The Active State of the Thin Filament Is Destabilized by an Internal Deletion in Tropomyosin*

(Received for publication, February 3, 1997, and in revised form, March 24, 1997)

Cheryl A. Landis Dagger , Alyona Bobkova §, Earl Homsher § and Larry S. Tobacman Dagger

From the Dagger  Departments of Internal Medicine and Biochemistry, The University of Iowa, Iowa City, Iowa 52242 and the § Department of Physiology, School of Medicine, University of California at Los Angeles, Los Angeles, California 90024

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The function of three of tropomyosin's sequential quasiequivalent regions was studied by deletion from skeletal muscle alpha -tropomyosin of internal residues 49-167. This deletion mutant tropomyosin spans four instead of the normal seven actins, and most of the tropomyosin region believed to interact with troponin is retained and uninterrupted in the mutant. The mutant tropomyosin was compared with a full-length control molecule that was modified to functionally resemble muscle tropomyosin (Monteiro, P. B., Lataro, R. C., Ferro, J. A., and Reinach, F. C. (1994) J. Biol. Chem. 269, 10461-10466). The tropomyosin deletion suppressed the actin-myosin subfragment 1 MgATPase rate and the in vitro sliding of thin filaments over a heavy meromyosin-coated surface. This inhibition was not reversed by troponin plus Ca2+. Comparable tropomyosin affinities for actin, regardless of the deletion, suggest that the deleted region has little interaction with actin in the absence of other proteins. Similarly, the deletion did not weaken binding of the troponin-tropomyosin complex to actin. Furthermore, Ca2+ had a 2-fold effect on troponin-tropomyosin's affinity for actin, regardless of the deletion. Notably, the deletion greatly weakened tropomyosin binding to myosin subfragment 1-decorated actin, with the full-length tropomyosin having a 100-fold greater affinity. The inhibitory properties resulting from the deletion are attributed to defective stabilization of the myosin-induced active state of the thin filament.


INTRODUCTION

Tropomyosin is an extended alpha -helical coiled-coil that spans seven actin monomers. Periodicity in tropomyosin's amino acid sequence and also in its modeled three-dimensional structure have led to the proposal that it contains 7 and possibly 14 quasiequivalent regions (1-4). Each <FR><NU>1</NU><DE>7</DE></FR> length region is approximately 39 <FR><NU>2</NU><DE>3</DE></FR> residues long and contains either one or, in the original proposal (1), a pair of putative actin binding motifs. Deletion of approximately one-half (i.e. 21 residues) of a long period repeat or one complete such region has little effect on troponin-tropomyosin's ability to bind actin or regulate the myosin S-11 thin filament ATPase rate. (4). However, deletion of <FR><NU>2</NU><DE>3</DE></FR> of a putative actin binding motif abolishes binding and therefore regulation (4). In the present study a mutant tropomyosin designated ralpha TmDelta (49-167) with three of the seven regions (regions 2, 3, and 4) deleted has been created. 119 amino acids are absent from this mutant, corresponding to approximately 3 × 39 <FR><NU>2</NU><DE>3</DE></FR> residues per region as defined by McLachlan and Stewart (1). ralpha TmDelta (49-167) is long enough to span only four actin monomers, yet the present report shows it to behave normally in many respects.

Recent structural data suggest that tropomyosin can be positioned in three different locations on the actin filament. These positions are dependent upon the presence or the absence of troponin, calcium, and myosin. Troponin in the absence of Ca2+ induces an "off" state in which only weak transitory myosin S-1 binding occurs (5, 6). Tropomyosin in this position blocks actin sites necessary for strong myosin S-1 binding as modeled by Rayment et al. and others (6-10). When either Ca2+ is added or troponin is removed, there is a 25 ° rotation of the tropomyosin from the off state, partially uncovering actin residues involved in myosin binding (7-11). An additional 10 ° rotation of tropomyosin about the actin filament occurs when myosin S-1 is bound to the actin (7, 9, 11, 12).

