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1.
Pflugers Arch ; 458(4): 761-76, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19255776

ABSTRACT

The effects of tropomyosin on muscle mechanics and kinetics were examined in skeletal myofibrils using a novel method to remove tropomyosin (Tm) and troponin (Tn) and then replace these proteins with altered versions. Extraction employed a low ionic strength rigor solution, followed by sequential reconstitution at physiological ionic strength with Tm then Tn. SDS-PAGE analysis was consistent with full reconstitution, and fluorescence imaging after reconstitution using Oregon-green-labeled Tm indicated the expected localization. Myofibrils remained mechanically viable: maximum isometric forces of myofibrils after sTm/sTn reconstitution (control) were comparable (~84%) to the forces generated by non-reconstituted preparations, and the reconstitution minimally affected the rate of isometric activation (k (act)), calcium sensitivity (pCa(50)), and cooperativity (n (H)). Reconstitutions using various combinations of cardiac and skeletal Tm and Tn indicated that isoforms of both Tm and Tn influence calcium sensitivity of force development in opposite directions, but the isoforms do not otherwise alter cross-bridge kinetics. Myofibrils reconstituted with Delta23Tm, a deletion mutant lacking the second and third of Tm's seven quasi-repeats, exhibited greatly depressed maximal force, moderately slower k (act) rates and reduced n (H). Delta23Tm similarly decreased the cooperativity of calcium binding to the troponin regulatory sites of isolated thin filaments in solution. The mechanisms behind these effects of Delta23Tm also were investigated using P ( i ) and ADP jumps. P ( i ) and ADP kinetics were indistinguishable in Delta23Tm myofibrils compared to controls. The results suggest that the deleted region of tropomyosin is important for cooperative thin filament activation by calcium.


Subject(s)
Muscle Contraction/physiology , Myofibrils/chemistry , Myofibrils/physiology , Tropomyosin/chemistry , Tropomyosin/physiology , Animals , Cells, Cultured , Female , Kinetics , Mechanics , Rabbits , Stress, Mechanical
2.
J Physiol ; 552(Pt 3): 917-31, 2003 Nov 01.
Article in English | MEDLINE | ID: mdl-12937281

ABSTRACT

The effects of the removal of fast skeletal troponin C (fsTnC) and its replacement by cardiac troponin C (cTnC) and the exchange of fast skeletal troponin (fsTn) for cardiac troponin (cTn) were measured in rabbit fast skeletal myofibrils. Electrophoretic analysis of myofibril suspensions indicated that replacement of fsTnC or exchange of fsTn with cTnC or cTn was about 90% complete in the protocols used. Mechanical measurements in single myofibrils, which were maximally activated by fast solution switching, showed that replacement of fsTnC with cTnC reduced the isometric tension, the rate of tension rise following a step increase in Ca2+ (kACT), and the rate of tension redevelopment following a quick release and restretch (kTR), but had no effect on the kinetics of the fall in tension when the concentration of inorganic phosphate (Pi) was abruptly increased (kPi(+)). These data suggest that the chimeric protein produced by cTnC replacement in fsTn alters those steps controlling the weak-to-strong crossbridge attachment transition. Inefficient signalling within the chimeric troponin may cause these changes. However, replacement of fsTn by cTn had no effect on maximal isometric tension, kACT or kTR, suggesting that these mechanics are largely determined by the isoform of the myosin molecule. Replacement of fsTn by cTn, on the other hand, shifted the pCa50 of the pCa-tension relationship from 5.70 to 6.44 and reduced the Hill coefficient from 3.3 to 1.4, suggesting that regulatory protein isoforms primarily alter Ca2+ sensitivity and the cooperativity of the force-generating mechanism.


