Steady-state kinetic studies on the actin activation of skeletal muscle heavy meromyosin subfragments: Effects of skeletal,smooth and non-muscle tropomyosins

The addition of either smooth muscle or brain tropomyosin to skeletal muscle actoheavy meromyosin (HMM) or acto-myosin subfragment-1 (SF1) produces an activation of the actin-activated ATPase activity up to 100%. This contrasts with the opposite, inhibitory effect produced by skeletal muscle tropomyosin. The degree of activation or inhibition depends on the ionic conditions, which influence the affinities of tropomyosin and HMM or SF1 for actin as well as on the molar ratio of actin to myosin.Enzyme kinetic analysis indicates that the inhibitory effect of skeletal muscle tropomyosin results from an approximately six- to tenfold increase in the apparent affinity (K_app) of the myosin head for the F-actin-tropomyosin complex with a concomitant six- to tenfold reduction in the maximal turnover rate (V_max). Thus, there is no direct competition of skeletal muscle tropomyosin and myosin for the same site on actin. Brain tropomyosin has an opposite effect, decreasing the apparent affinity with concomitant increase in the V_max.The effect of smooth muscle tropomyosin is more complex. At high ratios of myosin to actin this tropomyosin produces the same change in the K_app as skeletal muscle tropomyosin but yields a value of V_max that is about twofold higher. At lower molar ratios (below about 1 to 5 myosin subfragments to actin) the activating effect of this tropomyosin remains unchanged while the apparent affinity decreases to that observed for pure F-actin.On the basis of these data as well as from experiments carried out at fixed actin and varying SF1 concentrations, it is concluded that tropomyosins act in general as allosteric un-competitive inhibitors or activators of actomyosin by increasing or reducing the co-operative activation of myosin by actin at the level of product release.     

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.     

Crosslinking of actin filaments and inhibition of actomyosin subfragment-1 ATPase activity by streptococcal M6 protein

M proteins are antiphagocytic molecules on the surface of group A streptococci having physical characteristics similar to those of mammalian tropomyosin. Both are alpha-helical coiled-coil fibrous structures with a similar seven-residue periodicity of nonpolar and charged amino acids. To determine if M protein is functionally similar to tropomyosin we studied the interaction of M protein with F-actin. At low ionic strength, M protein binds to actin weakly with a stoichiometry different from that of tropomyosin. M protein does not compete with tropomyosin for the binding to actin, indicating that it is functionally different from tropomyosin. M protein does compete with myosin subfragment-1 for binding to actin and induces the formation of bundles of actin filaments. The formation of actin aggregates is associated with a sharp reduction in the rate of ATP hydrolysis by subfragment-1. Intact streptococci having M protein on their surface are shown to bind to actin.     

Caldesmon inhibits skeletal actomyosin subfragment-1 ATPase activity and the binding of myosin subfragment-1 to actin

Smooth muscle contraction is controlled in part by the state of phosphorylation of myosin. A recently discovered actin and calmodulin-binding protein, named caldesmon, may also be involved in regulation of smooth muscle contraction. Caldesmon cross-links actin filaments and also inhibits actin-activated ATP hydrolysis by myosin, particularly in the presence of tropomyosin. We have studied the effect of caldesmon on the rate of hydrolysis of ATP by skeletal muscle myosin subfragment-1, a system in which phosphorylation of the myosin is not important in regulation. Caldesmon is a very effective inhibitor of ATP hydrolysis giving up to 95% inhibition. At low ionic strength (approximately 20 mM) this effect does not require smooth muscle tropomyosin, whereas at high ionic strength (approximately 120 mM) tropomyosin enhances the inhibitory activity of caldesmon at low caldesmon concentrations. Cross-linking of actin is not essential for inhibition of ATP hydrolysis to occur since at high ionic strength there is very little cross-linking as determined by a low speed sedimentation assay. Under all conditions examined, the decrease in the rate of ATP hydrolysis is accompanied by a decrease in the binding of myosin subfragment-1 to actin. Furthermore, caldesmon weakens the equilibrium binding of myosin subfragment-1 to actin in the presence of pyrophosphate. We conclude that caldesmon has a general weakening effect on the binding of skeletal muscle myosin subfragment-1 to actin and that this weakening in binding may be responsible for inhibition of ATP hydrolysis.     

