©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Minimal Motor Domain from Chicken Skeletal Muscle Myosin (*)

Guillermina S. Waller , Greta Ouyang , James Swafford (§) , Peter Vibert , Susan Lowey (¶)

From the (1)Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254-9110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The myosin head (S1) consists of a wide, globular region that contains the actin- and nucleotide-binding sites and an -helical, extended region that is stabilized by the presence of two classes of light chains. The essential light chain abuts the globular domain, whereas the regulatory light chain lies near the head-rod junction of myosin. Removal of the essential light chain by a mild denaturant exposes the underlying heavy chain to proteolysis by chymotrypsin. The cleaved fragment, or ``motor domain'' (MD), migrates as a single band on SDS-polyacrylamide gel electrophoresis, with a slightly greater mobility than S1 prepared by papain or chymotrypsin. Three-dimensional image analysis of actin filaments decorated with MD reveals a structure similar to S1, but shorter by an amount consistent with the absence of a light chain-binding domain. The actin-activated MgATPase activity of MD is similar to that of S1 in V and K. But the ability of MD to move actin filaments in a motility assay is considerably reduced relative to S1. We conclude that the globular, active site region of the myosin head is a stable, independently folded domain with intrinsic motor activity, but the coupling efficiency between ATP hydrolysis and movement declines markedly as the light chain binding region is truncated.


INTRODUCTION

The introduction of in vitro motility assays in the 1980s established that the myosin head (subfragment-1, S1) is sufficient to move actin filaments and generate force (Toyoshima et al., 1987; Kishino and Yanagida, 1988). With the publication of the crystal structure of S1 from chicken skeletal muscle myosin (Rayment et al., 1993), it became clear that the myosin head consists of distinct domains, which do not correspond to the three fragments (25, 50, and 20-kDa) obtained by proteolytic digestion of flexible loops in S1 (Mornet et al., 1989). Instead, there is a globular domain containing the nucleotide- and actin-binding sites, from which extends a long -helix (the C-terminal half of the 20-kDa fragment) that is stabilized by the essential (ELC) and regulatory light chains (RLC).

A persistent aim in the motility field has been to isolate a minimal fragment that can function as a mechanochemical motor. In the case of kinesin, whose ATPase activity is stimulated by microtubules, as few as 340 amino acids from the N terminus are sufficient for enzymatic activity and for movement in a motility assay (Stewart et al., 1993). An N-terminal 75-kDa fragment from skeletal myosin was reported to have actin-activated ATPase activity (Okamoto and Sekine, 1987), but subsequent attempts to isolate such a proteolytic fragment proved unsuccessful. In light of the x-ray structure of S1, it is clear that any fragment devoid of the 20-kDa region will lack activity, since the latter polypeptide extends all the way from the C terminus of S1 to the nucleotide- and actin-binding sites (Rayment et al., 1993). With the advent of molecular genetics, it has been possible to produce a recombinant fragment in Dictyostelium that contains only the globular domain of S1 without the light chain binding region (Itakura et al., 1993). This truncated head can generate force and movement in a motility assay, although it displays very limited movement despite an unusually high ATPase activity (Itakura et al., 1993).

Here, a truncated S1 was prepared by proteolytic digestion of ELC-deficient myosin. We have shown recently that it is possible to strip myosin of its light chains by gel filtration in the presence of high concentrations of ammonium chloride (Lowey et al., 1993a). Unmasking the heavy chain sequence that interacts with ELC exposes sites susceptible to chymotrypsin, and thereby facilitates the isolation of a globular domain from S1. This fragment shares the same enzymatic activity as S1 prepared by papain or chymotrypsin, but its ability to generate movement is considerably reduced compared to S1. These studies support our earlier conclusion that the light chain-binding domain is essential for efficient coupling of ATP hydrolysis to movement.


MATERIALS AND METHODS

Protein Preparations

Chicken pectoralis myosin was prepared as described in Margossian and Lowey(1982) and stored at -20 °C in 0.6 M NaCl, 20 mM sodium phosphate, pH 7.0, 1 mM DTT, 3 mM NaN, 50% glycerol.

Actin was prepared from chicken pectoralis acetone powder (Pardee and Spudich, 1982) and stored at 4 °C as F-actin in 5 mM KCl, 5 mM imidazole, pH 7.5, 2 mM MgCl, 3 mM NaN.

Chicken RLC was prepared essentially as described in Katoh and Lowey (1989), but with further purification by ion-exchange chromatography. After precipitation of RLC with 26% ethanol, 120 mg of protein in 50 mM sodium phosphate, pH 6.2, 1 mM DTT, 3 mM NaN, was applied to a hydroxylapatite (Bio-Rad) column (1.5 20 cm) equilibrated with the same buffer, and eluted with a 50-500 mM sodium phosphate gradient (total volume = 350 ml). Pooled fractions containing pure RLC were dialyzed against 10 mM sodium phosphate, pH 7, 1 mM DTT and freeze-dried in the presence of an equal weight of sucrose.

