Modification of Lys-237 on actin by 2,4-pentanedione. Alteration of the interaction of actin with tropomyosin

It has been possible to specifically label rabbit skeletal muscle actin at Lys-237 with 2,4-pentanedione, producing an enamine. This reaction can be reversed with hydroxylamine. The modification can be carried out with actin in either the G- or F-forms and does not affect polymerization-depolymerization. The modification does affect, however, the interaction of tropomyosin (Tm) with the modified F-actin. In the absence of Ca2+ and Mg2+ (mu = 0.12), Tm failed to bind to the modified F-actin whereas it did bind to unmodified F-actin (1 Tm:7 actins). Tm binding could be restored under these conditions by the addition of either troponin (Tn), Mg2+, or Mg2+ and Ca2+. Under certain conditions, Tm alone has been shown to inhibit actin-activated heavy meromyosin (HMM)-Mg2+-ATPase. This inhibition did not occur with the modified F-actin even though Tm was bound (approximately 1 Tm:7 actins). Even when Tn was added to this system (in the absence of Ca2+), no inhibition of ATPase could be observed. Thus, this modification appears to prevent F-actin X Tm from assuming the "blocking" inhibitory position (conformation). In addition, Tn appears to enhance the activation of heavy meromyosin-Mg2+-ATPase by the modified F-actin X Tm complex whether Ca2+ is present or not. This state may be analogous to the potentiated state (Murray, J. M., Knox, M. K., Trueblood, C. E., and Weber, A. (1982) Biochemistry 27, 906-915) seen with myosin subfragment 1-saturated actin at low ATP levels. Thus, using modified and unmodified F-actin, it is possible to produce three Tm X actin states: off (F-actin X Tm), on (modified F-actin X Tm), and "potentiated" (modified F-actin X Tm X Tn).     

Interaction between Acanthamoeba actin and rabbit skeletal muscle tropomyosin.

The binding of 125I-labeled muscle tropomyosin to Acanthamoeba and muscle actin was studied by ultracentrifugation and by the effect of tropomyosin on the actin-activated muscle heavy meromyosin ATPase activity. Binding of muscle tropomyosin to Acanthamoeba actin was much weaker than its binding to muscle actin. For example, at 5 mM MgCl2, 2 mM ATP, and 5 micronM actin, tropomyosin bound strongly to muscle actin but not detectably to Acanthamoeba actin. When the concentration of actin was raised from 5 micronM to 24 micronM in the presence of 80 mM KCl, the binding of tropomyosin to Acanthamoeba actin approached its binding to muscle actin. As with muscle actin, the addition of muscle heavy meromyosin in the absence of ATP induced binding of tropomyosin in Acanthamoeba actin under conditions were binding would otherwise not have occurred. The most striking difference between the interactions of muscle tropomyosin with the two actins, however, was that under conditions where tropomyosin was found to both actins, its stimulated the Acanthamoeba actin-activated heavy meromyosin ATPase but inhibited the muscle actin-activated heavy meromyosin ATPase.     

Dynamics of the muscle thin filament regulatory switch: the size of the cooperative unit.

Actin thin filaments containing bound tropomyosin (Tm) or tropomyosin troponin (Tm.Tn) exist in two states ("off" and "on") with different affinities for myosin heads (S1), which results in the cooperative binding of S1. The rate of S1 binding to, and dissociating from, actin, Tm.actin, and Tm.Tn.actin, monitored by light scattering (LS), was compared with the rate of change in state, monitored by the excimer fluorescence (Fl) of a pyrene label attached to Tm. The ATP-induced S1 dissociation showed similar exponential decreases in LS for actin.S1, Tm.actin.S1, and Tm.Tn.actin.S1 +/- Ca2+. The Fl change, however, showed a delay that was greater for Tm.Tn.actin than Tm.actin, independent of Ca2+. The S1 binding kinetics gave observed rate constants for the S1-induced change in state that were 5-6 times the observed rate constants of S1 binding to Tm.actin, which were increased to 10-12 for Tm.Tn.actin, independent of Ca2+. The rate of the Fl signals showed that the on/off states were in rapid equilibrium. These data indicate that the apparent cooperative unit for Tm.actin is 5-6 actin subunits rather than the minimum structural unit size of 7, and is increased to 10-12 subunits for Tm.Tn.actin, independent of the presence of Ca2+. Thus, Tm appears semi-flexible, and Tn increases communication between neighboring structural units. A general model for the dynamic transitions involved in muscle regulation is presented.     

