Ca(2+)- and S1-induced movement of troponin T on reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy

Troponin T (TnT) is an essential component of troponin (Tn) for the Ca(2+)-regulation of vertebrate striated muscle contraction. TnT consists of an extended NH(2)-terminal domain that interacts with tropomyosin (Tm) and a globular COOH-terminal domain that interacts with Tm, troponin I (TnI), and troponin C (TnC). We have generated two mutants of a rabbit skeletal beta-TnT 25-kDa fragment (59-266) that have a unique cysteine at position 60 (N-terminal region) or 250 (C-terminal region). To understand the spatial rearrangement of TnT on the thin filament in response to Ca(2+) binding to TnC, we measured distances from Cys-60 and Cys-250 of TnT to Gln-41 and Cys-374 of F-actin on the reconstituted thin filament by using fluorescence resonance energy transfer (FRET). The distances from Cys-60 and Cys-250 of TnT to Gln-41 of F-actin were 39.5 and 30.0 A, respectively in the absence of Ca(2+), and increased by 2.6 and 5.8 A, respectively upon binding of Ca(2+) to TnC. The rigor binding of myosin subfragment 1 (S1) further increased these distances by 4 and 5 A respectively, when the thin filaments were fully decorated with S1. This indicates that not only the C-terminal but also the N-terminal region of TnT showed the Ca(2+)- and S1-induced movement, and the C-terminal region moved more than N-terminal region. In the absence of Ca(2+), the rigor S1 binding also increased the distances to the same extent as the presence of Ca(2+) when the thin filaments were fully decorated with S1. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding both in the presence and absence of Ca(2+). However, plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed hyperbolic curve in the presence of Ca(2+) but sigmoidal curve in the absence of Ca(2+). FRET measurement of the distances from Cys-60 and Cys-250 of TnT to Cys-374 of actin showed almost the same results as the case of Gln-41 of actin. The present FRET measurements demonstrated that not only TnI but also TnT change their positions on the thin filament corresponding to three states of thin filaments (relaxed, Ca(2+)-induced or closed, and S1-induced or open states).     

Ca(2+)-induced movement of tropomyosin in skeletal muscle thin filaments observed by multi-site FRET

To obtain information on Ca(2+)-induced tropomyosin (Tm) movement in Ca(2+)-regulated muscle thin filaments, frequency-domain fluorescence energy transfer data were collected between 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid at Cys-190 of Tm and phalloidin-tetramethylrhodamine B isothiocyanate bound to F-actin. Two models were used to fit the experimental data: an atomic coordinate (AC) model coupled with a search algorithm that varies the position and orientation of Tm on F-actin, and a double Gaussian distance distribution (DD) model. The AC model showed that little or no change in transfer efficiency is to be expected between different sites on F-actin and Tm if Ca(2+) causes azimuthal movement of Tm of the magnitude suggested by structural data (C. Xu, R. Craig, L. Tobacman, R. Horowitz, and W. Lehman. 1999. Biophys. J. 77:985-992). However, Ca(2+) produced a small but significant change in our phase/modulation versus frequency data, showing that changes in lifetime decay can be detected even when a change of the steady-state transfer efficiency is very small. A change in Tm azimuthal position of 17 on the actin filament obtained with the AC model indicates that solution data are in reasonable agreement with EM image reconstruction data. In addition, the data indicate that Tm also appears to rotate about its axis, resulting in a rolling motion over the F-actin surface. The DD model showed that the distance from one of the two chains of Tm to F-actin was mainly affected, further verifying that Ca(2+) causes Tm to roll over the F-actin surface. The width of the distance distributions indicated that the position of Tm in absence and in presence of Ca(2+) is well defined with appreciable local flexibility.     