Subject(s)
Isometric Contraction/physiology , Muscle, Skeletal/metabolism , Myocardium/metabolism , Myofibrils/physiology , Psoas Muscles/metabolism , Troponin/metabolism , Animals , Calcium/metabolism , Electrophoresis, Polyacrylamide Gel , Myofibrils/metabolism , Rabbits , Troponin C/metabolism
3.
Biophys J ; 85(2): 1046-52, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12885651

ABSTRACT

The sliding speed of unregulated thin filaments in motility assays is only about half that of the unloaded shortening velocity of muscle fibers. The addition of regulatory proteins, troponin and tropomyosin, is known to increase the sliding speed of thin filaments in the in vitro motility assay. To learn if this effect is related to the rate of MgADP dissociation from the acto-S1 cross-bridge head, the effects of regulatory proteins on nucleotide binding and release in motility assays were measured in the presence and absence of regulatory proteins. The apparent affinity of acto-heavy meromyosin (acto-HMM) for MgATP was reduced by the presence of regulatory proteins. Similarly, the regulatory proteins increase the concentration of MgADP required to inhibit sliding. These results suggest that regulatory proteins either accelerate the rate of MgADP release from acto-HMM-MgADP or slow its binding to acto-HMM. The reduction of temperature also altered the relationship between thin filament sliding speed and the regulatory proteins. At lower temperatures, the regulatory proteins lost their ability to increase thin filament sliding speed above that of unregulated thin filaments. It is hypothesized that structural changes in the actin portion of the acto-myosin interface are induced by regulatory protein binding to actin.


Subject(s)
Actins/chemistry , Adenosine Triphosphate/chemistry , Molecular Motor Proteins/chemistry , Motion , Myosin Subfragments/chemistry , Tropomyosin/chemistry , Kinetics , Nucleotides/chemistry , Protein Binding , Temperature
4.
J Biol Chem ; 276(37): 34832-9, 2001 Sep 14.
Article in English | MEDLINE | ID: mdl-11457840

ABSTRACT

Tropomyosin is an extended coiled-coil protein that influences actin function by binding longitudinally along thin filaments. The present work compares cardiac tropomyosin and the two tropomyosins from Saccharomyces cerevisiae, TPM1 and TPM2, that are much shorter than vertebrate tropomyosins. Unlike cardiac tropomyosin, the phase of the coiled-coil-forming heptad repeat of TPM2 is discontinuous; it is interrupted by a 4-residue deletion. TPM1 has two such deletions, which flank the 38-residue partial gene duplication that causes TPM1 to span five actins instead of the four of TPM2. Each of the three tropomyosin isoforms modulates actin-myosin interactions, with isoform-specific effects on cooperativity and strength of myosin binding. These different properties can be explained by a model that combines opposite effects, steric hindrance between myosin and tropomyosin when the latter is bound to a subset of its sites on actin, and also indirect, favorable interactions between tropomyosin and myosin, mediated by mutually promoted changes in actin. Both of these effects are influenced by which tropomyosin isoform is present. Finally, the tropomyosins have isoform-specific effects on in vitro sliding speed and on the myosin concentration dependence of this movement, suggesting that non-muscle tropomyosin isoforms exist, at least in part, to modulate myosin function.


Subject(s)
Drosophila Proteins , Fungal Proteins/pharmacology , Myosins/physiology , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/chemistry , Tropomyosin/pharmacology , Actins/metabolism , Amino Acid Sequence , Animals , Cattle , Molecular Sequence Data , Myosin Subfragments/metabolism , Protein Isoforms , Rabbits , Tropomyosin/chemistry , Tropomyosin/metabolism
5.
J Mol Biol ; 307(3): 739-44, 2001 Mar 30.
Article in English | MEDLINE | ID: mdl-11273697

ABSTRACT

The steric model of muscle regulation holds that at low Ca(2+) concentration, tropomyosin strands, running along thin filaments, are constrained by troponin in an inhibitory position that blocks myosin-binding sites on actin. Ca(2+) activation, releasing this constraint, allows tropomyosin movement, initiating actin-myosin interaction and contraction. Although the different positions of tropomyosin on the thin filament are well documented, corresponding information on troponin has been lacking and it has therefore not been possible to test the model structurally. Here, we show that troponin can be detected on thin filaments and demonstrate how its changing association with actin can control tropomyosin position in response to Ca(2+). To accomplish this, thin filaments were reconstituted with an engineered short tropomyosin, creating a favorable troponin stoichiometry and symmetry for three-dimensional analysis. We demonstrate that in the absence of Ca(2+), troponin bound to both tropomyosin and actin can act as a latch to constrain tropomyosin in a position on actin that inhibits actomyosin ATPase. In addition, we find that on Ca(2+) activation the actin-troponin connection is broken, allowing tropomyosin to assume a second position, initiating actomyosin ATPase and thus permitting contraction to proceed.