Dynamics of tropomyosin in muscle fibers as monitored by saturation transfer EPR of bi-functional probe

The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into "ghost muscle fibers". The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1.     

Anti-actin antibodies. An immunological approach to the myosin-actin and the tropomyosin-actin interfaces.

The topography of the rigor complex between subfragment-1 (S-1) of myosin and actin was investigated by using several specific antibodies directed to well-located sequences in actin. A major contact area for S-1 was characterized in the hydrophilic 18-28 constant sequence, and the variable 1-7 sequence was only found to be in close proximity to the interface. The C-terminal extremity of actin situated around Cys-374 appeared to be included in a region close to the S-1 heavy chain and the N-terminal part of actin. The interaction between tropomyosin and actin was also studied. Neither of the terminal parts of actin were involved in this interaction. Thus, the regions involved in the interactions of S-1 and tropomyosin with actin do not overlap.     

Pig platelet tropomyosin: interactions with the other thin-filament proteins

Pig platelet tropomyosin exhibits many of the functional activities of skeletal tropomyosin. At low ionic strength it forms end-to-end aggregates similar to those formed by skeletal tropomyosins. It forms a 1:1 complex with muscle troponin or with a troponin I-pig brain calmodulin complex, as well as a 1:6 association with platelet filamentous actin. Electron microscopy of paracrystals shows that the troponin binding site is slightly C-terminal of the unique cysteine, corresponding to position 190 of the rabbit skeletal alpha-tropomyosin sequence. The effect of a complex comprising platelet actin and tropomyosin on the ATPase activity of rabbit skeletal muscle myosin subfragment-1 was similar to that displayed by its skeletal muscle counterpart. Platelet tropomyosin decreased the activity by roughly half in a calcium-independent manner. Addition of troponin to the actin-tropomyosin in the absence of calcium results in further inhibition and allows the full activity of the complex to be restored by Ca2+. These results differ from those obtained by C?té & Smillie for horse platelet tropomyosin and this may reflect the different isomeric nature of pig platelet tropomyosin. These results suggest that the functional properties of non-muscle tropomyosins may differ when comparisons are made between proteins isolated from the same type of cell but in different species. Differences in self-association and actin-binding properties may be finely graded between different isoforms.     

The binding of skeletal muscle C-protein to F-actin, and its relation to the interaction of actin with myosin subfragment-1.

C-protein is a component of thick filaments of skeletal muscle myofibrils. It is bound to the assembly of myosin tails that forms the filament backbone. We report here that C-protein can also bind to F-actin, with a limiting stoichiometry of approximately one C-protein molecule per 3 to 5 actin subunits and a dissociation constant in the micromolar range at ionic strength 0·07. The binding is not significantly affected by ATP, calcium ions or temperature, or by the presence of tropomyosin on the actin, but it is weakened by increasing ionic strength. Myosin subfragment-1 (S-1) competes with C-protein for binding to actin. In the absence of ATP, S-1 displaces nearly all bound C-protein from actin, while in the presence of ATP, C-protein inhibits the actin activation of S-1 ATPase. Although there is no direct evidence that interaction of C-protein with actin is physiologically significant, the lenght of the C-protein molecule is sufficient so that it could make contact with the thin filaments in muscle while remaining attached to the thick filaments.     

Construction, expression and unexpected regulatory properties of a tropomyosin mutant with a 31-residue deletion at the C-terminus (exon 9)

The cDNA coding for human skeletal muscle beta-tropomyosin was expressed in Escherichia coli to produce an unacetylated beta-tropomyosin. This cDNA was deleted from the sequence corresponding to the exon 9 and expressed in E. coli to produce an unacetylated beta-tropomyosin mutant lacking the C-terminal residues 254-284. The main structural and functional properties of the two isolated proteins, designated tropomyosin-1 and des-(254-284)-tropomyosin, respectively, were characterized in comparison with those of the genuine rabbit skeletal muscle alpha beta-tropomyosin. The folding and thermal stability of the three tropomyosins were indistinguishable. Tropomyosin-1, but not des-(254-284)-tropomyosin, was polymerized in the presence of troponin and did bind to actin in the presence of the troponin complex. Despite its weak binding to actin, des-(254-284)-tropomyosin displayed a regulatory function in the presence of troponin with a marked activation of the actomyosin subfragment-1 ATPase in the presence of Ca2+ and low concentrations of subfragment-1. The data were interpreted in the light of the allosteric models of regulation and suggest the involvement of the sequence coded by exon 9 in the stabilization by tropomyosin of the off state of the thin filament.     