Ca2+ -induced tropomyosin movement in scallop striated muscle thin filaments

Striated muscle contraction in most animals is regulated at least in part by the troponin-tropomyosin (Tn-Tm) switch on the thin (actin-containing) filaments. The only group that has been suggested to lack actin-linked regulation is the mollusks, where contraction is regulated through the myosin heads on the thick filaments. However, molluscan gene sequence data suggest the presence of troponin (Tn) components, consistent with actin-linked regulation, and some biochemical and immunological data also support this idea. The presence of actin-linked (in addition to myosin-linked) regulation in mollusks would simplify our general picture of muscle regulation by extending actin-linked regulation to this phylum as well. We have investigated this question structurally by determining the effect of Ca²⁺ on the position of Tm in native thin filaments from scallop striated adductor muscle. Three-dimensional reconstructions of negatively stained filaments were determined by electron microscopy and single-particle image analysis. At low Ca²⁺, Tm appeared to occupy the “blocking” position, on the outer domain of actin, identified in earlier studies of regulated thin filaments in the low-Ca²⁺ state. In this position, Tm would sterically block myosin binding, switching off filament activity. At high Ca²⁺, Tm appeared to move toward a position on the inner domain, similar to that induced by Ca²⁺ in regulated thin filaments. This Ca²⁺-induced movement of Tm is consistent with the hypothesis that scallop thin filaments are Ca²⁺ regulated.     

Ca(2+)-induced switching of troponin and tropomyosin on actin filaments as revealed by electron cryo-microscopy

Muscle contraction is regulated by the intracellular Ca(2+ )concentration. In vertebrate striated muscle, troponin and tropomyosin on actin filaments comprise a Ca(2+)-sensitive switch that controls contraction. Ca(2+ )binds to troponin and triggers a series of changes in actin-containing filaments that lead to cyclic interactions with myosin that generate contraction. However, the precise location of troponin relative to actin and tropomyosin and how its structure changes with Ca(2+ )have been not determined. To understand the regulatory mechanism, we visualized the location of troponin by determining the three-dimensional structure of thin filaments from electron cryo-micrographs without imposing helical symmetry to approximately 35 A resolution. With Ca(2+), the globular domain of troponin was gourd-shaped and was located over the inner domain of actin. Without Ca(2+), the main body of troponin was shifted by approximately 30 A towards the outer domain and bifurcated, with a horizontal branch (troponin arm) covering the N and C-terminal regions of actin. The C-terminal one-third of tropomyosin shifted towards the outer domain of actin by approximately 35 A supporting the steric blocking model, however it is surprising that the N-terminal half of tropomyosin shifted less than approximately 12 A. Therefore tropomyosin shifted differentially without Ca(2+). With Ca(2+), tropomyosin was located entirely over the inner domain thereby allowing greater access of myosin for force generation. The interpretation of three-dimensional maps was facilitated by determining the three-dimensional positions of fluorophores labelled on specific sites of troponin or tropomyosin by applying probabilistic distance geometry to data from fluorescence resonance energy transfer measurements.     

Increased Ca2+ affinity of cardiac thin filaments reconstituted with cardiomyopathy-related mutant cardiac troponin I

To understand the molecular mechanisms whereby cardiomyopathy-related cardiac troponin I (cTnI) mutations affect myofilament activity, we have investigated the Ca2+ binding properties of various assemblies of the regulatory components that contain one of the cardiomyopahty-related mutant cTnI. Acto-S1 ATPase activities in reconstituted systems were also determined. We investigated R145G and R145W mutations from the inhibitory region and D190H and R192H mutations from the second actin-tropomyosin-binding site. Each of the four mutations sensitized the acto-S1 ATPase to Ca2+. Whereas the mutations from the inhibitory region increased the basal level of ATPase activity, those from the second actin-tropomyosin-binding site did not. The effects on the Ca2+ binding properties of the troponin ternary complex and the troponin-tropomyosin complex with one of four mutations were either desensitization or no effect compared with those with wild-type cTnI. All of the mutations, however, affected the Ca2+ sensitivities of the reconstituted thin filaments in the same direction as the acto-S1 ATPase activity. Also the thin filaments with one of the mutant cTnIs bound Ca2+ with less cooperativity compared with those with wild-type cTnI. These data indicate that the mutations found in the inhibitory region and those from the second actin-tropomyosin site shift the equilibrium of the states of the thin filaments differently. Moreover, the increased Ca2+ bound to myofilaments containing the mutant cTnIs may be an important factor in triggered arrhythmias associated with the cardiomyopathy.     