Subject(s)
Actin Cytoskeleton/ultrastructure , Troponin/metabolism , Troponin/ultrastructure , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Actins/metabolism , Actins/ultrastructure , Calcium/pharmacology , Image Processing, Computer-Assisted , Microscopy, Electron , Models, Molecular , Muscle Contraction/drug effects , Muscle, Skeletal/chemistry , Muscle, Skeletal/metabolism , Muscle, Skeletal/ultrastructure , Protein Conformation/drug effects , Protein Engineering , Sequence Deletion/genetics , Tropomyosin/chemistry , Tropomyosin/genetics , Tropomyosin/metabolism , Tropomyosin/ultrastructure , Troponin/chemistry
6.
J Biol Chem ; 276(23): 20245-51, 2001 Jun 08.
Article in English | MEDLINE | ID: mdl-11262388

ABSTRACT

Calcium controls the level of muscle activation via interactions with the troponin complex. Replacement of the native, skeletal calcium-binding subunit of troponin, troponin C, with mixtures of functional cardiac and mutant cardiac troponin C insensitive to calcium and permanently inactive provides a novel method to alter the number of myosin cross-bridges capable of binding to the actin filament. Extraction of skeletal troponin C and replacement with functional and mutant cardiac troponin C were used to evaluate the relationship between the extent of thin filament activation (fractional calcium binding), isometric force, and the rate of force generation in muscle fibers independent of the calcium concentration. The experiments showed a direct, linear relationship between force and the number of cross-bridges attaching to the thin filament. Further, above 35% maximal isometric activation, following partial replacement with mixtures of cardiac and mutant troponin C, the rate of force generation was independent of the number of actin sites available for cross-bridge interaction at saturating calcium concentrations. This contrasts with the marked decrease in the rate of force generation when force was reduced by decreasing the calcium concentration. The results are consistent with hypotheses proposing that calcium controls the transition between weakly and strongly bound cross-bridge states.


Subject(s)
Calcium/physiology , Muscle Contraction , Muscle, Skeletal/physiology , Mutation , Troponin C/physiology , Animals , Female , Rabbits , Troponin C/genetics
7.
J Biol Chem ; 276(23): 20788-94, 2001 Jun 08.
Article in English | MEDLINE | ID: mdl-11262409

ABSTRACT

Familial hypertrophic cardiomyopathy (FHC) is caused by missense or premature truncation mutations in proteins of the cardiac contractile apparatus. Mutant proteins are incorporated into the thin filament or thick filament and eventually produce cardiomyopathy. However, it has been unclear how the several, genetically identified defects in protein structure translate into impaired protein and muscle function. We have studied the basis of FHC caused by premature truncation of the most frequently implicated thin filament target, troponin T. Electron microscope observations showed that the thin filament undergoes normal structural changes in response to Ca(2+) binding. On the other hand, solution studies showed that the mutation alters and destabilizes troponin binding to the thin filament to different extents in different regulatory states, thereby affecting the transitions among states that regulate myosin binding and muscle contraction. Development of hypertrophic cardiomyopathy can thus be traced to a defect in the primary mechanism controlling cardiac contraction, switching between different conformations of the thin filament.


Subject(s)
Cardiomyopathies/genetics , Mutation , Troponin T/genetics , Actins/metabolism , Animals , Cattle , Microscopy, Electron , Protein Conformation , Rabbits , Troponin T/metabolism , Troponin T/ultrastructure
8.
Circulation ; 103(1): 65-71, 2001 Jan 02.
Article in English | MEDLINE | ID: mdl-11136687

