosin heads, actin filaments, troponin, and tropomyosin were consistent with the hypothesis that troponin bridges are the key agent of mechanical signal transduction. The time-resolved sequence of molecular changes suggests a mechanism for stretch activation, in which troponin bridges mechanically tug tropomyosin aside to relieve tropomyosin's steric blocking of myosin-actin binding. This enables subsequent force production, with cross-bridge targeting further enhanced by stretch-induced lattice compression and thick-filament twisting. Similar linkages may operate in other muscle systems, such as mammalian cardiac muscle, where stretch activation is thought to aid in cardiac ejection.
| S-EPMC3017141 | biostudies-literature
Project description:Contraction in striated and cardiac muscles is regulated by the motions of a Ca(2+)-sensitive tropomyosin/troponin switch. In contrast, troponin is absent in other muscle types and in nonmuscle cells, and actomyosin regulation is myosin-linked. Here we report an unusual crystal structure at 2.7 A of the C-terminal 31 residues of rat striated-muscle alpha-tropomyosin (preceded by a fragment of the GCN4 leucine zipper). The C-terminal 22 residues (263-284) of the structure do not form a two-stranded alpha-helical coiled coil as does the rest of the molecule, but here the alpha-helices splay apart and are stabilized by the formation of a tail-to-tail dimer with a symmetry-related molecule. The site of splaying involves a small group of destabilizing core residues that is present only in striated muscle tropomyosin isoforms. These results reveal a specific recognition site for troponin T and clarify the physical basis for the unique regulatory mechanism of striated muscles.
| S-EPMC124239 | biostudies-literature