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+.     

Study of reciprocal effects of cardiac myosin and tropomyosin isoforms on actin-myosin interaction with in vitro motility assay

Interaction of myosin with actin in striated muscle is controlled by Ca²⁺ via thin filament associated proteins: troponin and tropomyosin. In cardiac muscle there is a whole pattern of myosin and tropomyosin isoforms. The aim of the current work is to study regulatory effect of tropomyosin on sliding velocity of actin filaments in the in vitro motility assay over cardiac isomyosins. It was found that tropomyosins of different content of α- and β-chains being added to actin filament effects the sliding velocity of filaments in different ways. On the other hand the velocity of filaments with the same tropomyosins depends on both heavy and light chains isoforms of cardiac myosin.     

Identification of a Unique “Stability Control Region” that Controls Protein Stability of Tropomyosin: A Two-stranded α-Helical Coiled-coil

Nine recombinant chicken skeletal α-tropomyosin proteins were prepared, eight C-terminal deletion constructs and the full length protein (1-81, 1-92, 1-99, 1-105, 1-110, 1-119, 1-131, 1-260 and 1-284) and characterized by circular dichroism spectroscopy and analytical ultracentrifugation. We identified for the first time, a stability control region between residues 97 and 118. Fragments of tropomyosin lacking this region (1-81, 1-92, and 1-99) still fold into two-stranded α-helical coiled-coils but are significantly less stable (T_m between 26-28.5 °C) than longer fragments containing this region (1-119, 1-131, 1-260 and 1-284) which show a large increase in their thermal midpoints (T_m 40-43 °C) for a ΔT_m of 16-18 °C between 1-99 and 1-119. We further investigated two additional fragments that ended between residues 99 and 119, that is fragments 1-105 and 1-110. These fragments were more stable than 1-99 and less stable than 1-119, and showed that there were three separate sites that synergistically contribute to the large jump in protein stability (electrostatic clusters 97-104 and 112-118, and a hydrophobic interaction from Leu 110). All the residues involved in these stabilizing interactions are located outside the hydrophobic core a and d positions that have been shown to be the major contributor to coiled-coil stability. Our results show clearly that protein stability is more complex than previously thought and unique sites can synergistically control protein stability over long distances.     

Interaction of myosin subfragment-1 with actin. II. Location of the actin binding site in a fragment of subfragment-1 heavy chain

The heavy chain of subfragment-1 prepared by chymotrypsin treatment had a molecular weight of about 96K. The heavy chain was split into 26 K, 50 K, and 21 K fragments by trypsin. When the trypsin-treated subfragment-1 was cross-linked with dimethyl suberimidate, cross-linked products of 26 K, 50 K, and 21 K fragments and of 50 K and 21 K fragments appeared, but there was little cross-linked product of 26 K and 50 K fragments or of 26 K and 21 K fragments. When the cross-linking experiments were carried out in the presence of actin, a new band appeared and the amount of cross-linked product of 26 K, 50 K, and 21 K fragments decreased by about 50%. The molecular weight of the new band was lower than that of the cross-linked product of 26 K, 50 K, and 21 K fragments, and higher than that of the dimer of actin. Based on this and some other results, we suggest that this band represented a cross-linked product of actin and the 50 K fragment. We also suggest that the decrease in the amount of cross-linked product of 26 K, 50 K, and 21 K fragments reflected the conformational change in subfragment-1 due to the binding of actin.     

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.     

Purification and characterization of an exon 2-deleted human beta-tropomyosin constructed by the polymerase chain reaction.