Inhibitory region of troponin I: Ca(2+)-dependent structural and environmental changes in the troponin-tropomyosin complex and in reconstituted thin filaments

In muscle thin filaments, the inhibitory region (residues 96-117) of troponin I (TnI) is thought to interact with troponin C (TnC) in the presence of Ca(2+) and with actin in the absence of Ca(2+). To better understand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys-117 and labeled them with the thiol-specific fluorescent probe N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the microenvironments of the AEDANS labels on TnI in the presence and absence of Ca(2+) by measuring the extent of acrylamide quenching of fluorescence and lifetime-resolved anisotropy. In the troponin-tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys-96 and Cys-117 were less accessible to solvent and less flexible in the presence of Ca(2+), reflecting closer interactions with TnC under these conditions. In reconstituted thin filaments, the environment of the AEDANS on Cys-96 was not greatly affected by Ca(2+), while the AEDANS on Cys-117 was more accessible but significantly less flexible as it moved away from actin and interacted strongly with TnC in the presence of Ca(2+). We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANS on TnI Cys-96 or Cys-117 and 4-?[(dimethylamino)phenyl]azo?phenyl-4'-maleimide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca(2+), the mean distances were 40.2 A for Cys-96 and 35.2 A for Cys-117. In the presence of Ca(2+), Cys-96 moved away from actin Cys-374 by approximately 3.6 A, while Cys-117 moved away by approximately 8 A. This suggests the existence of a flexible "hinge" region near the middle of TnI, allowing amino acid residues in the N-terminal half of TnI to interact with TnC in a Ca(2+)-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca(2+) or to TnC in the presence of Ca(2+). This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.     

Effect of ethanol on tropomyosin in solution and in reconstituted thin filaments

The effects of ethanol at concentrations below 10% on the conformation of tropomyosin, its end-to-end polymerization, its binding to F-actin, and its effects on actomyosin ATPase activity were studied. Ethanol stabilized the tropomyosin conformation by shifting the helix thermal unfolding profile to higher temperatures, and increased the end-to-end polymerization of tropomyosin. Ethanol-induced changes in the excimer fluorescence of pyrene-tropomyosin indicated that its conformation was stabilized by ethanol both free and bound to F-actin. Effects of tropomyosin and tropomyosin-troponin on actomyosin ATPase activity were measured under conditions for which tropomyosin binding to F-actin increases the activity. Under conditions for which the binding of tropomyosin to F-actin is optimum, in the presence of tropomyosin, the actomyosin ATPase activity decreased as the ethanol concentration increased, further indicating that ethanol induces a structural change in the tropomyosin-F-actin complex. Under conditions for which the binding of tropomyosin to F-actin is weak (low salt or high temperature), addition of ethanol increased the ATPase activity due to increased binding of tropomyosin to F-actin. Thus, ethanol appears to modify actomyosin ATPase activity by increasing the binding of tropomyosin to F-actin and affecting the structure of tropomyosin in the tropomyosin-F-actin filament.     

Crosslinking of tropomyosin to myosin subfragment-1 in reconstituted rabbit skeletal thin filaments

Rabbit skeletal tropomyosin was labeled with the bifunctional photoactivatable crosslinker N-succinimidyl-6- (4'-azido-2'-nitrophenylamino)hexanoate. After irradiating the rigor complex composed of myosin subfragment-1, crosslinker-labeled tropomyosin, and F-actin, a crosslinked product was formed. This product was identified as a 1:1 adduct of tropomyosin and subfragment-1. This finding is in support of recent structural studies which suggest that tropomyosin and subfragment-1 are in close proximity to each other, and may be relevant to the mechanism of thin filament regulation.     

