Subunit flow in F-actin under steady-state conditions. Application of a novel method to determination of the rate of subunit exchange of F-actin at the terminals

We developed a novel method to determine the subunit exchange rates of F-actin at its terminals under quasi-steady-state conditions by using a powerful fluorescent probe, N-(1-pyrenyl)iodoacetamide. The applicability of the method was checked with regard to both theoretical and experimental aspects. We determined the rates of subunit exchange of F-actin and F-actin-tropomyosin complex under various ionic conditions. We found that: (i) KCl accelerated both on and off rates at each end, and lowered the critical concentration of the P-end while the critical concentration of the B-end was not affected; (ii) binding of tropomyosin drastically reduced the subunit flow in F-actin by suppressing the off rate principally of the P-end. It is therefore believed that tropomyosin exerts an anisotropic constraint on F-actin and regulates its dynamic polarity.     

Calcium-triggered movement of regulated actin in vitro. A fluorescence microscopy study

We previously reported setting up an in vitro system for the observation of actin filament sliding along myosin filaments. The system involved a minute amount of fluorescently labelled F-actin, and its movement was monitored by fluorescence microscopy. Here, we report observations of the Ca2+-dependent movement of F-actin complex with tropomyosin plus troponin (regulated actin) added to the movement system in place of pure F-actin. In a wide range of pCa (-log10[Ca2+]) between 3 and 5.5 at 30 degrees C, regulated actin filaments moved rapidly, and the average velocity depended little on the Ca2+ concentration (about 7.5 microns/s). However, when the Ca2+ concentration was decreased to pCa = 5.8 or lower, the filaments suddenly stopped moving. In striking contrast to these observations, unregulated actin moved rapidly within the whole pCa range examined, the average velocity (about 7.5 microns/s) being essentially Ca2+-independent. These observations indicate that (1) tropomyosin-troponin actually gave Ca2+-sensitivity to F-actin, and (2) the movement system was regulated by Ca2+ in an on-off fashion within a narrow range of Ca2+ concentration. In a pCa range between 5.8 and 6.0, regulated actin filaments did not exhibit thermal motion; instead, they had fixed positions in the specimen, possibly because they remained associated with myosin filaments in the background, without sliding past each other. Although regulated actin moved fast in the presence of 1 mM-CaCl2 (pCa = 3) at 30 degrees C, it became entirely non-motile as the temperature was decreased to 25 degrees C or lower. Such a sharp movement/temperature relation was never found for unregulated actin. We assayed regulated actin-activated myosin ATPase in the same conditions as used for microscopy, and found that the ATPase activity depended both on pCa and on the temperature considerably less than the movement of regulated actin. The results suggest that the sliding velocity in the in vitro system would not be proportional to the rate of actin-activated ATPase.     

Ca2+ regulation of rabbit skeletal muscle thin filament sliding: role of cross-bridge number

We investigated how strong cross-bridge number affects sliding speed of regulated Ca(2+)-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca(2+). Second, we varied the number of Ca(2+)-activatable troponin complexes at maximal Ca(2+) using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A,D63A), a mutant deficient in Ca(2+) binding at both N-terminal, low affinity Ca(2+)-binding sites (xxsTnC-sTn). Sliding speed decreased nonlinearly as the proportion of WT sTn decreased. Speed of regulated thin filaments varied with pCa when filaments contained WT sTn but filaments containing only xxsTnC-sTn did not move. pCa(50) decreased by 0.12-0.18 when either heavy meromyosin density was reduced to approximately 60% or the fraction of Ca(2+)-activatable regulatory units was reduced to approximately 33%. Third, we exchanged mixtures of sTnC and xxsTnC into single, permeabilized fibers from rabbit psoas. As the proportion of xxsTnC increased, unloaded shortening velocity decreased nonlinearly at maximal Ca(2+). These data are consistent with unloaded filament sliding speed being limited by the number of cycling cross-bridges so that maximal speed is attained with a critical, low level of actomyosin interactions.     