The structural studies above suggest that tropomyosin-actin interactions are highly diverse. Accordingly, several conditions were examined to characterize the deletion tropomyosin of the present report. The deletion was found not to alter tropomyosin-actin binding under almost all conditions. However, the deletion had a major effect on this process in the presence of myosin S-1. Furthermore, ralpha TmDelta (49-167) was found to inhibit in vitro motility and thin filament-myosin S-1 MgATPase activity. The functional significance of these interrelated observations is discussed.


MATERIALS AND METHODS

Construction of Recombinant Tropomyosins

One consideration in designing these experiments was that bacterially expressed forms of striated muscle tropomyosin are unacetylated and therefore will not bind actin without troponin (13-15). Monteiro et al. (16) have shown that a dipeptide (Ala-Ser) at the NH2 terminus can substitute for acetylation and restore actin binding and polymerization. Therefore, full-length and deletion tropomyosins were both modified at the 5' end so that the recombinant forms expressed in Escherichia coli would mimic acetylated bovine cardiac tropomyosin. A control molecule, designated ralpha Tm, was created using rat alpha  tropomyosin as a PCR template. Oligo 1 (5'-GCGCTCGAGCCATGGCTAGTATGGACGCCATCAAG-3') added a tripeptide, Met-Ala-Ser, to the 5' end as well as a XhoI and NcoI restriction sites for cloning purposes. The methionine is removed during processing in the cell (16). PCR oligo 2 (5'-GCGTCTAGATCTTTATATGGAAGTC-ATATCC-3') contained an error and would have caused a mistake in Asn281, but the proofreading mechanisms of Vent DNA polymerase (New England Biolab) corrected the PCR product to match the template as was confirmed by sequencing.

The internal deletion was accomplished by two PCR reactions that were cloned individually and later ligated together. Oligo 1 from above and oligo 3 (5'-ATGCGCTGCAGCGCGCCACCAGTGACACCAGCTC-3') were used to create the actin binding region 1-containing portion of ralpha TmDelta (49-167). Oligo 3 introduces a BSSH II site without altering the amino acid sequence. The other PCR product, encoding putative actin binding regions 5, 6, and 7, was constructed using oligo 2 and the BSSHII-containing oligo 4 (5'-ATGCGGCGCGCAAGCTGGTCATCATC-3'). The number of amino acids deleted was a multiple of seven, thus allowing proper assembly of the coiled-coil (17). The PCR products were cloned into pSP72 for confirmation by sequencing and then cloned into pET-3D for expression in E. coli (DE3).

Protein Preparation

Bovine cardiac troponin and tropomyosin (18), rabbit skeletal muscle F-actin (19), and rabbit skeletal myosin S-1 (20) were purified as described previously. Recombinant rat striated muscle alpha  tropomyosins were purified as described (15) with revisions. A 40% (242 mg/ml)/70% (231.8 mg/ml) ammonium sulfate fractionation was done. The protein was dialyzed, 6 M urea was added, and the protein was loaded on a 80-ml DEAE-cellulose column at 4 °C. The column had been equilibrated with 50 mM Tris (pH 8.0), 0.01% NaN3, 0.5 mM EDTA, 0.5 mM DTT, and 6 M urea. This same buffer was used to wash the column after protein loading. The protein was then eluted using a 0-300 mM NaCl gradient of 360 ml. The pooled fractions were dialyzed against 10 mM Tris (pH 7.5), 1 mM DTT, 0.01% NaN3. Protein concentrations were determined using extinction coefficients as determined by Gill and von Hippel (21). The coefficient for ralpha TmDelta (49-167) was 1.024 × 104 M-1 cm-1. The full-length ralpha Tm and bovine cardiac tropomyosin were calculated to have an extinction coefficient of 1.5360 × 104 M-1 cm-1.

MgATPase Rate and Binding Assays

The release of phosphate from [gamma -32P]ATP (DuPont NEN) in the presence of myosin S-1 was measured (22) at six 2-min intervals. Conditions and protein concentrations are as described in the legend to Fig. 5.