Ca2+-induced rolling of tropomyosin in muscle thin filaments: the alpha- and beta-band hypothesis revisited

Tropomyosin is a filamentous coiled-coil protein directly involved in the regulation of the actomyosin interaction responsible for muscle contraction: it transmits the local calcium-induced conformational change in troponin to the helical array of myosin-binding sites on the surface of the actin filament. McLachlan and Stewart (McLachlan, A. D., and Stewart, M. (1976) J. Mol. Biol. 103, 271-298) proposed that the tropomyosin coiled-coil structure can be divided into 14 alternating 19- to 20-residue "alpha- and beta-bands," which could act as alternate 7-fold sets of sites for specific binding to actin in the different conformational states of the regulated thin filament. Here we present the first direct experimental evidence in support of the alpha- and beta-band hypothesis: we analyze the acrylamide quenching of the fluorescence of mutant tropomyosins containing 5-hydroxytryptophan residues at different positions along the coiled-coil structure under a variety of conditions (alone, complexed with actin, and complexed with actin and troponin with or without Ca(2+)). We show that fluorescent probes placed in the alpha-bands become less solvent-exposed in the absence of calcium, whereas those in the beta-bands become less solvent-exposed in the presence of calcium. A model in which the tropomyosin coiled-coil rolls across the actin surface in response to Ca(2+)-binding to troponin most easily explains these observations.     

Akazara scallop troponin C: Ca(2+)-induced conformational change and interaction with rabbit troponin subunits.

The number of specific Ca2+ bound to Akazara scallop troponin C was estimated to be 0.7 with an apparent binding constant of 5 x 10(5) M-1 (T. Ojima and K. Nishita, 1986, J. Biol. Chem. 261, 16749-16754). In the present paper, we report on the Ca(2+)-induced conformational changes in the troponin C and the interaction of the troponin C with rabbit troponin subunits. The Ca2+ binding to the troponin C caused a marked change in difference uv absorption spectra and a retardation of elution on Sephacryl S-200 gel filtration. However, its circular dichroism spectrum was hardly changed by the Ca2+ binding. These results suggest that the Ca2+ binding to the troponin C induced changes predominantly in tertiary structure rather than in secondary structure. Akazara scallop troponin C was shown to be able to bind to rabbit troponin I-Cellulofine affinity column, but the affinity was not greatly increased by Ca2+ unlike the case of rabbit troponin C. On hybridizing with rabbit troponin T and I, Akazara scallop troponin C was shown to be incapable of substituting rabbit troponin C; i.e., the hybrid troponin strongly inhibited the Mg-ATPase activity of rabbit actomyosin-tropomyosin irrespective of the presence or absence of Ca2+, thus recovering no Ca2+ sensitivity.     

Ca(2+) activation of myofilaments from transgenic mouse hearts expressing R92Q mutant cardiac troponin T

The functional consequences of the R92Q mutation in cardiac troponin T (cTnT), linked to familial hypertrophic cardiomyopathy in humans, are not well understood. We have studied steady- and pre-steady-state mechanical activity of detergent-skinned fiber bundles from a transgenic (TG) mouse model in which 67% of the total cTnT in the heart was replaced by the R92Q mutant cTnT. TG fibers were more sensitive to Ca(2+) than nontransgenic (NTG) fibers [negative logarithm of half maximally activating molar Ca(2+) (pCa(50)) = 5.84 +/- 0.01 and 6.12 +/- 0.01 for NTG and TG fibers, respectively]. The shift in pCa(50) caused by increasing the sarcomere length from 1.9 to 2.3 microm was significantly higher for TG than for NTG fibers (DeltapCa(50) = 0.13 +/- 0.01 and 0.29 +/- 0.02 for NTG and TG fibers, respectively). The relationships between rate of ATP consumption and steady-state isometric tension were linear, and the slopes were the same in NTG and TG fibers. Rate of tension redevelopment was more sensitive to Ca(2+) in TG than in NTG fibers (pCa(50) = 5.71 +/- 0.02 and 6.07 +/- 0.02 for NTG and TG fibers, respectively). We concluded that overall cross-bridge cycling kinetics are not altered by the R92Q mutation but that altered troponin-tropomyosin interactions could be responsible for the increase in myofilament Ca(2+) sensitivity in TG myofilaments.     