The regulation of subtilisin-cleaved actin by tropomyosin/troponin

Vertebrate striated muscle contraction is regulated in a Ca(2+)-dependent fashion by tropomyosin (Tm) and troponin (Tn). This regulation involves shifts in the position of Tm and Tn on actin filaments and may include conformational changes in actin that are then communicated to myosin subfragment 1 (S1). To determine whether subdomain 2 of actin plays a role in this regulation, the DNase-I loop 38-52 of this subdomain was cleaved by subtilisin between residues Met(47) and Gly(48). Despite impaired unregulated function, the potentiation and regulation of cleaved actin movement in the in vitro motility assay was not significantly different from that of uncleaved actin. Stopped-flow measurements of ADP release from regulated and unregulated cleaved acto-S1 showed a marked increase in ADP release from acto-S1 in the presence of the regulatory complex. The enhancement of the actin affinity for S1 in the presence of regulatory proteins was greater for uncleaved than for cleaved F-actin. Finally, both cleaved and uncleaved actins protect myosin loop 1 from papain cleavage equally well. Our results suggest that the potentiation of actin function in the in vitro motility assay by regulatory proteins stems from changes in cross-bridge cycle kinetics. In addition, the unimpaired calcium-sensitive regulation of cleaved actin indicates that subdomain 2 conformation does not play an essential role in the regulation process.     

Actomyosin kinetics and in vitro motility of wild-type Drosophila actin and the effects of two mutations in the Act88F gene.

Two missense mutations of the flight muscle-specific actin gene of Drosophila melanogaster, Act88F, assemble into normally structured myofibrils but affect the flight ability of flies and the mechanical kinetics of isolated muscle fibers. We describe the isolation of actin from different homozygous Act88F strains, including wild-type, an Act88F null mutant (KM88), and two Act88F single point mutations (E316K and G368E), their biochemical interactions with rabbit myosin subfragment 1 (S1), and behavior with rabbit myosin and heavy meromyosin in in vitro motility assays. The rabbit and wild-type Drosophila actins have different association rate constants with S1 (2.64 and 1.77 microM-1 s-1, respectively) and in vitro motilities (2.51, 1.60 microns s-1) clearly demonstrating an isoform-specific difference. The G368E mutation shows a reduced affinity for rabbit S1 compared with the wild type (increasing from 0.11 to 0.17 microM) and a reduced velocity in vitro (reduced by 19%). The E316K mutant actin has no change in affinity for myosin S1 or in vitro motility with heavy meromyosin but does have a reduced in vitro motility (15%) with myosin. These results are discussed with respect to the recently published atomic models for the actomyosin structure and our findings that G368E fibers show a reduced rate constant for delayed tension development and increased fiber stiffness. We interpret these results as possibly caused either by effects on A1 myosin light chain binding or conformational changes within the subdomain 1 of actin, which contains the myosin binding site. E316K is discussed with respect to its likely position within the tropomyosin binding site of actin.     

The mechanism of Ca2+ regulation of vascular smooth muscle thin filaments by caldesmon and calmodulin

The interactions of vascular smooth muscle caldesmon with actin, tropomyosin, and calmodulin were determined under conditions in which the four proteins can form reconstituted Ca2+-sensitive smooth muscle thin filaments. Caldesmon bound to actin in a complex fashion with high affinity sites (K = 10(7) M-1) saturating at a stoichiometry of 1 per 28 actins, and lower affinity sites at 1 per 7 actins. The affinity of binding was increased in the presence of tropomyosin, and this could be attributed to a direct interaction between caldesmon and tropomyosin which was demonstrated using caldesmon cross-linked to Sepharose. In the presence of tropomyosin, occupancy of the high affinity sites was associated with inhibition of actin-activated myosin MgATPase activity. Caldesmon was found to bind to calmodulin in the presence of Ca2+, with an affinity of 10(6) M-1. The binding of Ca2+ X calmodulin to caldesmon was associated with the neutralization of inhibition of actin-tropomyosin. Ca2+ X calmodulin binding reduced but did not abolish the binding of caldesmon to actin-tropomyosin. From this data we have proposed a model for smooth muscle thin filaments in which Ca2+ regulates activity by converting the inhibited actin-tropomyosin-caldesmon complex to the active complexes, actin-tropomyosin-caldesmon-calmodulin X Ca2+ and actin-tropomyosin.     

Calcium regulation of thin filament movement in an in vitro motility assay.