ABSTRACT

BACKGROUND: We report hypertrophic cardiomyopathy (HCM) in a Spanish-American family caused by a novel alpha-tropomyosin (TPM1) mutation and examine the pathogenesis of the clinical disease by characterizing functional defects in the purified mutant protein. METHODS AND RESULTS: HCM was linked to the TPM1 gene (logarithm of the odds [LOD] score 3.17). Sequencing and restriction digestion analysis demonstrated a TPM1 mutation V95A that cosegregated with HCM. The mutation has been associated with 13 deaths in 26 affected members (11 sudden deaths and 2 related to heart failure), with a cumulative survival rate of 73+/-10% at the age of 40 years. Left ventricular wall thickness (mean 16+/-6 mm) and disease penetrance (53%) were similar to those for the ss-myosin mutations L908V and G256E previously associated with a benign prognosis. Left ventricular hypertrophy was milder than with the ss-myosin mutation R403Q, but the prognosis was similarly poor. With the use of recombinant tropomyosins, we identified several functional alterations at the protein level. The mutation caused a 40% to 50% increase in calcium affinity in regulated thin filament-myosin subfragment-1 (S1) MgATPase assays, a 20% decrease in MgATPase rates in the presence of saturating calcium, a 5% decrease in unloaded shortening velocity in in vitro motility assays, and no change in cooperative myosin S1 binding to regulated thin filaments. CONCLUSIONS: In contrast to other reported TPM1 mutations, V95A-associated HCM exhibits unusual features of mild phenotype but poor prognosis. Both myosin cycling and calcium binding to troponin are abnormal in the presence of the mutant tropomyosin. The genetic diagnosis afforded by this mutation will be valuable in the management of HCM.


Subject(s)
Calcium/metabolism , Cardiomyopathy, Hypertrophic/genetics , Myosins/metabolism , Tropomyosin/genetics , Troponin/metabolism , Adult , Amino Acid Substitution/genetics , Ca(2+) Mg(2+)-ATPase/metabolism , Cardiomyopathy, Hypertrophic/diagnosis , Cardiomyopathy, Hypertrophic/epidemiology , Cardiomyopathy, Hypertrophic/metabolism , DNA Mutational Analysis , Death, Sudden, Cardiac/epidemiology , Death, Sudden, Cardiac/etiology , Female , Genetic Linkage , Genetic Testing , Hispanic or Latino/genetics , Humans , Hypertrophy, Left Ventricular/epidemiology , Hypertrophy, Left Ventricular/etiology , Incidence , Lod Score , Male , Mutation, Missense , Pedigree , Penetrance , Phenotype , Prognosis , Survival Rate , Tropomyosin/metabolism
9.
J Mol Biol ; 302(3): 593-606, 2000 Sep 22.
Article in English | MEDLINE | ID: mdl-10986121

ABSTRACT

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.


Subject(s)
Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Actins/metabolism , Actins/ultrastructure , Tropomyosin/metabolism , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/genetics , Actins/chemistry , Actins/genetics , Animals , Binding, Competitive , Calcium/metabolism , Calcium/pharmacology , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fungal Proteins/ultrastructure , Microscopy, Electron , Models, Molecular , Movement/drug effects , Muscle, Skeletal/chemistry , Muscle, Skeletal/ultrastructure , Mutation , Myosins/metabolism , Myosins/pharmacology , Protein Binding/drug effects , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/ultrastructure , Protein Structure, Quaternary/drug effects , Rabbits , Thermodynamics , Tropomyosin/chemistry , Tropomyosin/genetics , Tropomyosin/ultrastructure , Troponin/metabolism , Troponin/pharmacology , Yeasts
10.
J Biol Chem ; 275(36): 27587-93, 2000 Sep 08.
Article in English | MEDLINE | ID: mdl-10864931

ABSTRACT

Cooperative myosin binding to the thin filament is critical to regulation of cardiac and skeletal muscle contraction. This report delineates and fits to experimental data a new model of this process, in which specific tropomyosin-actin interactions are important, the tropomyosin-tropomyosin polymer is continuous rather than disjointed, and tropomyosin affects myosin-actin binding by shifting among three positions as in recent structural studies. A myosin- and tropomyosin-induced conformational change in actin is proposed, rationalizing the approximately 10,000-fold strengthening effect of myosin on tropomyosin-actin binding. Also, myosin S1 binding to regulated filaments containing mutant tropomyosins with internal deletions exhibited exaggerated cooperativity, implying an allosteric effect of tropomyosin on actin and allowing the effect's measurement. Comparisons among the mutants suggest the change in actin is promoted much more strongly by the middle of tropomyosin than by its ends. Regardless of calcium binding to troponin, this change in actin facilitates the shift in tropomyosin position to the actin inner domain, which is required for tight myosin-actin association. It also increases myosin-actin affinity 7-fold compared with the absence of troponin-tropomyosin. Finally, initiation of a shift in tropomyosin position is 100-fold more difficult than is its extension from one actin to the next, producing the myosin binding cooperativity that underlies cooperative activation of muscle contraction.