Ca2+- and S1-induced conformational changes of reconstituted skeletal muscle thin filaments observed by fluorescence energy transfer spectroscopy: structural evidence for three States of thin filament

Rabbit skeletal muscle alpha-tropomyosin (Tm) and the deletion mutant (D234Tm) in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056] were used to investigate the interaction between actin and tropomyosin or actin and troponin (Tn) by means of fluorescence resonance energy transfer (FRET). FRET between Cys-190 of D234Tm and Gln-41 or Cys-374 of actin did not cause any significant Ca2+-induced movement of D234Tm, as reported previously for native Tm [Miki et al. (1998) J. Biochem. 123, 1104-1111]. FRET did not show any significant S1-induced movement of Tm and D234Tm on thin filaments either. The distances between Cys-133 of TnI, and Gln-41 and Cys-374 of actin on thin filaments reconstituted with D234Tm (mutant thin filaments) were almost the same as those on thin filaments with native Tm (wild-type thin filaments) in the absence of Ca2+. Upon binding of Ca2+ to TnC, these distances on mutant thin filaments increased by approximately 10 A in the same way as on wild-type thin filaments, which corresponds to a Ca2+-induced conformational change of thin filaments [Miki et al. (1998) J. Biochem. 123, 324-331]. The rigor binding of myosin subfragment 1 (S1) further increased these distances by approximately 7 A on both wild-type and mutant thin filaments when the thin filaments were fully decorated with S1. This indicates that a further conformational change on thin filaments was induced by S1 rigor-binding (S1-induced or open state). Plots of the extent of S1-induced conformational change vs. molar ratio of S1 to actin showed that the curve for wild-type thin filaments is hyperbolic, whereas that for mutant thin filaments is sigmoidal. This suggests that the transition to the S1-induced state on mutant thin filaments is depressed with a low population of rigor S1. In the absence of Ca2+, the distance also increased on both wild-type and mutant thin filaments close to the level in the presence of Ca2+ as the molar ratio of S1 to actin increased up to 1. The curves are sigmoidal for both wild-type and mutant thin filaments. The addition of ATP completely reversed the changes in FRET induced by rigor S1 binding. For mutant thin filaments, the transition from the closed state to the open state in the presence of ATP is strongly depressed, which results in the inhibition of acto-myosin ATPase even in the presence of Ca2+. The present FRET measurements provide structural evidence for three states of thin filaments (relaxed, Ca2+-induced or closed, and S1-induced or open states) for the regulation mechanism of skeletal muscle contraction.     

The second half of the fourth period of tropomyosin is a key region for Ca(2+)-dependent regulation of striated muscle thin filaments

Rabbit skeletal muscle alpha-tropomyosin (Tm), a 284-residue dimeric coiled-coil protein, spans seven actin monomers and contains seven quasiequivalent periods. X-ray analysis of cocrystals of Tm and troponin (Tn) placed the Tn core domain near residues 150-180 of Tm. To identify the Ca(2+)-sensitive Tn interaction site on Tm, we generated three Tm mutants to compare the consequences of sequence substitution inside and outside of the Tn core domain-binding region. Residues 152-165 and 156-162 in the second half of period 4 were replaced by corresponding residues 33-46 and 37-43 in the second half of period 1, respectively (termed mTm152-165 and mTm156-162, respectively), and residues 134-147 in the first half of period 4 were replaced with residues 15-28 in the first half of period 1 (mTm134-147). Recombinant Tms designed with an additional tripeptide, Ala-Ala-Ser, at the N-terminus were expressed in Escherichia coli. Both mTm152-165 and mTm156-162 suppressed the actin-activated myosin subfragment-1 Mg(2+)-ATPase rate regardless of whether Ca(2+) and Tn were present. On the other hand, mTm134-147 retained the normal Ca(2+)-sensitive regulation, although the actin binding of mTm alone was significantly impaired. Differential scanning calorimetry showed that the sequence substitution in the second half of period 4 affected the thermal stability of the complete Tm molecule and also the actin-induced stabilization. These results suggest that the second half of period 4 of Tm is a key region for inducing conformational changes of the regulated thin filament required for its fully activated state.     