Fig. 5. Effect of myosin S-1 on the binding of ralpha Tm and ralpha TmDelta (49-167) to actin. A, control molecule ralpha Tm (squares versus circles) exhibited a dramatic increase in binding upon the addition of rabbit skeletal muscle myosin S-1. This effect was absent for ralpha TmDelta (49-167), which showed no binding regardless of the presence of myosin S-1 (diamonds and triangles). There is at least a 100-fold effect of the deletion when myosin is present. The conditions were 25 °C, 10 mM NaH2PO4 (pH 7.0), 10 mM Tris (pH 7.5), 5 mM MgCl2, 0.1 mM DTT, 1 mM EGTA, 300 mM KCl, 5 µM actin, with or without 5 µM myosin S-1. B, to determine whether myosin S1 promotes or inhibits the association of ralpha TmDelta (49-167) to actin, the experiment in A was repeated in the presence of a lower KCl concentration, 60 mM. (Binding of ralpha Tm to actin-myosin S1 is too tight to measure under these conditions.) The squares show data for ralpha TmDelta (49-167) obtained in the presence of myosin S-1. For comparison, the curve representing binding in the absence of myosin S-1 is also shown, using K0 and y for ralpha TmDelta (49-167) from Fig. 2. Conditions were the same as in A except for the KCl concentration. The figure indicates that ralpha TmDelta (49-167) had a 6-fold increase in actin affinity when rabbit skeletal myosin S-1 was included.
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Tropomyosins used in binding assays were radiolabeled on Cys190 with [3H]iodoacetic acid (23). Radioactivity from before and after centrifugation at 35,000 rpm in a TLA100 rotor for 30 min at 25 °C were compared (24). Binding curves of tropomyosins or troponin-tropomyosin complexes to actin were fit to the McGhee-Von Hippel equation (23-25). This equation evaluates the binding of a long ligand to a linear lattice. The equation deals with the problem of random gaps in the lattice that are too short for a ligand to fit and explicitly considers cooperative interactions between adjacent ligands that counteract this parking problem. K0 is defined as the affinity of a ligand for an isolated site on the lattice. Any cooperativity between ligands results in a y-fold increase in affinity for a singly adjacent site. The overall binding affinity, Kapp, approximately equals the product yK0.

In Vitro Motility Assays

The movement of rhodamine phalloidin-labeled thin filaments over rabbit fast skeletal muscle-heavy meromyosin-coated coverslips was observed by video epifluorescence microscopy and analyzed as described previously (26, 27). The experiments were performed at an ionic strength of 0.1 M in the presence of 0.5% methyl cellulose. The temperature of the assay was held constant at 25 °C. Protein concentrations and other conditions are described under "Results" or in Table I.

Table I. Deletion tropomyosin inhibits movement in motility assays

2 µM rhodamine-phalloidin F-actin was incubated overnight with varying concentrations (0.5, 0.75, and 1.0 µM) of ralpha TmDelta (49-167), designated as "in preincubation" row below. The labeled F-actin was then diluted 100-fold and applied to a motility slide, designated by "after dilution" in the table. The slides were then exposed to motility buffers containing varying concentrations of ralpha TmDelta (49-167) (designated by "Additional [ralpha TmDelta (49-167)] in motility buffer" in the table. In all cases at least 100 filaments were analyzed for mean speed ± S.D. and for the fraction of filaments moving continuously (27). 2 µM rhodamine-phalloidin F-actin was incubated overnight with varying concentrations (0.5, 0.75, and 1.0 µM) of ralpha TmDelta (49-167), designated as "in preincubation" row below. The labeled F-actin was then diluted 100-fold and applied to a motility slide, designated by "after dilution" in the table. The slides were then exposed to motility buffers containing varying concentrations of ralpha TmDelta (49-167) (designated by "Additional [ralpha TmDelta (49-167)] in motility buffer" in the table. In all cases at least 100 filaments were analyzed for mean speed ± S.D. and for the fraction of filaments moving continuously (27).

ralpha TmDelta (49-167) in preincubation 0.5 µM 0.75 µM 1.0 µM
ralpha TmDelta (49-167) after dilution 5 nM
7.5 nM
10 nM
Additional ralpha TmDelta (49-167) in motility buffer Speeda %b Speed % Speed %

0 nM 4.97  ± 0.9 78.2 4.78  ± 0.8 60 4.90  ± 1.0 80
25 nM 4.00  ± 1.0 70.0 3.40  ± 0.9 57 2.80  ± 0.7 49
50 nM 2.88  ± 0.9 55  2.80  ± 0.9 35.3 2.80  ± 0.8 33
100 nM 0 0 0 0  0

a Sliding speed µm/s.
b % of filaments moving uniformly.