Interaction of rabbit skeletal muscle troponin T and F-actin at physiological ionic strength

Troponin T has been shown to interact significantly with F-actin at 150 mM KC1 by using an F-actin pelleting assay and 125I-labeled proteins. While troponin T fragment T1 (residues 1-158) fails to pellet with F-actin, fragment T2 (residues 159-259) mimics the binding properties of the intact molecule. The weak competition of T2 binding to F-actin, shown by subfragments of T2, indicates that the interaction site(s) encompass(es) an extensive segment of troponin T. The extent of pelleting of troponin T (or T2) with F-actin is only marginally altered in the binary complex troponin IT (or T2), indicating that the direct interactions either of troponin T (or T2) or of troponin I, or both, with F-actin are weakened when these components are incorporated into a binary complex. The binding of troponin T (or T2) is moderately (-Ca2+) or more extensively reduced (+Ca2+) in the presence of troponin C. The pelleting of Tn-T seen in the presence of Tn-C (-Ca2+) and Tn-I was further reduced when either Tn-I or Tn-C (-Ca2+) was added, respectively, to form a fully reconstituted Tn complex. As noted by others, whole troponin shows little sensitivity to Ca2+ in its binding to F-actin (-tropomyosin). These and other observations, taken together with the restoration of troponin IC (+/- Ca2+) binding to F-actin by troponin T, implicate a role for the interaction of troponin T and F-actin in the thin filament assembly.     

Interaction between troponin and myosin enhances contractile activity of myosin in cardiac muscle

Ca(2+) signaling in striated muscle cells is critically dependent upon thin filament proteins tropomyosin (Tm) and troponin (Tn) to regulate mechanical output. Using in vitro measurements of contractility, we demonstrate that even in the absence of actin and Tm, human cardiac Tn (cTn) enhances heavy meromyosin MgATPase activity by up to 2.5-fold in solution. In addition, cTn without Tm significantly increases, or superactivates sliding speed of filamentous actin (F-actin) in skeletal motility assays by at least 12%, depending upon [cTn]. cTn alone enhances skeletal heavy meromyosin's MgATPase in a concentration-dependent manner and with sub-micromolar affinity. cTn-mediated increases in myosin ATPase may be the cause of superactivation of maximum Ca(2+)-activated regulated thin filament sliding speed in motility assays relative to unregulated skeletal F-actin. To specifically relate this classical superactivation to cardiac muscle, we demonstrate the same response using motility assays where only cardiac proteins were used, where regulated cardiac thin filament sliding speeds with cardiac myosin are >50% faster than unregulated cardiac F-actin. We additionally demonstrate that the COOH-terminal mobile domain of cTnI is not required for this interaction or functional enhancement of myosin activity. Our results provide strong evidence that the interaction between cTn and myosin is responsible for enhancement of cross-bridge kinetics when myosin binds in the vicinity of Tn on thin filaments. These data imply a novel and functionally significant molecular interaction that may provide new insights into Ca(2+) activation in cardiac muscle cells.     

Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry. IV. Conformational changes in the region containing fluorescence-labeled sulfhydryl group(s) of troponin.

Conformational changes associated with the functional states of the molecule of troponin were studied using SH-direct fluorogenic reagents, N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM) and N-(1-anilinonaphthyl-4) maleimide (ANM). 1. The fluorescence parameters of ANM-troponin, intensity, and polarization, did not change on combining it with tropomyosin alone, but markedly changed when F-actin was further added to the system. 2. The conformation around the dye-labeled sulfhydryl group(s) was shown to be susceptible to Ca2+ in terms of fluorescence intensity of the label, thermal transition of the conformation, and the microenvironment near the label. 3. On addition of Ca2+, the fluorescence characteristics of the two systems, ANM-troponin . tropomyosin and ANM-troponin . tropomyosin . F-actin complexes, were altered in opposite directions. When BIPM was used in place of ANM, similar changes were observed: a simple decrease in the intensity when pCa was decreased from 7.4 to 5.5 in the system without F-actin and a sigmoidal increase in the range from pCa 7 to 6 in the system with F-actin. Heavy meromyosin, when added to the latter complex (the reconstituted thin filaments), made the profile of its Ca2+ concentration dependence of fluorescence similar to that of the former complex. When tropomyosin was labeled in place of troponin, similar results were obtained. The data obtained imply that the Ca2+-induced conformational changes of troponin are markedly modified when detached from actin, and that heavy meromyosin weakens the interaction of the troponin . tropomyosin complex with F-actin.     

Evidence that F-actin can hydrolyze ATP independent of monomer-polymer end interactions

The rate of ATP hydrolysis in solutions of F-actin at steady state in 50 mM KC1, 0.1 mM CaC12 was inhibited by AMP and ADP. The inhibition was competitive with ATP (Km of about 600 microM) with Ki values of 9 microM for AMP and 44 microM for ADP. ATP hydrolysis was inhibited greater than 95% by 1 mM AMP. AMP had no effect on the time course of actin polymerization, ATP hydrolysis during polymerization, or the critical actin concentration. Simultaneous measurements of G-actin/F-actin subunit exchange and nucleotide exchange showed that nucleotide exchange occurred much more rapidly than subunit exchange; during the experiment over 50% of the F-actin-bound nucleotide was replaced when less than 1% of the F-actin subunits had exchanged. When AMP was present it was incorporated into the polymer, preventing incorporation of ADP from ATP in solution. F-actin with bound Mg2+ was much less sensitive to AMP than F-actin with bound Ca2+. These data provide evidence for an ATP hydrolysis cycle associated with direct exchange of F-actin-bound ADP for ATP free in solution independent of monomer-polymer end interactions. This exchange and hydrolysis of nucleotide may be enhanced when Ca2+ is bound to the F-actin protomers.     