RyR3 amplifies RyR1-mediated Ca(2+)-induced Ca(2+) release in neonatal mammalian skeletal muscle

The neonatal mammalian skeletal muscle contains both type 1 and type 3 ryanodine receptors (RyR1 and RyR3) located in the sarcoplasmic reticulum membrane. An allosteric interaction between RyR1 and dihydropyridine receptors located in the plasma membrane mediates voltage-induced Ca(2+) release (VICR) from the sarcoplasmic reticulum. RyR3, which disappears in adult muscle, is not involved in VICR, and the role of the transiently expressed RyR3 remains elusive. Here we demonstrate that RyR1 participates in both VICR and Ca(2+)-induced Ca(2+) release (CICR) and that RyR3 amplifies RyR1-mediated CICR in neonatal skeletal muscle. Confocal measurements of intracellular Ca(2+) in primary cultured mouse skeletal myotubes reveal active sites of Ca(2+) release caused by peripheral coupling between dihydropyridine receptors and RyR1. In myotubes lacking RyR3, the peripheral VICR component is unaffected, and RyR1s alone are able to support inward CICR propagation in most cells at an average speed of approximately 190 microm/s. With the co-presence of RyR1 and RyR3 in wild-type cells, unmitigated radial CICR propagates at 2,440 microm/s. Because neonatal skeletal muscle lacks a well developed transverse tubule system, the RyR3 reinforcement of CICR seems to ensure a robust, uniform, and synchronous activation of Ca(2+) release throughout the cell body. Such functional interplay between RyR1 and RyR3 can serve important roles in Ca(2+) signaling of cell differentiation and muscle contraction.     

Removal of tropomyosin overlap modifies cooperative binding of myosin S-1 to reconstituted thin filaments of rabbit striated muscle

Cooperative binding of myosin S-1.ADP to regulated F-actin was previously reported and has been interpreted by a two-state model in which an important source of cooperativity is nearest neighbor interactions between the 7-actin.tropomyosin (TM).troponin units (functional units) (Hill, T.L., Eisenberg, E., and Greene, L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186-3190). It has been postulated that the head-to-tail overlap between adjacent TM molecules is the structural basis of the nearest neighbor interactions. We tested the hypothesis by examining S-1.ADP binding to reconstituted regulated F-actin containing either intact TM or nonpolymerizable TM from which the COOH-terminal 11 residues were removed. In the absence of Ca2+, substitution of nonpolymerizable TM for TM reduced significantly the slope of the steeply rising phase of the sigmoidal S-1.ADP binding curve. Nevertheless, considerable residual cooperativity remained. Analysis of the data using the two-state model of Hill et al. suggests that removal of TM overlap abolishes nearest neighbor interactions, while the concerted change of the state of 7 actins in a functional unit can account for the residual cooperativity.     

Ca(2+)-dependent, myosin subfragment 1-induced proximity changes between actin and the inhibitory region of troponin I.

In order to help understand the spatial rearrangements of thin filament proteins during the regulation of muscle contraction, we used fluorescence resonance energy transfer (FRET) to measure Ca(2+)-dependent, myosin-induced changes in distances and fluorescence energy transfer efficiencies between actin and the inhibitory region of troponin I (TnI). We labeled the single Cys-117 of a mutant TnI with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS) and Cys-374 of actin with 4-dimethylaminophenylazophenyl-4'-maleimide (DABmal). These fluorescent probes were used as donor and acceptor, respectively, for the FRET measurements. We reconstituted a troponin-tropomyosin (Tn-Tm) complex which contained the AEDANS-labeled mutant TnI, together with natural troponin T (TnT), troponin C (TnC) and tropomyosin (Tm) from rabbit fast skeletal muscle. Fluorescence titration of the AEDANS-labeled Tn-Tm complex with DABmal-labeled actin, in the presence and absence of Ca(2+), resulted in proportional, linear increases in energy transfer efficiency up to a 7:1 molar excess of actin over Tn-Tm. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased from 37.9 A to 44.1 A when Ca(2+) bound to the regulatory sites of TnC. Titration of reconstituted thin filaments, containing AEDANS-labeled Tn-Tm and DABmal-labeled actin, with myosin subfragment 1 (S1) decreased the energy transfer efficiency, in both the presence and absence of Ca(2+). The maximum decrease occurred at well below stoichiometric levels of S1 binding to actin, showing a cooperative effect of S1 on the state of the thin filaments. S1:actin molar ratios of approximately 0.1 in the presence of Ca(2+), and approximately 0.3 in the absence of Ca(2+), were sufficient to cause a 50% reduction in normalized transfer efficiency. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased by approximately 7 A in the presence of Ca(2+) and by approximately 2 A in the absence of Ca(2+) when S1 bound to actin. Our results suggest that TnI's interaction with actin inhibits actomyosin ATPase activity by modulating the equilibria among active and inactive states of the thin filament. Structural rearrangements caused by myosin S1 binding to the thin filament, as detected by FRET measurements, are consistent with the cooperative behavior of the thin filament proteins.     