RESULTS

Binding of Recombinant Tropomyosins to Actin at Various Ionic Strengths

Bacterially expressed skeletal muscle tropomyosin lacks the NH2-terminal acetylation necessary for tropomyosin-actin binding in the absence of troponin (13, 28). However, a fusion chicken alpha -tropomyosin with an NH2-terminal dipeptide binds to actin normally (16). In this study an analogous rat fusion alpha -tropomyosin, ralpha Tm, was created and compared with bovine cardiac muscle tropomyosin and to ralpha TmDelta (49-167), which contains the NH2-terminal dipeptide and has 119 internal residues deleted. For purposes of comparison, concentrations of both full-length tropomyosins (0.8 µM) and of ralpha TmDelta (49-167) (1.6 µM) were chosen so that 50% of each would bind to 5 µM actin at low ionic strength (Fig. 1). As reported previously for the analogous chicken fusion tropomyosin, ralpha Tm (Fig. 1, squares) functioned very similarly to muscle tropomyosin (Fig. 1, circles). More significantly, ralpha TmDelta (49-167) (Fig. 1, triangles) did not act differently from ralpha Tm. All three tropomyosins had decreases in affinity for actin as the salt concentration increased. Beyond 0.15 M both recombinant tropomyosins did not bind detectably to actin, whereas bovine cardiac tropomyosin appeared to maintain weak actin binding (see also Refs. 23 and 24).


Fig. 1. Effect of ionic strength on binding of various tropomyosins to actin. The binding of various forms of tropomyosin to actin was assessed by cosedimentation, as the KCl concentration was varied from 0 to 0.3 M. Bovine cardiac muscle tropomyosin and ralpha Tm concentrations were constant at 0.8 µM for all KCl concentrations. ralpha TmDelta (49-167), which binds to actin with a different stoichiometry, was held constant at 1.6 µM. The ordinate shows the fraction of total added tropomyosin that bound. Raising the ionic strength had similar effects on ralpha Tm (squares), ralpha TmDelta (49-167) (triangles), and bovine cardiac tropomyosin (circles). The conditions were 25 °C, 10 mM NaH2PO4 (pH 7.0), 10 mM Tris (pH 7.5), 5 mM MgCl2, 0.1 mM DTT, 1 mM EGTA, 5 µM F-actin.
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Recombinant Tropomyosin Binds Actin with Normal Affinity and Cooperativity

Fig. 1 suggests that the tropomyosin internal deletion has relatively little effect on tropomyosin-actin binding. To assess this more precisely, tropomyosin-actin binding was analyzed as a function of the tropomyosin concentration, with representative data in Fig. 2. These results demonstrate that the stoichiometry of binding was as expected. Both full-length tropomyosins span seven actins, whereas ralpha TmDelta (49-167) spans four. Fig. 2 shows that ralpha Tm plateaued at approximately 1.5 µM of tropomyosin bound. Due to the difference in length, 1.75 times as much ralpha TmDelta (49-167) would be needed to saturate the same amount of actin. This was the case with a value of approximately 2.6 µM tropomyosin bound. Under the conditions presented in the figure (60 mM KCl), the ralpha Tm had a Kapp for actin of 1.6 × 106 M-1. Remarkably the Kapp for ralpha TmDelta (49-167), 0.88 × 105 M-1, was only slightly lower than that of the full-length molecule. Each Kapp value is the product of two terms, K0 and y (see "Materials and Methods"). The 1.8-fold change in Kapp is within the variation observed from preparation to preparation (23, 24). For the paired preparations shown in Fig. 2, the cooperativity parameters were almost identical (ralpha TmDelta (49-167), y = 35; control ralpha Tm, y = 42). The deletion tropomyosin had a slightly lower K0 (affinity of tropomyosin for an isolated site on F-actin) compared with full-length tropomyosin (2.5 versus 3.7 × 104 M-1), a 1.5-fold difference. Bovine cardiac tropomyosin (not shown) bound with an intermediate binding affinity to the recombinant forms. These results parallel findings by Monteiro et al. (16), who first determined that the fusion dipeptide resulted in normal tropomyosin function.