Subject(s)
Actins/chemistry , Actins/metabolism , Myosins/chemistry , Myosins/metabolism , Tropomyosin/metabolism , Animals , Cattle , Kinetics , Models, Biological , Models, Chemical , Models, Molecular , Muscle Fibers, Fast-Twitch/physiology , Muscle, Skeletal/physiology , Myosin Subfragments/chemistry , Myosin Subfragments/metabolism , Protein Conformation , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Deletion , Tropomyosin/chemistry
11.
J Biol Chem ; 275(29): 22470-8, 2000 Jul 21.
Article in English | MEDLINE | ID: mdl-10801864

ABSTRACT

Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca(2+) to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca(2+) showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosin-actin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.


Subject(s)
Actin Cytoskeleton/metabolism , Actins/genetics , Actins/metabolism , Tropomyosin/metabolism , Troponin/metabolism , Actin Cytoskeleton/ultrastructure , Animals , Binding Sites , Cattle , Muscle Contraction , Mutation , Protein Binding , Protein Folding , Saccharomyces cerevisiae
12.
J Physiol ; 524 Pt 1: 233-43, 2000 Apr 01.
Article in English | MEDLINE | ID: mdl-10747195

ABSTRACT

1. Measurements of the unloaded sliding speed of and isometric force exerted on single thin filaments in in vitro motility assays were made to evaluate the role of regulatory proteins in the control of unloaded thin filament sliding speed and isometric force production. 2. Regulated actin filaments were reconstituted from rabbit F-actin, native bovine cardiac tropomyosin (nTm), and either native bovine cardiac troponin (nTn), troponin containing a TnC mutant, CBMII, in which the sole regulatory site in cardiac TnC (site II) is inactivated (CBMII-Tn), or troponin containing a point mutation in TnT (I79N, where isoleucine at position 79 is replaced with asparagine) associated with familial hypertrophic cardiomyopathy (FHC). 3. Addition of regulatory proteins to the thin filament increases both the unloaded sliding speed and the isometric force exerted by myosin heads on the thin filaments. 4. Variation of thin filament activation by varying [Ca2+] or the fraction of CBMII/TnC bound to the thin filament at pCa 5, had little effect on the unloaded filament sliding speed until the fraction of the thin filament containing calcium bound to TnC was less than 0.15. These results suggest that [Ca2+] primarily affects the number of attached and cycling crossbridges. 5. The presence of the FHC TnT mutant increased the thin filament sliding speed but reduced the isometric force that heavy meromyosin exerted on regulated thin filaments. These latter results, together with the increased sliding speed and isometric force seen in the presence of regulatory proteins, suggest that thin filament regulatory proteins exert significant allosteric effects on the interaction of crossbridges with the thin filament.


Subject(s)
Actins/physiology , Muscle, Skeletal/physiology , Tropomyosin/physiology , Troponin/physiology , Actins/chemistry , Adenine Nucleotides/metabolism , Amino Acid Substitution , Animals , Cattle , Heart/physiology , Isometric Contraction , Models, Biological , Movement , Muscle, Skeletal/chemistry , Myocardium/chemistry , Point Mutation , Rabbits , Tropomyosin/chemistry , Troponin/chemistry , Troponin C/chemistry , Troponin C/physiology
13.
Biophys J ; 78(2): 908-17, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10653803