The ability of calcium to regulate thin filament sliding velocity was studied in an in vitro motility assay system using cardiac troponin and tropomyosin and rhodamine-phalloidin-labeled skeletal actin and skeletal heavy meromyosin to propel the filaments. Measurements showed that both the number of thin filaments sliding and their sliding speed (Sf) were dependent on the calcium concentration in the range of pCa 5 to 9. Thin filament motility was completely inhibited only if troponin and tropomyosin were added at a concentration of 100 nM to the motility assay solution and the pCa was more than 8. The filament sliding speed was dependent on the pCa in a noncooperative fashion (Hill coefficient = 1) and reached maximum at 5 microns/s at a pCa of 5. The number of filaments moving uniformly decreased from > 90% at pCa 5-6 to near zero in less than 1 pCa unit. This behavior may be explained by a hypothesis in which the regulatory proteins control the number of cross-bridge heads interacting with the thin filaments rather than the rate at which they individually hydrolyze ATP or translocate the thin filaments.     

Alteration of actin-tropomyosin interaction in 2,4-pentanedione-treated rabbit skeletal myofibrils

In previous work, we (El-Saleh, S., Theiret, R., Johnson, P., and Potter, J. D. (1984) J. Biol. Chem. 259, 11014-11021) presented evidence that Ca2+ activation of skeletal myofilaments depends on a specific actin domain. We showed that rabbit skeletal thin filaments reconstituted with actin modified at Lys-237 activate heavy meromyosin X Mg2+-ATPase activity independently of the Ca2+ ion concentration. The modification, which apparently blocks the inhibitory effects of troponin-tropomyosin (Tn X Tm), on acto-heavy meromyosin X Mg2+-ATPase activity, consisted of conversion of Lys-237 to an enamine by reaction of purified actin with 2,4-pentanedione (PD). In experiments reported here, we have treated myofibrils with PD with the idea of altering actin in its native state within the myofilament lattice. Preparations of native and Tn X Tm free ("desensitized") myofibrils were incubated with PD (100 mol/mol of actin lysine) under rigorous conditions (10 mM 4-morpholinepropanesulfonic acid, pH 7.0, 2.0 nM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.4 mM dithiothreitol, and 0.15 mM NaN3). Actin isolated from PD X myofibrils contained 0.5 mol of enamine/mol. In the presence of Ca2+, the Mg2+-ATPase activity of PD-treated myofibrils was 110-120% of the maximum Ca2+-stimulated Mg2+-ATPase activity of untreated control myofibrils. In low free Ca2+ (pCa greater than 8), the Mg2+-ATPase activity of the PD-treated myofibrils was not suppressed and remained at 100-106% of the maximum activity of the control myofibrils. Ca2+ sensitivity of the PD-treated myofibrils was restored following treatment with hydroxylamine, which hydrolyzes enamine's products. Preparations of desensitized myofibrils reconstituted with PD-modified or unmodified Tn X Tm demonstrated the same Ca2+-sensitive ATPase activities. On the other hand, preparations reconstituted with unmodified or PD-modified Tn X Tm and PD-modified desensitized myofibrils were insensitive to Ca2+ ion concentration. The Mg2+-ATPase activity of preparations of myosin treated with PD was not activated by modified or unmodified actin. Our results indicate that is is possible to produce an active state(s) of the myofibrils in the absence and presence of Ca2+ by specific alteration of the actin X Tm interaction following modification of myofibrillar actin most likely at Lys-237.     

Two-step ligand binding and cooperativity. A model to describe the cooperative binding of myosin subfragment 1 to regulated actin.

The binding of actin to myosin subfragment 1 (S1) has been shown to occur as a two-step reaction. In the first step actin is weakly bound and then the complex isomerizes to the "rigor type" acto-S1 complex (Coates, J. H., A. H. Criddle, and M. A. Geeves, 1985 Biochem. J., 232:351-356). We propose here a model in which troponin/tropomyosin (Tn/Tm) controls the actin-S1 interaction by inhibiting the isomerization step. In this model the (actin)7 Tn/Tm unit is assumed to exist in two states: open and closed. S1 can bind to either of the two states but only the open form allows the isomerization reaction to take place. We demonstrate that this model can account for the cooperative binding of S1 and S1 nucleotide complexes to actin. The model provides a way of integrating both the effects of calcium and nucleotide on actin-S1 interactions.     

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.     