Fig. 2. Actin binding of recombinant forms of tropomyosin in the absence of troponin. Bacterially expressed striated muscle tropomyosin binds very poorly to actin in the absence of troponin (13-15). However, binding occurs if a fusion dipeptide is present at the NH2 terminus (ralpha Tm, squares). Binding also occurred for a tropomyosin molecule containing the fusion peptide but lacking 119 internal residues that were deleted (ralpha TmDelta (49-167), triangles). As expected from its shorter length, ralpha TmDelta (49-167) bound to actin with a higher stoichiometry. The conditions were 25 °C, 10 mM NaH2PO4 (pH 7.0), 10 mM Tris (pH 7.5), 5 mM MgCl2, 0.1 mM DTT, 1 mM EGTA, 60 mM KCl, and 10 µM F-actin. The solid lines are theoretical curves corresponding to values of Kapp, K0, and y as described in the text.
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If each quasiequivalent region of tropomyosin contributed equal binding energy, then removing three of seven such regions would result in <FR><NU>4</NU><DE>7</DE></FR> the binding energy. This would mean that K0 for ralpha TmDelta (49-167) would be 4.1 × 102 M-1 ((ralpha Tm K0)4/7 = (3.7 × 104 M-1)4/7 = 4.1 × 102 M-1). This would be a 90-fold effect of the deletion on K0 and the same 90-fold effect on Kapp (because Kapp = yK0). This calculation is meant as a general estimate for the expected effects of the deletion, not an exact prediction. However, the observed effects (<2-fold) are much smaller than estimated, suggesting that putative actin binding regions 2, 3, and 4 do not interact with actin under these conditions.

Effect of Deletion Tropomyosin on MgATPase Rate

In the presence of EGTA, increasing concentrations of bovine cardiac troponin plus either ralpha TmDelta (49-167) or ralpha Tm caused inhibition of the actin-myosin S1 MgATPase rate (Fig. 3, triangles and circles). Upon the addition of CaCl2, the activity remained inhibited in samples with ralpha TmDelta (49-167) (Fig. 3, diamonds). This indicates that the troponin-deletion tropomyosin complex can inhibit normally but that this is not reversed by the addition of Ca2+. By comparison, saturating amounts of troponin-ralpha Tm activated the ATPase rate approximately 16-fold when CaCl2 was present (Fig. 3, squares), as expected with normal regulation. This verifies that dipeptide fusion tropomyosin molecules are capable of activation as well as inhibition.


Fig. 3. Troponin-ralpha TmDelta (49-167) inhibits actin-myosin S-1 MgATPase activity even upon the addition of calcium. Increasing total concentrations of ralpha TmDelta (49-167) plus troponin were inhibitory to actin-myosin S1 MgATPase activity in the presence of either Ca2+ (triangles) or EGTA (diamonds). In contrast, troponin-full-length ralpha Tm resulted in a MgATPase rate that was activated in the presence of Ca2+ (squares) but inhibited without Ca2+ (circles). The conditions were 25 °C, 10 mM NaH2PO4 (pH 7.0), 5 mM MgCl2, 1 mM DTT, 1 mM ATP, 1 µM rabbit skeletal S-1, 4 µM F-actin, and either 0.5 mM EGTA or 0.1 mM CaCl2. Tropomyosin concentrations ranged from 0 to 1.0 µM with troponin in 0.1 µM excess of tropomyosin.
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Other results indicated that when tropomyosin-saturated actin was studied in the absence of troponin, the shortened tropomyosin inhibited the MgATPase rate to a greater degree than did full-length recombinant tropomyosin. In MgATPase assays (not shown) with 1 µM myosin S-1, 4 µM actin, 3.5 µM ralpha Tm, and other conditions as in Fig. 3, the MgATPase rate was 0.16 s-1, whereas it was 0.06 s-1 with ralpha TmDelta (49-167). By comparison actin and myosin S-1 alone had a value of 0.6 s-1. The inhibition of control ralpha Tm was 4-fold, similar to observations with cardiac tropomyosin (Ref. 18; see also Refs. 29 and 30). Shortened ralpha TmDelta (49-167), however, produced a 10-fold inhibition of the MgATPase rate.