The hill coefficient for the Ca2+-activation of striated muscle contraction

The following arguments are presented for the observation that curves relating free Ca2+ and force development of thin filament regulated myofilaments of skinned muscle fibers have Hill coefficient (n) greater than 4, which is the number of Ca2+ binding sites on troponin: Activation of the myofilaments is a process relaxing to a nonequilibrium steady state or stationary state. Systems operating at nonequilibrium stationary states are known to display Hill coefficients greater than the number of interacting sites and similar results have been obtained for Ca2+ activation of myofilament isometric force. The size of the basic subunit of thin filament regulated muscle may be the entire thin filament rather than seven actins, one tropomyosin, and one troponin. In this case the number of interacting sites may be on the order of hundreds. Hysteresis in the Ca2+ activation of isometric force might result from multiple stationary states and also might give rise to Hill coefficients greater than 4.     

Interaction of phosphorylase kinase with thin filament proteins of rabbit skeletal muscles

The binding of phosphorylase kinase to thin filaments and their effects on the enzyme activity as well as the contribution of the enzyme to contractile protein phosphorylation have been studied. The data obtained suggest that the kinase binding to thin filaments is controlled by the regulatory proteins, troponin and tropomyosin. The bulk of the enzyme is bound to the F-actin-tropomyosin-troponin complex which activates the enzyme in a far greater degree than each of its constituent components. Ca2+ and ATP control the kinase binding to F-actin. ATP increases the enzyme binding 6-fold; Ca2+ decrease the S0.5 value for F-actin 5-fold. In acetone powder extracts phosphorylase kinase phosphorylates thin filament-bound phosphorylase b, troponin T and troponin I as well as 51-58 kDa and 114 kDa proteins. These results suggest that phosphorylase kinase plays a role in the mechanism of synchronization of glycogenolysis and muscle contraction rates.     

Calcium regulation of skeletal muscle thin filament motility in vitro.

Using an in vitro motility assay, we have investigated Ca2+ regulation of individual, regulated thin filaments reconstituted from rabbit fast skeletal actin, troponin, and tropomyosin. Rhodamine-phalloidin labeling was used to visualize the filaments by epifluorescence, and assays were conducted at 30 degrees C and at ionic strengths near the physiological range. Regulated thin filaments exhibited well-regulated behavior when tropomyosin and troponin were added to the motility solutions because there was no directed motion in the absence of Ca2+. Unlike F-actin, the speed increased in a graded manner with increasing [Ca2+], whereas the number of regulated thin filaments moving was more steeply regulated. With increased ionic strength, Ca2+ sensitivity of both the number of filaments moving and their speed was shifted toward higher [Ca2+] and was steepest at the highest ionic strength studied (0.14 M gamma/2). Methylcellulose concentration (0.4% versus 0.7%) had no effect on the Ca2+ dependence of speed or number of filaments moving. These conclusions hold for five different methods used to analyze the data, indicating that the conclusions are robust. The force-pCa relationship (pCa = -log10[Ca2+]) for rabbit psoas skinned fibers taken under similar conditions of temperature and solution composition (0.14 M gamma/2) paralleled the speed-pCa relationship for the regulated filaments in the in vitro motility assay. Comparison of motility results with the force-pCa relationship in fibers suggests that relatively few cross-bridges are needed to make filaments move, but many have to be cycling to make the regulated filament move at maximum speed.     

Fluorescence polarization study on Ca2+-sensitivity of conformational changes in F-actin induced by the formation of F-actin-subfragment-1 complex

Ca2+-dependent conformational changes in F-actin during myosin subfragment-1 binding with thin filament (in the absence of troponin and tropomyosin) were found in myosin-free ghost fibres by polarized UV microscopy. The pattern of the conformational changes in F-actin changed cooperatively within the range of free Ca2+ concentrations from 10(-7) mol/l to 10(-6) mol/l. It should be suggested that in skeletal muscle of vertebrates there exists a myosin-linked modulation of contraction by Ca2+.     

Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T

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.     