Effect of hypertrophic cardiomyopathy-linked troponin C mutations on the response of reconstituted thin filaments to calcium upon troponin I phosphorylation

The objective of this work was to investigate the effect of hypertrophic cardiomyopathy-linked A8V and E134D mutations in cardiac troponin C (cTnC) on the response of reconstituted thin filaments to calcium upon phosphorylation of cardiac troponin I (cTnI) by protein kinase A. The phosphorylation of cTnI at protein kinase A sites was mimicked by the S22D/S23D double mutation in cTnI. Our results demonstrate that the A8V and E134D mutations had no effect on the extent of calcium desensitization of reconstituted thin filaments induced by cTnI pseudophosphorylation. However, the A8V mutation enhanced the effect of cTnI pseudophosphorylation on the rate of dissociation of calcium from reconstituted thin filaments and on the calcium dependence of actomyosin ATPase. Consequently, while the A8V mutation still led to a slower rate of dissociation of calcium from reconstituted thin filaments upon pseudophosphorylation of cTnI, the ability of the A8V mutation to decrease the rate of calcium dissociation was weakened. In addition, the ability of the A8V mutation to sensitize actomyosin ATPase to calcium was weakened after cTnI was replaced by the phosphorylation mimetic of cTnI. Consistent with the hypothesis that the E134D mutation is benign, it exerted a minor to no effect on the rate of dissociation of calcium from reconstituted thin filaments or on the calcium sensitivity of actomyosin ATPase, regardless of the cTnI phosphorylation status. In conclusion, our study enhances our understanding of how cardiomyopathy-linked cTnC mutations affect the response of reconstituted thin filaments to calcium upon cTnI phosphorylation.     

Conformation of spin-labeled tropomyosin in reconstituted muscle thin filaments in response to calcium ion and heavy meromyosin

Tropomyosin (TM) exists in thermal equilibrium between a highly structured N state, a partially unfolded X state, and a completely unfolded D state, i.e., N in equilibrium X in equilibrium D. The strongly immobilized electron spin resonance (ESR) spectral component of spin-labeled TM corresponds to TM in the N state and the weakly immobilized component to TM in the X state below the main unfolding transition and to TM in the D state above this transition [Graceffa, P., & Lehrer, S. S. (1984) Biochemistry 23, 2606-2612]. The addition of actin, troponin (TN), and heavy meromyosin (HMM) to spin-labeled TM reduces the ratio of weakly to strongly immobilized labels, indicating a shift in the N in equilibrium X in equilibrium D equilibrium toward the N state. At 37 degrees C, for spin-labeled TM alone K (=X/N) greater than 1.0 with some TM in the D state, K = 0.8 for spin-labeled TM bound to actin, and K less than 0.05 for spin-labeled TM bound to actin + TN +/- Ca2+, actin + HMM + TN +/- Ca2+, and actin + HMM. Thus, actin + TN dramatically shifts the TM structure to the N conformation with little further effect upon addition of Ca2+ or HMM. The temperature at which spin-labeled TM begins to dissociate from a protein complex was determined from the temperature dependence of the ESR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)