Deletion Tropomyosin Inhibits in Vitro Motility in the Absence and the Presence of Troponin

Rhodamine-phalloidin labeled F-actin (2 µM) was incubated overnight with various concentrations of ralpha TmDelta (49-167). The skeletal muscle heavy meromyosin-propelled movement of these filaments (diluted to low actin concentration with supplemental ralpha TmDelta (49-167) in the motility buffer) was studied as in Ref. 27. Regardless of the overnight conditions, increases in the final ralpha TmDelta (49-167) concentration caused a decrease in speed and the number of moving filaments (Table I). In contrast, control ralpha Tm had no effect on movement (not shown). The prevention of all filament movement by sufficient concentrations of ralpha TmDelta (49-167) (last line of Table I) is consistent with the ATPase results. The deletion tropomyosin blocks productive actin-myosin interactions. Significantly, the reduction in speed at intermediate concentrations of ralpha TmDelta (49-167) indicates that it does not completely block actin-myosin binding. Rather, it must permit weak actin-myosin interactions that exert a drag on the actin filament. In all cases, the addition of 100 nM ralpha TmDelta (49-167) resulted in no movement. This concentration is lower than expected from Fig. 2, perhaps due to methylcellulose in the motility buffer.

Motility assays using reconstituted actin-tropomyosin-troponin thin filaments were also done. As expected, both bovine cardiac tropomyosin and ralpha Tm conferred Ca2+-sensitive regulation in the presence of troponin. The results with ralpha TmDelta (49-167) resembled the MgATPase results. 100 nM concentrations of troponin plus ralpha TmDelta (49-167) resulted in no movement, regardless whether Ca2+ was added (data not shown).

Effect of Bovine Cardiac Troponin and Calcium on Tropomyosin-Actin Binding

To understand the inhibitory effect of the deletion, thin filament assembly was studied in the presence of troponin with or without CaCl2. Ionic conditions were chosen (150 mM KCl) so that binding to actin was weak in the absence of troponin and in a stronger but still measurable range in the presence of troponin. Troponin caused ralpha TmDelta (49-167) to bind actin at least as tightly as the control ralpha Tm (Fig. 4). (The deletion mutant retains most of the tropomyosin region believed to interact with troponin (31-34).) Full-length and <FR><NU>4</NU><DE>7</DE></FR> length tropomyosins in the presence of troponin bound to actin with very similar Kapp (2.2 × 106 M-1, 2.6 × 106 M-1, respectively). Interestingly, the addition of Ca2+ caused a decrease in binding affinity of both complexes to actin, a 2-fold change in each case. This change is comparable with that seen with cardiac muscle tropomyosin (23). Although the deletion abolishes regulation, it does not alter this effect of Ca2+ on thin filament assembly.


Fig. 4. Similar thin filament assembly for ralpha Tm and ralpha TmDelta (49-167) in the presence of troponin. The triangles and diamonds show representative actin binding curves for troponin-ralpha TmDelta (49-167) plus and minus Ca2+, respectively. The presence and the absence of Ca2+ with control ralpha Tm are represented by circles and squares, respectively. Both recombinant molecules exhibited a similar shift upon the addition of Ca2+. The conditions were 25 °C, 10 mM NaH2PO4 (pH 7.0), 10 mM Tris (pH 7.5), 5 mM MgCl2, 0.1 mM DTT, 0.15 M KCl, 5 µM F-actin, and either 0.1 mM CaCl2 or 1 mM EGTA. The total tropomyosin concentrations were varied from 0 to 3.4 µM. Troponin was varied in parallel and maintained at a 0.1 µM excess relative to the tropomyosin concentration. Theoretical curves were obtained as described in Ref. 23.
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Effect of Myosin S-1 on the Binding of Recombinant Tropomyosin and Mutant Tropomyosin to Actin

