Role of residues 230 and 236 of actin in myosin-ATPase activation by actin-tropomyosin

The Dictyostelium/Tetrahymena-chimeric actin (Q228K/T229A/A230Y) showed higher Ca(2+)-activation of myosin S1 ATPase in the presence of tropomyosin-troponin. The crystal structure of the chimeric actin is almost the same as that of wild-type except the conformation of the side chain of Leu236. Here, we introduced an additional mutation (L236A), in which the side chain of Leu236 was truncated, into the chimeric actin (Q228K/T229A/A230Y/L236A). Without regulatory proteins, the new mutant actin showed normal myosin S1 activation and normal sliding velocity. However, in the presence of tropomyosin, the new mutant actin activated myosin S1 ATPase higher than the wild-type actin and showed higher velocities in in vitro motility assay at low HMM concentrations. These results suggest that the mutations of A230Y and L236A in the actin subdomain-4 facilitate the transition of thin filaments from a "closed" state to an "open" state.     

Structural basis for the higher Ca(2+)-activation of the regulated actin-activated myosin ATPase observed with Dictyostelium/Tetrahymena actin chimeras

Replacement of residues 228-230 or 228-232 of subdomain 4 in Dictyostelium actin with the corresponding Tetrahymena sequence (QTA to KAY replacement: half chimera-1; QTAAS to KAYKE replacement: full chimera) leads to a higher Ca(2+)-activation of the regulated acto-myosin subfragment-1 ATPase activity. The ratio of ATPase activation in the presence of tropomyosin-troponin and Ca(2+) to that without tropomyosin-troponin becomes about four times as large as the ratio for the wild-type actin. To understand the structural basis of this higher Ca(2+)-activation, we have determined the crystal structures of the 1:1 complex of Dictyostelium mutant actins (half chimera-1 and full chimera) with gelsolin segment-1 to 2.0 A and 2.4 A resolution, respectively, together with the structure of wild-type actin as a control. Although there were local changes on the surface of the subdomain 4 and the phenolic side-chain of Tyr230 displaced the side-chain of Leu236 from a non-polar pocket to a more solvent-accessible position, the structures of the actin chimeras showed that the mutations in the 228-232 region did not introduce large changes in the overall actin structure. This suggests that residues near position 230 formed part of the tropomyosin binding site on actin in actively contracting muscle. The higher Ca(2+)-activation observed with A230Y-containing mutants can be understood in terms of a three-state model for thin filament regulation in which, in the presence of both Ca(2+) and myosin heads, the local changes of actin generated by the mutation (especially its phenolic side-chain) facilitate the transition of thin filaments from a "closed" state to an "open" state. Between 394 and 469 water molecules were identified in the different structures and it was found that actin recognizes hydrated forms of the adenine base and the Ca ion in the nucleotide binding site.     

Three-dimensional reconstruction of thin filaments containing mutant tropomyosin

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

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)     

Interaction of maleimidobenzoyl actin with myosin subfragment 1 and tropomyosin-troponin.

A chemical modification of G-actin with (m-maleimidobenzoyl)-N-hydroxysuccinimide ester (MBS) impairs actin polymerization [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. MBS-actin recovers the ability to polymerize when a 2-fold molar excess of phalloidin is added in 30 mM KCl/2 mM MgCl2/20 mM Tris-HCl (pH 7.6). The resulting polymer (MBS-P-actin) is highly potentiated so that it activates the Mg(2+)-ATPase of S1 more strongly than native F-actin. The affinity of MBS-P-actin for S1 in the presence of ATP (KATPase) is about four times higher than that of native F-actin, although the maximum velocity at infinite actin concentration (Vmax) is almost the same. This high activation is not due to a cross-linking between MBS-P-actin and the S1 heavy chain, since no substantial amount of cross-linking was observed in SDS gel electrophoresis. Direct binding studies and ATPase measurements showed that the modification of actin with MBS impairs the binding of tropomyosin. Tropomyosin binding can be improved considerably by the addition of troponin. However, the regulation mechanism of the acto-S1 ATPase activity by troponin-tropomyosin is damaged. The addition of troponin-tropomyosin reduces the S1 ATPase activation by MBS-P-actin to the same level as that of native F-actin in 30 mM KCl/2.5 mM ATP/2 mM MgCl2, but there is no difference in the ATPase activation in the presence and absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin

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

Differential interaction of cardiac, skeletal muscle, and yeast tropomyosins with fluorescent (pyrene235) yeast actin