Solution structure of the chicken skeletal muscle troponin complex via small-angle neutron and X-ray scattering

Troponin is a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle by participating in a series of conformational events within the actin-based thin filament. Troponin is a heterotrimeric complex consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT). Ternary troponin complexes have been produced by assembling recombinant chicken skeletal muscle TnC, TnI and the C-terminal portion of TnT known as TnT2. A full set of small-angle neutron scattering data has been collected from TnC-TnI-TnT2 ternary complexes, in which all possible combinations of the subunits have been deuterated, in both the +Ca2+ and -Ca2+ states. Small-angle X-ray scattering data were also collected from the same troponin TnC-TnI-TnT2 complex. Guinier analysis shows that the complex is monomeric in solution and that there is a large change in the radius of gyration of TnI when it goes from the +Ca2+ to the -Ca2+ state. Starting with a model based on the human cardiac troponin crystal structure, a rigid-body Monte Carlo optimization procedure was used to yield models of chicken skeletal muscle troponin, in solution, in the presence and in the absence of regulatory calcium. The optimization was carried out simultaneously against all of the scattering data sets. The optimized models show significant differences when compared to the cardiac troponin crystal structure in the +Ca2+ state and provide a structural model for the switch between +Ca2+ and -Ca2+ states. A key feature is that TnC adopts a dumbbell conformation in both the +Ca2+ and -Ca2+ states. More importantly, the data for the -Ca2+ state suggest a long extension of the troponin IT arm, consisting mainly of TnI. Thus, the troponin complex undergoes a large structural change triggered by Ca2+ binding.     

The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase

The cardiac troponin (Tn) complex, consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca2+-binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca2+ concentration. Cardiac Tn binds 3 mol Ca2+/mol and contains two Ca2+-binding sites with a binding constant of 3 X 10(8) M-1 and one binding site with a binding constant of 2 X 10(6) M-1. In the presence of 4 mM MgC12, the binding constant of the sites of higher affinity is reduced to 3 X 10(7) M-1, while Ca2+ binding to the site at the lower affinity is unaffected. The two high affinity Ca2+-binding sites of cardiac Tn are analogous to the two Ca2+-Mg2+ sites of skeletal Tn, while the single low affinity site is similar to the two Ca2+-specific sites of skeletal Tn (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4625-5633). The Ca2+-binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca2+/mol and contains two sites with a binding constant of 1 X 10(7) M-1 and a single site with a binding constant of 2 X 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the high affinity sites of TnC and Tn, the binding constants for Mg2+ were 0.7 and 3.0 X 10(3) M-1, respectively. The Ca2+ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison by the Ca2+-binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca2+] and at millimolar [Mg2+] suggests that activation of the ATPase occurs over the same range of [Ca2+] where the Ca2+-specific site of cardiac Tn binds Ca2+.     

Chymotryptic subfragments of troponin T from rabbit skeletal muscle. Interaction with tropomyosin, troponin I and troponin C

The binding of the chymotryptic troponin T subfragments to tropomyosin, troponin I, and troponin C was semiquantitatively examined by using affinity chromatography, and also by co-sedimentation with F-actin and polyacrylamide gel electrophoresis in 14 mM Tris/90 mM glycine. Circular dichroism spectra of the subfragments were measured to confirm that the subfragments retained their conformational structures. Based on these results, the binding sites of tropomyosin, troponin I, and troponin C on the troponin T sequence were elucidated. Tropomyosin bound mainly to the region of troponin T1 (residues 1-158) with the same binding strength as to the original troponin T. The C-terminal region of troponin T (residues 243-259) was the second binding site to tropomyosin under physiological conditions. The binding site of troponin I was concluded to be the region including residues 223-227. The binding of troponin C was dependent on Ca2+ ion concentration. The C-terminal region of troponin T2 (residues 159-259) was indicated to be the Ca2+-independent troponin C-binding site and the N-terminal side of troponin T2 to be the Ca2+-dependent site.     

Properties of the gamma subunit of phosphorylase kinase

Enzymatic properties of the isolated, active gamma subunit of phosphorylase kinase were characterized. Kinetic parameters indicated that the gamma subunit binds the substrates MgATP and phosphorylase b as well as the holoenzyme with a Km (MgATP) of 98 microM and a Km (phosphorylase b) of 80 microM at pH 8.2, but maximal velocities are significantly lower than the holoenzyme's. Unlike the gamma-calmodulin complex, the gamma subunit activity is dependent on pH in the range of pH 6.2-9.0, with a ratio of activity at pH 6.8 to activity at pH 8.2 of 0.5-0.6. Calmodulin activates the gamma subunit more at low pH than at high pH. ADP inhibits the gamma subunit in a competitive manner with a Ki of 60 microM. Free Mg2+ stimulates gamma subunit activity 3.5-fold at both pH 6.8 and 8.2. MnATP is equivalent to MgATP as a substrate for the enzyme, but free Mn2+ inhibits gamma subunit activity. Several protein substrates of holophosphorylase kinase were found also to be phosphorylated by the gamma subunit. These included kappa-casein, myelin basic protein, the troponin complex, and troponin T alone. In the troponin complex, the proportion of 32P incorporated by the gamma subunit into troponin I compared with troponin T was not Ca2+ dependent, but with the holoenzyme, this proportion was changed greatly by Ca2+ concentration.     