Myosin S-1 and heavy meromyosin greatly strengthen the affinity of tropomyosin for actin (24, 35, 36). Muscle tropomyosin binding to actin-myosin S-1 is so tight that it is difficult to measure, even in the presence of high ionic strength (24). Fig. 5A shows that a similar effect was observed for ralpha Tm. This full-length recombinant tropomyosin did not substantially bind to actin in the absence of myosin S-1 in the presence of 0.3 M KCl, but the addition of myosin S-1 increased binding greatly to a Kapp of 9.1 × 106 M-1 (Fig. 5A, squares versus circles). The effect of myosin S-1 on ralpha Tm-actin binding is at least 100-fold and probably greater. In contrast, ralpha TmDelta (49-167) did not bind to actin measurably under the same conditions, regardless of the presence of myosin S-1 (Fig. 5A, diamonds versus triangles). Deletion of the internal tropomyosin region had a major 100-fold or larger effect on tropomyosin binding to actin-myosin S1.

To investigate any effect of myosin S-1 on ralpha TmDelta (49-167) binding to actin, positive or negative, these experiments were repeated at lower ionic strength. At 60 mM KCl, myosin S-1 had a 6-fold strengthening effect on the Kapp of ralpha TmDelta (49-167) binding to actin (Fig. 5B). This effect of myosin S-1 is attributable to an increase in tropomyosin's affinity for an isolated site rather than to a change in cooperativity (the semilog curves are parallel). Binding of ralpha Tm to actin was too tight to measure under these conditions.


DISCUSSION

Tropomyosin has been shown by several investigators to have different affinities for actin depending upon the presence or the absence of myosin, troponin, and Ca2+ (reviewed in Ref. 37). The addition of troponin increases tropomyosin's affinity for actin (23), perhaps due to troponin-actin interactions (37), and causes it to shift position when Ca2+ is added (7-11). Myosin S-1 also increases tropomyosin's affinity for actin (24, 35, 36), and emerging results (9, 11, 12) indicate that it induces tropomyosin to shift to a third location on the actin, different from either the relaxed state or the Ca2+ state. These different quaternary structures for the thin filament may correspond to the blocked, closed, and opened states proposed in Ref. 38. Our results suggest that the deleted region, tropomyosin regions 2, 3, and 4, is required for stabilizing the myosin-induced state but is not required for other conformational states. The idea that the ends rather than the middle of tropomyosin are most important for actin binding in the absence of myosin is supported by several lines of evidence. First, in electron microscopy data obtained under conditions (0.5 mM MgCl2) poorly favoring tropomyosin-actin binding, Mabuchi (39) observes tropomyosin molecules tethered by one end to the actin filament. Second, the use of carboxypeptidase-A-digested tropomyosin or unacetylated tropomyosin severely decreases tropomyosin's affinity for actin (13, 40), and this effect is not due to diminished cooperativity (15, 41). Finally, Hitchcock-DeGregori and An (17) showed that deletion of tropomyosin regions 2 and 3 had only a 2-fold effect on troponin-tropomyosin binding to actin. This prior study differed from the present results, because two rather than three of the quasiequivalent regions were deleted and because the fusion dipeptide at the NH2 terminus was absent. This latter difference compensates for the absence of acetylation in bacterially expressed tropomyosin and permits experiments in the absence of troponin. Such experiments now demonstrate that the deleted region does not contribute significantly to actin binding when troponin is absent.

The deletion of tropomyosin regions 2, 3, and 4 also had no effect on troponin-mediated changes in thin filament assembly. For both full-length and deletion tropomyosins, troponin promoted binding to actin (compare Fig. 4 with the 150 mM KCl data in Fig. 1). In these assays the Kapp values for full-length tropomyosin-troponin and <FR><NU>4</NU><DE>7</DE></FR> length tropomyosin-troponin are very close in value. This parallels published findings that deletion of regions 2 and 3 had little effect (17). Fig. 4 also shows both recombinant tropomyosin-troponin complexes have a decreased affinity for actin upon the addition of Ca2+. The decrease in affinity is comparable between full-length and shortened tropomyosins, suggesting that the deletion of these three regions does not interfere with Ca2+-induced changes in the thin filament conformation. This would be very significant because it suggests that the shortened tropomyosin is capable of undergoing calcium-induced changes yet inhibits MgATPase activity (Fig. 3) and in vitro motility (Table I). Other data will be needed to establish this point definitively, however.