The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity

In cardiac and skeletal muscles tropomyosin binds to the actin outer domain in the absence of Ca(2+), and in this position tropomyosin inhibits muscle contraction by interfering sterically with myosin-actin binding. The globular domain of troponin is believed to produce this B-state of the thin filament (Lehman, W., Hatch, V., Korman, V. L., Rosol, M., Thomas, L. T., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593-606) via troponin I-actin interactions that constrain the tropomyosin. The present study shows that the B-state can be promoted independently by the elongated tail region of troponin (the NH(2) terminus (TnT-(1-153)) of cardiac troponin T). In the absence of the troponin globular domain, TnT-(1-153) markedly inhibited both myosin S1-actin-tropomyosin MgATPase activity and (at low S1 concentrations) myosin S1-ADP binding to the thin filament. Similarly, TnT-(1-153) increased the concentration of heavy meromyosin required to support in vitro sliding of thin filaments. Electron microscopy and three-dimensional reconstruction of thin filaments containing TnT-(1-153) and either cardiac or skeletal muscle tropomyosin showed that tropomyosin was in the B-state in the complete absence of troponin I. All of these results indicate that portions of the troponin tail domain, and not only troponin I, contribute to the positioning of tropomyosin on the actin outer domain, thereby inhibiting muscle contraction in the absence of Ca(2+).     

Effects of Ca2+ and Mg2+ on the actomyosin adenosine-5'-triphosphatase of stably phosphorylated gizzard myosin

There are conflicting reports on the effect of Ca2+ on actin activation of myosin adenosine-triphosphatase (ATPase) once the light chain is fully phosphorylated by a calcium calmodulin dependent kinase. Using thiophosphorylated gizzard myosin, Sherry et al. [Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., & Hartshorne, D. J. (1978) Biochemistry 17, 4417-4418] observed that the actin activation of ATPase was not inhibited by the removal of Ca2+. Hence, it was suggested that the regulation of actomyosin ATPase activity of gizzard myosin by calcium occurs only via phosphorylation. In the present study, phosphorylated and thiophosphorylated myosins were prepared free of kinase and phosphatase activity; hence, the ATPase activity could be measured at various concentrations of Ca2+ and Mg2+ without affecting the level of phosphorylation. The ATPase activity of myosin was activated either by skeletal muscle or by gizzard actin at various concentrations of Mg2+ and either at pCa 5 or at pCa 8. The activation was sensitive to Ca2+ at low Mg2+ concentrations with both actins. Tropomyosin potentiated the actin-activated ATPase activity at all Mg2+ and Ca2+ concentrations. The calcium sensitivity of phosphorylated and thiophosphorylated myosin reconstituted with actin and tropomyosin was most pronounced at a free Mg2+ concentration of about 3 mM. The binding of 125I-tropomyosin to actin showed that the calcium sensitivity of ATPase observed at low Mg2+ concentration is not due to a calcium-mediated binding of tropomyosin to F-actin. The actin activation of both myosins was insensitive to Ca2+ when the Mg2+ concentration was increased above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tropomyosin exon 6b is troponin-specific and required for correct acto-myosin regulation

The specificity of tropomyosin (Tm) exon 6b for interaction with and functioning of troponin (Tn) has been studied using recombinant fibroblast Tm isoforms 5a and 5b. These isoforms differ internally by exons 6a/6b and possess non-muscle exons 1b/9d at the termini, hence they lack the primary TnT(1)-tropomyosin interaction, allowing study of exon 6 exchange in isolation from this. Using kinetic techniques to measure regulation of myosin S1 binding to actin and fluorescently labeled Tm to directly measure Tn binding, we show that binding of Tn to both isoforms is similar (0.1-0.5 microm) and both produce well regulated systems. Calcium has little effect on Tn binding to the actin.Tm complex and both exons produce a 3-fold reduction in the S1 binding rate to actin.Tm.Tn in its absence. This confirms previous results that show exon 6 has little influence on Tn affinity to actin.Tm or its ability to fully inhibit the acto-myosin interaction. Thin filaments reconstituted with Tn and Tm5a or skeletal Tm (containing exon 6b) show nearly identical calcium dependence of acto-myosin regulation. However, Tm5b produces a dramatic increase in calcium sensitivity, shifting the activation mid-point by almost an order of magnitude. This shows that exon 6 sequence and, hence, Tm structure in this region have a significant effect upon the calcium regulation of Tn. This finding supports evidence that familial hypertrophic cardiomyopathy mutations occurring adjacent to this region can effect calcium regulation.     