Conformational change of troponin T induced by calcium binding to troponin C

The skeletal muscle troponin complex, the troponin T subunit of which was labeled with 2-((4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid, showed a fluorescence titration curve with a midpoint of around pCa 6.75. Addition of 2 mM MgCl2 had no effect on the fluorescence titration curve. Therefore, we conclude that Ca2+ binding to the low affinity Ca2+-binding sites of troponin C induces a conformational change of troponin T, but Ca2+ binding to the high affinity Ca2+-binding sites does not.     

Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin

Troponin and tropomyosin on actin filaments constitute a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle through a series of conformational changes within the actin-based thin filament. Troponin consists of three subunits: an inhibitory subunit (TnI), a Ca2+-binding subunit (TnC), and a tropomyosin-binding subunit (TnT). Ca2+-binding to TnC is believed to weaken interactions between troponin and actin, and triggers a large conformational change of the troponin complex. However, the atomic details of the actin-binding sites of troponin have not been determined. Ternary troponin complexes have been reconstituted from recombinant chicken skeletal TnI, TnC, and TnT2 (the C-terminal region of TnT), among which only TnI was uniformly labelled with 15N and/or 13C. By applying NMR spectroscopy, the solution structures of a "mobile" actin-binding domain (approximately 6.1 kDa) in the troponin ternary complex (approximately 52 kDa) were determined. The mobile domain appears to tumble independently of the core domain of troponin. Ca2+-induced changes in the chemical shift and line shape suggested that its tumbling was more restricted at high Ca2+ concentrations. The atomic details of interactions between actin and the mobile domain of troponin were defined by docking the mobile domain into the cryo-electron microscopy (cryo-EM) density map of thin filament at low [Ca2+]. This allowed the determination of the 3D position of residue 133 of TnI, which has been an important landmark to incorporate the available information. This enabled unique docking of the entire globular head region of troponin into the thin filament cryo-EM map at a low Ca2+ concentration. The resultant atomic model suggests that troponin interacted electrostatically with actin and caused the shift of tropomyosin to achieve muscle relaxation. An important feature is that the coiled-coil region of troponin pushed tropomyosin at a low Ca2+ concentration. Moreover, the relationship between myosin and the mobile domain on actin filaments suggests that the latter works as a fail-safe latch.     

An atomic model of the thin filament in the relaxed and Ca2+-activated states

Contraction of striated muscles is regulated by tropomyosin strands that run continuously along actin-containing thin filaments. Tropomyosin blocks myosin-binding sites on actin in resting muscle and unblocks them during Ca2+-activation. This steric effect controls myosin-crossbridge cycling on actin that drives contraction. Troponin, bound to the thin filaments, couples Ca2+-concentration changes to the movement of tropomyosin. Ca2+-free troponin is thought to trap tropomyosin in the myosin-blocking position, while this constraint is released after Ca2+-binding. Although the location and movements of tropomyosin are well known, the structural organization of troponin on thin filaments is not. Its mechanism of action therefore remains uncertain. To determine the organization of troponin on the thin filament, we have constructed atomic models of low and high-Ca2+ states based on crystal structures of actin, tropomyosin and the "core domain" of troponin, and constrained by distances between filament components and by their location in electron microscopy (EM) reconstructions. Alternative models were also built where troponin was systematically repositioned or reoriented on actin. The accuracy of the different models was evaluated by determining how well they corresponded to EM images. While the initial low and high-Ca2+ models fitted the data precisely, the alternatives did not, suggesting that the starting models best represented the correct structures. Thin filament reconstructions were generated from the EM data using these starting models as references. In addition to showing the core domain of troponin, the reconstructions showed additional detail not present in the starting models. We attribute this to an extension of TnI linking the troponin core domain to actin at low (but not at high) Ca2+, thereby trapping tropomyosin in the OFF-state. The bulk of the core domain of troponin appears not to move significantly on actin, regardless of Ca2+ level. Our observations suggest a simple model for muscle regulation in which troponin affects the charge balance on actin and hence tropomyosin position.