ABSTRACT

Interactions of the components of reconstituted thin filaments were investigated using a tropomyosin internal deletion mutant, D234, in which actin-binding pseudo-repeats 2, 3, and 4 are missing. D234 retains regions of tropomyosin that bind troponin and form end-to-end tropomyosin bonds, but has a length to span only four instead of seven actin monomers. It inhibits acto-myosin subfragment 1 ATPase (acto-S-1 ATPase) and filament sliding in vitro in both the presence and absence of Ca(2+) (, J. Biol. Chem. 272:14051-14056) and lowers the affinity of S-1.ADP for actin while increasing its cooperative binding. Electron microscopy and three-dimensional reconstruction of reconstituted thin filaments containing actin, troponin, and wild-type or D234 tropomyosin were carried out to determine if Ca(2+)-induced movement of D234 occurred in the filaments. In the presence and absence of Ca(2+), the D234 position was indistinguishable from that of the wild-type tropomyosin, demonstrating that the mutation did not affect normal tropomyosin movement induced by Ca(2+) and troponin. These results suggested that, in the presence of Ca(2+) and troponin, D234 tropomyosin was trapped on filaments in the Ca(2+)-induced position and was unable to undergo a transition to a completely activated position. By adding small amounts of rigor-bonded N-ethyl-maleimide-treated S-1 to mutant thin filaments, thus mimicking the myosin-induced "open" state, inhibition could be overcome and full activation restored. This myosin requirement for full activation provides support for the existence of three functionally distinct thin filament states (off, Ca(2+)-induced, myosin-induced; cf.;, J. Mol. Biol. 266:8-14). We propose a further refinement of the three-state model in which the binding of myosin to actin causes allosteric changes in actin that promote the binding of tropomyosin in an otherwise energetically unfavorable "open" state.


Subject(s)
Actin Cytoskeleton/ultrastructure , Tropomyosin/genetics , Actins/ultrastructure , Allosteric Regulation , Animals , Calcium/pharmacology , Ethylmaleimide/pharmacology , Microscopy, Electron , Models, Biological , Models, Molecular , Muscle, Skeletal/ultrastructure , Mutation , Myosins/ultrastructure , Protein Binding , Rats , Tropomyosin/ultrastructure , Troponin/ultrastructure
14.
J Biol Chem ; 274(44): 31279-85, 1999 Oct 29.
Article in English | MEDLINE | ID: mdl-10531325

ABSTRACT

Striated muscle tropomyosin spans seven actin monomers and contains seven quasi-repeating regions with loose sequence similarity. Each region contains a hypothesized actin binding motif. To examine the functions of these regions, full-length tropomyosin was compared with tropomyosin internal deletion mutants spanning either five or four actins. Actin-troponin-tropomyosin filaments lacking tropomyosin regions 2-3 exhibited calcium-sensitive regulation in in vitro motility and myosin S1 ATP hydrolysis experiments, similar to filaments with full-length tropomyosin. In contrast, filaments lacking tropomyosin regions 3-4 were inhibitory to these myosin functions. Deletion of regions 2-4, 3-5, or 4-6 had little effect on tropomyosin binding to actin in the presence of troponin or troponin-Ca(2+), or in the absence of troponin. However, all of these mutants inhibited myosin cycling. Deletion of the quasi-repeating regions diminished the prominent effect of myosin S1 on tropomyosin-actin binding. Interruption of this cooperative, myosin-tropomyosin interaction was least severe for the mutant lacking regions 2-3 and therefore correlated with inhibition of myosin cycling. Regions 3, 4, and 5 each contributed about 1.5 kcal/mol to this process, whereas regions 2 and 6 contributed much less. We suggest that a myosin-induced conformational change in actin facilitates the azimuthal repositioning of tropomyosin which is an essential part of regulation.


Subject(s)
Muscle, Skeletal/physiology , Myofibrils/physiology , Peptide Fragments/metabolism , Tropomyosin/metabolism , Actins/metabolism , Ca(2+) Mg(2+)-ATPase/metabolism , Calcium , Movement , Myosin Subfragments/metabolism , Peptide Fragments/genetics , Protein Binding , Sequence Deletion , Thermodynamics , Tropomyosin/genetics , Troponin/metabolism
15.
J Biol Chem ; 274(40): 28363-70, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10497196