Fluorescence probe studies of the state of tropomyosin in reconstituted muscle thin filaments

The monomer fluorescence of N-(1-pyrenyl)maleimide-labeled tropomyosin bound to F-actin (PTm-actin) increases when myosin subfragment 1 (S1) binds to actin and is half complete when only approximately 1 S1 is bound to 7 actin subunits [Ishii, Y., & Lehrer, S. S. (1985) Biochemistry 24, 6631-6638]. Similar studies of the binding of S1 and S1-ADP to fully reconstituted thin filaments [PTm-actin-troponin (Tn)] are now reported. The pyrene monomer fluorescence change was half complete when approximately 0.5 S1/7 actin subunits and approximately 1.5 S1/7 actin subunits were bound in the presence and absence of Ca2+, respectively. In the presence of Mg2+-ADP, when S1 binding is weakened, the S1 binding profiles and fluorescence changes were sigmoidal, with the cooperative transitions occurring at lower [S1] in the presence of Ca2+ as first shown by Greene and Eisenberg for S1 binding [Greene, L., & Eisenberg, E. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2616-2620]. It was possible to fit both the binding and fluorescence data with the same parameters of a two-state (weak and strong S1 binding) cooperative binding model [Hill, T., Eisenberg, E., & Greene, L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186-3190] for each Ca2+ situation if the fluorescence change is interpreted as the fraction of tropomyosin (Tm) units in the strong S1 binding state. These data indicate that the fluorescence change is a direct measure of the S1-induced change of state of Tm in the fully reconstituted thin filament.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of muscle contraction: bindings of troponin and its components to actin and tropomyosin.

The bindings of troponin components to actin and tropomyosin has been studied by cosedimentation with actin and affinity chromatography. It is shown that troponin binds to actin and tropomyosin in the presence and absence of calcium but the binding to actin is sensitive to ionic strength. Troponin-I + C binds to actin-tropomyosin in the absence of calcium but not to actin or tropomyosin alone. Troponin-I binds to actin and the binding is improved in the presence of tropomyosin even though troponin-I does not bind to tropomyosin alone. Troponin-C does not bind to actin or tropomyosin. The results suggest that the binding of troponin by actin is influenced by tropomyosin. A model of regulation by troponin is proposed.     

Cooperative binding of tropomyosin to muscle and Acanthamoeba actin.

Analyses of the binding of tropomyosin to muscle and Acanthamoeba actin by the use of Scatchard plots indicate that the binding exhibits strong positive cooperativity in the presence of Mg2+. The cooperative nature of the binding is not affected by the presence of 80 mm KCl, but appears to decrease somewhat in the presence of heavy meromyosin or subfragment-1. Heavy meromyosin, subfragment-1, and KCl each increase the binding affinity of actin for tropomyosin; depending on the experimental condition and the type of actin involved, the apparent binding constant, Kapp, is in the range of 1 to 4 x 10(6) M-1. Muscle actin cross-linked with glutaraldehyde failed to bind tropomyosin even when heavy meromyosin, subfragment-1, or KCl were added as inducers, although the cross-linked actin still markedly activated the heavy meromyosin ATPase.     

The effects of smooth muscle caldesmon on actin filament motility.

The movement of reconstituted thin filaments over an immobilized surface of thiophosphorylated smooth muscle myosin was examined using an in vitro motility assay. Reconstituted thin filaments contained actin, tropomyosin, and either purified chicken gizzard caldesmon or the purified COOH-terminal actin-binding fragment of caldesmon. Control actin-tropomyosin filaments moved at a velocity of 2.3 +/- 0.5 microns/s. Neither intact caldesmon nor the COOH-terminal fragment, when maintained in the monomeric form by treatment with 10 mM dithiothreitol, had any effect on filament velocity; and yet both were potent inhibitors of actin-activated myosin ATPase activity, indicating that caldesmon primarily inhibits myosin binding as reported by Chalovich et al. (Chalovich, J. M., Hemric, M. E., and Velaz, L. (1990) Ann. N. Y. Acad. Sci. 599, 85-99). Inhibition of filament motion was, however, observed under conditions where cross-linking of caldesmon via disulfide bridges was present. To determine if monomeric caldesmon could "tether" actin filaments to the myosin surface by forming an actin-caldesmon-myosin complex as suggested by Chalovich et al., we looked for caldesmon-dependent filament binding and motility under conditions (80 mM KCl) where filament binding to myosin is weak and motility is not normally seen. At caldesmon concentrations > or = 0.26 microM, actin filament binding was increased and filament motion (2.6 +/- 0.6 microns/s) was observed. The enhanced motility seen with intact caldesmon was not observed with the addition of up to 26 microM COOH-terminal fragment. Moreover, a molar excess of the COOH-terminal fragment competitively reversed the enhanced binding seen with intact caldesmon. These results show that tethering of actin filaments to myosin by the formation of an actin-caldesmon-myosin complex enhanced productive acto-myosin interaction without placing a significant mechanical load on the moving filaments.     