To monitor binding of tropomyosin to yeast actin, we mutated S235 to C and labeled the actin with pyrene maleimide at both C235 and the normally reactive C374. Saturating cardiac tropomyosin (cTM) caused about a 20% increase in pyrene fluorescence of the doubly labeled F-actin but no change in WT actin C374 probe fluorescence. Skeletal muscle tropomyosin caused only a 7% fluorescence increase, suggesting differential binding modes for the two tropomyosins. The increased cTM-induced fluorescence was proportional to the extent of tropomyosin binding. Yeast tropomyosin (TPM1) produced less increase in fluorescence than did cTM, whereas that caused by yeast TPM2 was greater than either TPM1 or cTM. Cardiac troponin largely reversed the cTM-induced fluorescence increase, and subsequent addition of calcium resulted in a small fluorescence recovery. An A230Y mutation, which causes a Ca(+2)-dependent hypercontractile response of regulated thin filaments, did not change probe235 fluorescence of actin alone or with tropomyosin +/- troponin. However, addition of calcium resulted in twice the fluorescence recovery observed with WT actin. Our results demonstrate isoform-specific binding of different tropomyosins to actin and suggest allosteric regulation of the tropomyosin/actin interaction across the actin interdomain cleft.     

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)     

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.     

Mutations in actin subdomain 3 that impair thin filament regulation by troponin and tropomyosin.

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

Mapping the domain of troponin T responsible for the activation of actomyosin ATPase activity. Identification of residues involved in binding to actin

The in vitro Ca(2+) regulation of the actomyosin Mg(2+)-ATPase at physiological ratios of actin, tropomyosin, and troponin occurs only in the presence of troponin T. We have previously demonstrated that a polypeptide corresponding to the first 191 amino acids of troponin T (TnT-(1-191)) activates the actomyosin Mg(2+)-ATPase in the presence of tropomyosin. In order to further characterize this activation domain, we constructed troponin T fragments corresponding to residues 1-157 (TnT-(1-157)), 1-76 (TnT-(1-76)), 77-157 (TnT-(77-157)), 77-191 (TnT-(77-191)), and 158-191 (TnT-(158-191)). Assays using these fragments demonstrated the following: (a) residues 1-76 do not bind to tropomyosin or actin; (b) residues 158-191 bind to actin cooperatively but not to tropomyosin; (c) the sequence 77-157 is necessary for troponin interaction with residue 263 of tropomyosin; (d) TnT-(77-191) on its own activates the actomyosin ATPase activity as described previously for TnT-(1-191). TnT-(1-157), TnT-(1-76), TnT-(77-157), TnT-(158-191), and combinations of TnT-(158-191) with TnT-(1-157) or TnT-(77-157) showed no effect on the ATPase activity. We conclude that the activation of actomyosin ATPase activity is mediated by a direct interaction between amino acids 77 and 191 of troponin T, tropomyosin, and actin.     

Effects of cardiac thin filament Ca2+: statistical mechanical analysis of a troponin C site II mutant.

Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.     

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments

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

Drosophila muscle regulation characterized by electron microscopy and three-dimensional reconstruction of thin filament mutants

Wild-type and mutant thin filaments were isolated directly from "myosinless" Drosophila indirect flight muscles to study the structural basis of muscle regulation genetically. Negatively stained filaments showed tropomyosin with periodically arranged troponin complexes in electron micrographs. Three-dimensional helical reconstruction of wild-type filaments indicated that the positions of tropomyosin on actin in the presence and absence of Ca(2+) were indistinguishable from those in vertebrate striated muscle and consistent with a steric mechanism of regulation by troponin-tropomyosin in Drosophila muscles. Thus, the Drosophila model can be used to study steric regulation. Thin filaments from the Drosophila mutant heldup(2), which possesses a single amino acid conversion in troponin I, were similarly analyzed to assess the Drosophila model genetically. The positions of tropomyosin in the mutant filaments, in both the Ca(2+)-free and the Ca(2+)-induced states, were the same, and identical to that of wild-type filaments in the presence of Ca(2+). Thus, cross-bridge cycling would be expected to proceed uninhibited in these fibers, even in relaxing conditions, and this would account for the dramatic hypercontraction characteristic of these mutant muscles. The interaction of mutant troponin I with Drosophila troponin C is discussed, along with functional differences between troponin C from Drosophila and vertebrates.     