ABSTRACT

Missense mutations in the cardiac thin filament protein troponin T (TnT) are a cause of familial hypertrophic cardiomyopathy (FHC). To understand how these mutations produce dysfunction, five TnTs were produced and purified containing FHC mutations found in several regions of TnT. Functional defects were diverse. Mutations F110I, E244D, and COOH-terminal truncation weakened the affinity of troponin for the thin filament. Mutation DeltaE160 resulted in thin filaments with increased calcium affinity at the regulatory site of troponin C. Mutations R92Q and F110I resulted in impaired troponin solubility, suggesting abnormal protein folding. Depending upon the mutation, the in vitro unloaded actin-myosin sliding speed showed small increases, showed small decreases, or was unchanged. COOH-terminal truncation mutation resulted in a decreased thin filament-myosin subfragment 1 MgATPase rate. The results indicate that the mutations cause diverse immediate effects, despite similarities in disease manifestations. Separable but repeatedly observed abnormalities resulting from FHC TnT mutations include increased unloaded sliding speed, increased or decreased Ca(2+) affinity, impairment of folding or sarcomeric integrity, and decreased force. Enhancement as well as impairment of contractile protein function is observed, suggesting that TnT, including the troponin tail region, modulates the regulation of cardiac contraction.


Subject(s)
Cardiomyopathy, Hypertrophic/genetics , Mutation , Troponin T/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , DNA, Complementary , Molecular Sequence Data , Troponin T/genetics
16.
J Biol Chem ; 274(32): 22191-6, 1999 Aug 06.
Article in English | MEDLINE | ID: mdl-10428784

ABSTRACT

Thin filament-mediated regulation of striated muscle contraction involves conformational switching among a few quaternary structures, with transitions induced by binding of Ca(2+) and myosin. We establish and exploit Saccharomyces cerevisiae actin as a model system to investigate this process. Ca(2+)-sensitive troponin-tropomyosin binding affinities for wild type yeast actin are seen to closely resemble those for muscle actin, and these hybrid thin filaments produce Ca(2+)-sensitive regulation of the myosin S-1 MgATPase rate. Yeast actin filament inner domain mutant K315A/E316A depresses Ca(2+) activation of the MgATPase rate, producing a 4-fold weakening of the apparent Ca(2+) affinity and a 50% decrease in the MgATPase rate at saturating Ca(2+) concentration. Observed destabilization of troponin-tropomyosin binding to actin in the presence of Ca(2+), a 1.4-fold effect, provides a partial explanation. Despite the decrease in apparent MgATPase Ca(2+) affinity, there was no detectable change in the true Ca(2+) affinity of the thin filament, measured using fluorophore-labeled troponin. Another inner domain mutant, E311A/R312A, decreased the MgATPase rate but did not change the apparent Ca(2+) affinity. These results suggest that charged residues on the surface of the actin inner domain are important in Ca(2+)- and myosin-induced thin filament activation.


Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Tropomyosin/metabolism , Troponin/metabolism , Actins/genetics , Alanine/genetics , Animals , Ca(2+) Mg(2+)-ATPase/metabolism , Calcium/metabolism , Cattle , Models, Chemical , Muscle Contraction/physiology , Mutation , Myosin Subfragments/metabolism , Osmolar Concentration , Protein Binding , Titrimetry , Troponin C/metabolism
17.
J Biol Chem ; 274(11): 7157-64, 1999 Mar 12.
Article in English | MEDLINE | ID: mdl-10066775

ABSTRACT

Striated muscle contraction is regulated by Ca2+ binding to troponin, which has a globular domain and an elongated tail attributable to the NH2-terminal portion of the bovine cardiac troponin T (TnT) subunit. Truncation of the bovine cardiac troponin tail was investigated using recombinant TnT fragments and subunits TnI and TnC. Progressive truncation of the troponin tail caused progressively weaker binding of troponin-tropomyosin to actin and of troponin to actin-tropomyosin. A sharp drop-off in affinity occurred with NH2-terminal deletion of 119 rather than 94 residues. Deletion of 94 residues had no effect on Ca2+-activation of the myosin subfragment 1-thin filament MgATPase rate and did not eliminate cooperative effects of Ca2+ binding. Troponin tail peptide TnT1-153 strongly promoted tropomyosin binding to actin in the absence of TnI or TnC. The results show that the anchoring function of the troponin tail involves interactions with actin as well as with tropomyosin and has comparable importance in the presence or absence of Ca2+. Residues 95-153 are particularly important for anchoring, and residues 95-119 are crucial for function or local folding. Because striated muscle regulation involves switching among the conformational states of the thin filament, regulatory significance for the troponin tail may arise from its prominent contribution to the protein-protein interactions within these conformations.