Actin from Saccharomyces cerevisiae.

Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth.     

Myofibrillar troponin exists in three states and there is signal transduction along skeletal myofibrillar thin filaments

Activation of striated muscle contraction is a highly cooperative signal transduction process converting calcium binding by troponin C (TnC) into interactions between thin and thick filaments. Once calcium is bound, transduction involves changes in protein interactions along the thin filament. The process is thought to involve three different states of actin-tropomyosin (Tm) resulting from changes in troponin's (Tn) interaction with actin-Tm: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interactions and favored by calcium binding to Tn, and an open or M state allowing strong myosin interactions. This was tested by measuring the apparent rate of Tn dissociation from rigor skeletal myofibrils using labeled Tn exchange. The location and rate of exchange of Tn or its subunits were measured by high-resolution fluorescence microscopy and image analysis. Three different rates of Tn exchange were observed that were dependent on calcium concentration and strong cross-bridge binding that strongly support the three-state model. The rate of Tn dissociation in the non-overlap region was 200-fold faster at pCa 4 (C-state region) than at pCa 9 (B-state region). When Tn contained engineered TnC mutants with weakened regulatory TnI interactions, the apparent exchange rate at pCa 4 in the non-overlap region increased proportionately with TnI-TnC regulatory affinity. This suggests that the mechanism of calcium enhancement of the rate of Tn dissociation is by favoring a TnI-TnC interaction over a TnI-actin-Tm interaction. At pCa 9, the rate of Tn dissociation in the overlap region (M-state region) was 100-fold faster than the non-overlap region (B-state region) suggesting that strong cross-bridges increase the rate of Tn dissociation. At pCa 4, the rate of Tn dissociation was twofold faster in the non-overlap region (C-state region) than the overlap region (M-state region) that likely involved a strong cross-bridge influence on TnT's interaction with actin-Tm. At sub-maximal calcium (pCa 6.2-5.8), there was a long-range influence of the strong cross-bridge on Tn to enhance its dissociation rate, tens of nanometers from the strong cross-bridge. These observations suggest that the three different states of actin-Tm are associated with three different states of Tn. They also support a model in which strong cross-bridges shift the regulatory equilibrium from a TnI-actin-Tm interaction to a TnC-TnI interaction that likely enhances calcium binding by TnC.     

The regulation of myosin binding to actin filaments by Lethocerus troponin

Lethocerus indirect flight muscle has two isoforms of troponin C, TnC-F1 and F2, which are unusual in having only a single C-terminal calcium binding site (site IV, isoform F1) or one C-terminal and one N-terminal site (sites IV and II, isoform F2). We show here that thin filaments assembled from rabbit actin and Lethocerus tropomyosin (Tm) and troponin (Tn) regulate the binding of rabbit myosin to rabbit actin in much the same way as the mammalian regulatory proteins. The removal of calcium reduces the rate constant for S1 binding to regulated actin about threefold, independent of which TmTn is used. This is consistent with calcium removal causing the TmTn to occupy the B or blocked state to about 70% of the total. The mid point pCa for the switch differed for TnC-F1 and F2 (pCa 6.9 and 6.0, respectively) consistent with the reported calcium affinities for the two TnCs. Equilibrium titration of S1 binding to regulated actin filaments confirms calcium regulated binding of S1 to actin and shows that in the absence of calcium the three actin filaments (TnC-F1, TnC-F2 and mammalian control) are almost indistinguishable in terms of occupancy of the B and C states of the filament. In the presence of calcium TnC-F2 is very similar to the control with approximately 80% of the filament in the C-state and 10-15% in the fully on M-State while TnC-F1 has almost 50% in each of the C and M states. This higher occupancy of the M-state for TnC-F1, which occurs above pCa 6.9, is consistent with this isoform being involved in the calcium activation of stretch activation. However, it leaves unanswered how a C-terminal calcium binding site of TnC can activate the thin filament.