A comparison of muscle thin filament models obtained from electron microscopy reconstructions and low-angle X-ray fibre diagrams from non-overlap muscle

The regulation of striated muscle contraction involves changes in the interactions of troponin and tropomyosin with actin thin filaments. In resting muscle, myosin-binding sites on actin are thought to be blocked by the coiled-coil protein tropomyosin. During muscle activation, Ca2+ binding to troponin alters the tropomyosin position on actin, resulting in cyclic actin-myosin interactions that accompany muscle contraction. Evidence for this steric regulation by troponin-tropomyosin comes from X-ray data [Haselgrove, J.C., 1972. X-ray evidence for a conformational change in the actin-containing filaments of verterbrate striated muscle. Cold Spring Habor Symp. Quant. Biol. 37, 341-352; Huxley, H.E., 1972. Structural changes in actin and myosin-containing filaments during contraction. Cold Spring Habor Symp. Quant. Biol. 37, 361-376; Parry, D.A., Squire, J.M., 1973. Structural role of tropomyosin in muscle regulation: analysis of the X-ray diffraction patterns from relaxed and contracting muscles. J. Mol. Biol. 75, 33-55] and electron microscope (EM) data [Spudich, J.A., Huxley, H.E., Finch, J., 1972. Regulation of skeletal muscle contraction. II. Structural studies of the interaction of the tropomyosin-troponin complex with actin. J. Mol. Biol. 72, 619-632; O'Brien, E.J., Gillis, J.M., Couch, J., 1975. Symmetry and molecular arrangement in paracrystals of reconstituted muscle thin filaments. J. Mol. Biol. 99, 461-475; Lehman, W., Craig, R., Vibert, P., 1994. Ca2+-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature 368, 65-67] each with its own particular strengths and limitations. Here we bring together some of the latest information from EM analysis of single thin filaments from Pirani et al. [Pirani, A., Xu, C., Hatch, V., Craig, R., Tobacman, L.S., Lehman, W. (2005). Single particle analysis of relaxed and activated muscle thin filaments. J. Mol. Biol. 346, 761-772], with synchrotron X-ray data from non-overlapped muscle fibres to refine the models of the striated muscle thin filament. This was done by incorporating current atomic-resolution structures of actin, tropomyosin, troponin and myosin subfragment-1. Fitting these atomic coordinates to EM reconstructions, we present atomic models of the thin filament that are entirely consistent with a steric regulatory mechanism. Furthermore, fitting the atomic models against diffraction data from skinned muscle fibres, stretched to non-overlap to preclude crossbridge binding, produced very similar results, including a large Ca2+-induced shift in tropomyosin azimuthal location but little change in the actin structure or apparent alteration in troponin position.     

Role of tropomyosin in smooth muscle contraction: effect of tropomyosin binding to actin on actin activation of myosin ATPase

The binding of gizzard tropomyosin to gizzard F-actin is highly dependent on free Mg2+ concentration. At 2 mM free Mg2+, a concentration at which actin-activated ATPase activity was shown to be Ca2+ sensitive, a molar ratio of 1:3 (tropomyosin:actin monomer) is required to saturate the F-actin with tropomyosin to the stoichiometric ratio of 1 mol of tropomyosin to 7 mol of actin monomer. Increasing the Mg2+ could decrease the amount of tropomyosin required for saturating the F-actin filament to the stoichiometric level. Analysis of the binding of smooth muscle tropomyosin to smooth muscle actin by the use of Scatchard plots indicates that the binding exhibits strong positive cooperativity at all Mg2+ concentrations. Calcium has no effect on the binding of tropomyosin to actin, irrespective of the free Mg2+ concentration. However, maximal activation of the smooth muscle actomyosin ATPase in low free Mg2+ requires the presence of Ca2+ and stoichiometric binding of tropomyosin to actin. The lack of effect of Ca2+ on the binding of tropomyosin to actin shows that the activation of actomyosin ATPase by Ca2+ in the presence of tropomyosin is not due to a calcium-mediated binding of tropomyosin to actin.     

Characterization of carbethoxylated actin

The carbethoxylation of histidine residues in G-actin impairs actin polymerization. The histidine residue essential for polymerization was identified as histidine-40 [Hegyi, G., Premecz, G., Sain, B., & Mühlrad, A. (1974) Eur. J. Biochem. 44, 7-12]. Non-polymerizable actin was separated from the polymerizable fraction after partial carbethoxylation. The non-polymerizable actin recovered the ability to polymerize following addition of phalloidin. Taking into account the evidence that phalloidin does not bind to G-actin in the absence of salt, the results indicate that the actin monomer undergoes a conformational change and subsequently binds phalloidin before polymerization. The resulting polymers activated S1 ATPase activity as effectively as control F-actin. In the presence of tropomyosin and troponin, a strong inhibition of actin-activated ATPase activity was observed in the absence of Ca2+, although no inhibition was observed in the presence of Ca2+. These results indicate that His-40 is not directly involved in a myosin binding site nor in a tropomyosin-troponin binding site.     