Subject(s)
Muscle Fibers, Skeletal/metabolism , Mutation , Myocardium/metabolism , Troponin T/metabolism , Actins/metabolism , Animals , Cattle , DNA, Complementary , Energy Metabolism , Protein Binding , Sequence Deletion , Tropomyosin/metabolism , Troponin T/genetics
19.
Biophys J ; 73(4): 1763-70, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9336171

ABSTRACT

New features of the structure and interactions of troponin T and tropomyosin have been revealed by electron microscopy of so-called double-diamond co-crystals. These co-crystals were formed using rabbit alpha2 tropomyosin complexed with troponin T from either skeletal or cardiac muscle, which have different lengths in the amino-terminal region, as well as a bacterially expressed skeletal muscle troponin T fragment of 190 residues that lacks the amino-terminal region. Differences in the images of the co-crystals have allowed us to establish the polarities of both the troponin T subunit and tropomyosin in the projected lattice. Moreover, in agreement with their sequences, the amino-terminal region of a bovine cardiac muscle troponin T isoform appears to be longer than that from the rabbit skeletal muscle troponin T isoform and to span more of the amino terminus of tropomyosin at the head-to-tail filament joints. Images of crystals tilted relative to the electron beam also reveal the supercoiling of the tropomyosin filaments in this lattice. Based on these results, a three-dimensional model of the double-diamond lattice has been constructed.


Subject(s)
Tropomyosin/chemistry , Troponin/chemistry , Animals , Binding Sites , Biophysical Phenomena , Biophysics , Cattle , Crystallization , In Vitro Techniques , Microscopy, Electron , Models, Molecular , Muscle, Skeletal/chemistry , Myocardium/chemistry , Protein Binding , Protein Conformation , Rabbits , Tropomyosin/ultrastructure , Troponin/ultrastructure , Troponin T
20.
J Biol Chem ; 272(22): 14051-6, 1997 May 30.
Article in English | MEDLINE | ID: mdl-9162027

ABSTRACT

The function of three of tropomyosin's sequential quasiequivalent regions was studied by deletion from skeletal muscle alpha-tropomyosin of internal residues 49-167. This deletion mutant tropomyosin spans four instead of the normal seven actins, and most of the tropomyosin region believed to interact with troponin is retained and uninterrupted in the mutant. The mutant tropomyosin was compared with a full-length control molecule that was modified to functionally resemble muscle tropomyosin (Monteiro, P. B., Lataro, R. C., Ferro, J. A., and Reinach, F. C. (1994) J. Biol. Chem. 269, 10461-10466). The tropomyosin deletion suppressed the actin-myosin subfragment 1 MgATPase rate and the in vitro sliding of thin filaments over a heavy meromyosin-coated surface. This inhibition was not reversed by troponin plus Ca2+. Comparable tropomyosin affinities for actin, regardless of the deletion, suggest that the deleted region has little interaction with actin in the absence of other proteins. Similarly, the deletion did not weaken binding of the troponin-tropomyosin complex to actin. Furthermore, Ca2+ had a 2-fold effect on troponin-tropomyosin's affinity for actin, regardless of the deletion. Notably, the deletion greatly weakened tropomyosin binding to myosin subfragment 1-decorated actin, with the full-length tropomyosin having a 100-fold greater affinity. The inhibitory properties resulting from the deletion are attributed to defective stabilization of the myosin-induced active state of the thin filament.


Subject(s)
Actin Cytoskeleton/chemistry , Muscle, Skeletal/chemistry , Tropomyosin/chemistry , Actin Cytoskeleton/ultrastructure , Animals , Cattle , Gene Deletion , Muscle, Skeletal/ultrastructure , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/ultrastructure , Tropomyosin/genetics , Tropomyosin/ultrastructure
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