We deleted exon 2 in human skeletal beta-tropomyosin (h beta-SK tropomyosin) using an improved adaptation of polymerase chain reaction (PCR) technology. The first PCR product was used to prime the full-length cDNA, leading to an exon 2-deleted h beta-SK tropomyosin. This new protein, des-(39-80)-tropomyosin, could then be expressed in Escherichia coli and purified to homogeneity. At the nucleotide level, the junction between exons 1 and 3 has been precisely made in the PCR product. The mutated protein was purified using high-performance liquid chromatography. Des-(39-80)-tropomyosin revealed new immunological properties but was still recognized by certain antitropomyosin antibodies. Furthermore, the structural characteristics of the mutated tropomyosin fit those of the full-length tropomyosin. This new adaptation of PCR technology appears to be suitable for every kind of mutation inside a cloned DNA molecule, and one mutation primer per mutation is sufficient.     

Cooperativity of actin-activated ATPase of gizzard heavy meromyosin in the presence of gizzard tropomyosin

The mechanism for the potentiation of the actin-activated ATPase of smooth muscle myosin by tropomyosin is investigated using smooth muscle actin, tropomyosin, and heavy meromyosin. In the presence of tropomyosin, an increase in Vmax occurs with no effect on KATPase and Kbinding at 20 mM ionic strength. Utilizing N-ethylmaleimide-treated subfragment-1, which forms rigor complexes with actin in the presence of ATP but does not have ATPase activity, experiments were carried out to determine if the tropomyosin-actin complex exists in both the turned-off and turned-on forms as in the skeletal muscle system. At both 60 and 100 mM ionic strengths, the presence of rigor complexes on the smooth muscle actin filament containing bound tropomyosin causes a 2-3-fold increase in Vmax and about a 3-fold increase in KATPase, resulting in about a 4-fold increase in ATPase activity at moderate actin concentration. The increase in KATPase is correlated with an increase in Kbinding. The finding that rigor complexes increase Vmax and the binding constant for heavy meromyosin to tropomyosin-actin at an ionic strength close to physiological conditions indicates that the tropomyosin-actin complex can be turned on by rigor complexes in a cooperative manner. However, in contrast to the situation in the skeletal muscle system, the increase in KATPase is associated with a corresponding increase in Kbinding. Furthermore, there is only a 3-fold increase in KATPase in the smooth muscle system rather than a 10-fold increase as in the skeletal muscle system.     

Characterization of f-actin tryptophan phosphorescence in the presence and absence of tryptophan-free myosin motor domain

The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140-293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (tau(1) and tau(2)) and increasing by approximately 20% the longest component (tau(3)). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (tau(4), twice as long as tau(3)) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique.     

Effect of avidin binding to SH1 on the interface between subfragment-1 and F-actin

The subfragment-1-avidin complex, in which avidin is attached to a well defined thiol group called SH1, was purified by CM cellulose column chromatography or affinity chromatography using lipoic acid agarose. The interaction of the purified complex with F-actin was compared to that of normal subfragment-1 using chemical cross-linking and limited tryptic digestion techniques. It was found that the binding of avidin to SH1 lowered the extent of cross-linking between the subfragment-1 heavy chain and actin. The amount of the 175K product decreased to about 50% of the normal level and that of the 165K product decreased to about 35%. It was also found that the binding of avidin abolished the protective effect of F-actin on the 50K-22K junction of the S-1 heavy chain against tryptic attack. Since more than 95% of the S-1-avidin complex was attached to F-actin under our experimental conditions, these changes are due to an alteration of the S-1-actin interface. Considering the facts that SH1 is located on the side of S-1 facing the F-actin, in the tertiary structure, and is close to the cross-linked site and to the 50K-22K junction, in the primary structure, it is quite likely that avidin bound to SH1 causes these effects by sterically preventing the close contact of S-1 and actin.     

A jump in an Arrhenius plot can be the consequence of a phase transition. The binding of ATP to myosin subfragment 1

The temperature dependence of the kinetics of the binding of ATP to myosin subfragment-1 was studied by an ATP chase technique in a rapid-flow-quench apparatus: (formula; see text) A temperature range of 30 degrees C to -15 degrees C was obtained with ethylene glycol as antifreeze. The Arrhenius plot of k2 is discontinuous with a jump at 12 degrees C. Above the jump delta H+ = 9.5 kcal/mol, below delta H+ = 28.5 kcal/mol. Few such Arrhenius plots are recorded in the literature but they are predicted from theory. Thus, we explain our results as a phase change of the subfragment 1-ATP system at 12 degrees C. This is in agreement with certain structural studies.