Phosphorylation of an actin.tropomyosin.troponin complex from human skeletal muscle.

A human skeletal actin.tropomyosin.troponin complex was phosphorylated in the presence of [gamma-32 P]ATP, Mg2+, adenosine 3':5'-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 microM cyclic AMP. In the presence of 10(-7) M Ca2+ and protein kinase 0.1 mole of [32P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [32P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca2+, 5.10(-5) M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [32P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [32P]phosphate. Increasing the Ca2+ concentration did not significantly decrease the [32P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstitute human skeletal actomyosin made with the [32P] phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca2+.     

Ca(2+)- and s1-induced movement of troponin T on mutant thin filaments reconstituted with functionally deficient mutant tropomyosin

The deletion mutant (D234Tm) of rabbit skeletal muscle alpha-tropomyosin, in which internal actin-binding pseudo-repeats 2, 3, and 4 are missing, inhibits the thin filament activated myosin-ATPase activity whether Ca(2+) ion is present or not [Landis et al. (1997) J. Biol. Chem. 272, 14051-14056]. Fluorescence resonance energy transfer (FRET) showed substantial changes in distances between Cys-60 or 250 of troponin T (TnT) and Gln-41 or Cys-374 of actin on wild-type thin filaments corresponding to three states of thin filaments [Kimura et al. (2002) J. Biochem. 132, 93-102]. Troponin T movement on mutant thin filaments reconstituted with D234Tm was compared with that on wild-type thin filaments to understand from which the functional deficiency of mutant thin filaments derives. The Ca(2+)-induced changes in distances between Cys-250 of TnT and Gln-41 or Cys-374 of F-actin were smaller on mutant thin filaments than on wild-type thin filaments. On the other hand, the distances between Cys-60 of TnT and Gln-41 or Cys-374 of F-actin on mutant thin filaments did not change at all regardless of whether Ca(2+) was present. Thus, FRET showed that the Ca(2+)-induced movement of TnT was severely impaired on mutant thin filaments. The rigor binding of myosin subfragment 1 (S1) increased the distances when the thin filaments were fully decorated with S1 in the presence and absence of Ca(2+). However, plots of the extent of S1-incuced movement of TnT against molar ratio of S1 to actin in the presence and absence of Ca(2+) showed that the S1-induced movement of TnT was also impaired on mutant thin filaments. The deficiency of TnT movement on mutant thin filaments causes the altered S1-induced movement of TnI, and mutant thin filaments consequently fail to activate the myosin-ATPase activity even in the presence of Ca(2+).     

Regulatory properties of recombinant tropomyosins containing 5-hydroxytryptophan: Ca2+-binding to troponin results in a conformational change in a region of tropomyosin outside the troponin binding site.

We have introduced tryptophan codons at different positions of the chicken alpha-tropomyosin cDNA (Monteiro, P. B., Lataro, R. C., Ferro, J. A., and Reinach, F. C. (1994) J. Biol. Chem. 269, 10461-10466) and employed a trp auxotrophic Escherichia coli strain to express the proteins in media containing either normal tryptophan, 5-hydroxytrptophan, or 7-azatryptophan. The fluorescence of these latter two tryptophan analogues is excitable at 312-315 nm at which the natural fluorescence of other thin filament proteins (actin, troponin) is not excited. The recombinant tropomyosins have tryptophans or analogues located at amino acid positions 90, 101, 111, 122, or 185 of the protein, all on the external surface of the tropomyosin coiled-coil (positions "c" or "f" of the hydrophobic heptad repeat). The first four mutations are located within the third actin-binding zone of tropomyosin, a region not expected to interact directly with troponin or with neighboring tropomyosin molecules in muscle thin filaments, while position 185 is located in a region that has been implicated in interactions with the globular domain of troponin. The fluorescence intensity of the mutant containing 5-hydroxytryptophan at position 122 (5OH122W) is sensitive to actin binding and sensitive to Ca2+-binding to thin filaments reconstituted with troponin. Assuming that the globular domain of troponin binds to a site between residues 150 and 190 of tropomyosin, the distance between the troponin-binding site and the fluorescent probes at position 122 can be estimated to be 4.2-10.2 nm. While X-ray diffraction and electron micrograph reconstitution studies have provided evidence of Ca2+-induced changes in tropomyosin's interactions in the thin filament, their resolution was not sufficient to distinguish between changes involving the whole tropomyosin molecule or only that region directly interacting with troponin. Here we provide a clear demonstration that Ca2+-binding to troponin results in a conformational change in a region of tropomyosin outside the troponin binding site which is probably associated with a changed interaction with actin.