Full-length ralpha Tm binds actin at least 100-fold more tightly in the presence than in the absence of myosin S-1 and permits actin-myosin S-1 MgATPase activity and in vitro filament movement. Shortened ralpha TmDelta (49-167) exhibits none of these properties. Because myosin S-1 slightly promotes ralpha TmDelta (49-167)-actin binding (Fig. 5B), it can be concluded that myosin S-1 and ralpha TmDelta (49-167) can bind simultaneously to F-actin. The reduced thin filament sliding speed and fraction of smoothly moving filaments at intermediate concentrations of ralpha TmDelta (49-167) (Table I) suggests the presence of a drag force, perhaps from myosin S-1 attachment to a portion of the thin filament where tropomyosin is bound. Nevertheless, the tropomyosin internal deletion results in destabilization of the actin-myosin S-1-tropomyosin structure relative to what is found with normal tropomyosin. Furthermore, the thin filament assembly data in the absence of myosin indicate that this destabilization is specific for the myosin-induced conformation of the thin filament.

Strongly bound myosin cross-bridges create an activated state of tropomyosin-containing thin filaments, with increased actomyosin ATPase rates (42-45), troponin affinity for Ca2+ (46-51), actin-myosin affinity (46, 52, 53), and actin-tropomyosin affinity (24, 35, 36). Solution studies of contractile proteins (38, 45) and muscle fiber investigations (54-58) have been explainable in terms of a cooperative transition to one active state for the thin filament, which presumably corresponds to this potentiated state (43). Recent structural data suggest that regulated thin filament have at least three conformations but that only one of these conformations has tropomyosin in a position to allow cross-bridge cycling (11, 59). These results and recent solution studies of actin-myosin S1 interactions in the absence (38) and the presence (60) of ATP point toward the conclusion that there is essentially one active state for the thin filament. One way to test this concept would be to specifically destabilize the myosin-induced or potentiated state of the thin filament and then evaluate the effect on regulation under low myosin, high MgATP conditions. Unexpectedly, tropomyosin with regions 2, 3, and 4 deleted has properties that provide such a test of the mechanism of regulation. The tropomyosin internal deletion destabilizes the actin-myosin-tropomyosin complex and does not change the Ca2+-sensitive assembly of the actin-myosin-tropomyosin-troponin complex, yet the deletion is profoundly inhibitory to actin-myosin cycling. Unless the tropomyosin binds very strongly to actin-myosin S-1, it is inhibitory.

Finally, a relevant prior report is the 1990 study by Hitchcock-DeGregori and Varnell, which showed that an internal deletion of tropomyosin did not prevent Ca2+-sensitive regulation of the myosin S-1 MgATPase rate (4). This shortened tropomyosin had only region 2 deleted and was <FR><NU>6</NU><DE>7</DE></FR> the normal length, so there is no conflict between this earlier data and the present report. Rather, the combined results suggest the possibility that a specific tropomyosin sequence within region 3 and/or 4 is necessary for thin filament activation. It may be functionally important, for example, that ralpha TmDelta (49-167) is missing half of the tropomyosin region (residues 150-180) believed to interact with the globular region of troponin (31-33, 61-64). However, loss of a specific interaction with troponin would not explain the inhibitory properties of ralpha TmDelta (49-167) in the absence of troponin. An alternative possibility is that there are a critical number of internal tropomyosin regions needed to stabilize the myosin S-1 induced "on" state or otherwise to restore regulation. In other words, the crucial difference may be that a <FR><NU>6</NU><DE>7</DE></FR> length tropomyosin permits thin filament activation and a <FR><NU>4</NU><DE>7</DE></FR> length tropomyosin does not, with the specific regions relatively unimportant. Further experiments may be able to distinguish among these different explanations.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed.
1   The abbreviations used are: S-1, subfragment 1; DTT, dithiothreitol; ralpha Tm, rat striated muscle recombinant Ala-Ser-alpha -tropomyosin; ralpha TmDelta (49-167), same as ralpha Tm except residues 49-167 of the rat muscle isoform are deleted; PCR, polymerase chain reaction.

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