Myosin without essential light chains (ELC-deficient myosin) was prepared as follows: myosin (5 mg/ml) was incubated in 4.7 M NHCl, 50 mM sodium phosphate, pH 7.5, 1 mM MgATP, 0.1 mM EGTA, 8 mM DTT, 3 mM NaN, at 4 °C for 10 min. Myosin heavy chain was separated from dissociated light chains by applying 1 ml to a Superose 6 column (1.6 30 cm; FLPC, Pharmacia) at 21-22 °C, equilibrated with 4.5 M NHCl, 50 mM sodium phosphate, 7.5, 2 mM EDTA, 0.5 mM ATP, 3 mM NaN, at a flow rate of 1 ml/min. Inclusion of ATP is essential to preserve the ATPase activity. Myosin heavy chain was eluted in the void volume 30 min after protein application and was collected on ice in the presence of 20 mM DTT, 2 mM MgATP. RLC (6 µM) was added immediately to myosin heavy chain (2 µM heads) to stabilize the protein. NHCl was removed by dialysis in the cold, first against 0.4 M NaCl, 5 mM MgCl, 20 mM sodium phosphate, pH 7.2, 1 mM DTT, 3 mM NaN, 0.5 mM ATP (4-5 h), and then overnight against low salt buffer (5 mM sodium phosphate, pH 6.2, 5 mM MgCl, 3 mM NaN, 1 mM DTT, 0.5 mM ATP). Excess light chain was removed from precipitated ELC-deficient myosin by centrifugation at 120,000 g for 30 min. The washed pellet was resuspended in a minimum volume of high salt buffer (0.6 M NaCl, 20 mM sodium phosphate, pH 7.5, 3 mM NaN, 2 mM DTT) and clarified at 150,000 g for 10 min (Beckman TL-100) to remove aggregated material.

Myosin Subfragments

Motor domain was prepared by proteolytic digestion of ELC-deficient myosin (4-5 mg/ml) in 0.12 M NaCl, 1 mM EDTA, 1 mM DTT, 20 mM imidazole, pH 7.0, 3 mM NaN with 30 µg/ml chymotrypsin (Worthington Biomedical) for 15 min at RT, and stopped with 2 mM phenylmethylsulfonyl fluoride (Sigma). Motor domain was separated from insoluble material by centrifugation at 320,000 g for 20 min (Beckman TL-100).

Chymotryptic S1 was prepared from myosin under the same conditions as described above for motor domain.

Papain S1 was prepared essentially as described in Margossian and Lowey (1982). Myosin (10 mg/ml) in 0.2 M ammonium acetate, pH 7.2, 2 mM MgCl, 1 mM DTT, was digested with 7 µg/ml papain (Cooper Biomedical) for 10 min at RT, and stopped with 2 mM 1-chloro-3-tosylamido-7-amino-L-2-heptanone (Sigma). Insoluble material was removed by centrifugation at 320,000 g for 20 min (Beckman TL-100).

Each of the subfragments was further purified by gel filtration at RT on a Superose 12 column (1 30 cm; FPLC, Pharmacia) equilibrated with 150 mM NaCl, 1 mM DTT, 3 mM NaN, 10 mM sodium phosphate, pH 7.2 (PBS), 0.1 mM ATP, at a flow rate of 0.5 ml/min. The order of elution on this column was papain S1, chymotrytic S1, and motor domain, consistent with their expected sizes.

Light chain isoforms of S1 (S1A1 and S1A2) were separated by ion-exchange chromatography on DEAE-Sephacel (Pharmacia). About 300 mg of chymotryptic S1 in 30 mM NaCl, 50 mM imidazole, pH 7.5, 3 mM Na, 0.5 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride was applied to a column (2.5 30 cm) equilibrated in the same buffer and eluted with a 30-150 mM NaCl gradient (total volume = 800 ml). Pools containing the fractionated isoforms were concentrated by dialysis against saturated ammonium sulfate, pH 7.3, 10 mM EDTA. The precipitated protein was collected by centrifugation, dialyzed againt PBS, and was stored at -20 °C in 50% glycerol.

Protein concentrations were determined by measuring absorbance at 280 nm using the following extinction coefficients (1 mg/ml): 0.55, 1.1, 0.75, 0.83, and 0.5 for myosin, actin, chymotryptic S1, papain S1, and RLC, respectively. The concentration of ELC-deficient myosin and motor domain was determined by the Bradford method(1976) using myosin or chymotryptic S1 as a standard.

ATPase Measurements

The actin-activated MgATPase activity was determined at 25 °C as described in Lowey et al. (1993b) except that the KCl and imidazole concentrations were lowered to 5 mM each. Phosphate release was measured by the colorimetric method of White(1982).

In Vitro Motility Assays

Either cleaned glass or nitrocellulose-coated coverslips were used for the flow-through microchamber. For motility on glass, coverslips were soaked overnight in a solution containing 50% ethanol, 1.5 N HCl, washed thoroughly with distilled water, and air-dried. The assay was performed essentially as described in Warshaw et al.(1990). To remove rigor heads, samples were spun 320,000 g for 20 min (Beckman TL-100) with equimolar concentrations of actin in the presence of MgATP just prior to the assay. Subfragments in PBS were perfused through flow cells at 0.2-0.4 mg/ml. More concentrated samples were necessary compared to myosin (50 µg/ml), in order to have smooth movement. A higher percentage (60%) of filaments moved on glass than nitrocellulose when subfragments were used.

In some motility experiments, papain S1 was bound to the coverslip via attachment to a anti-RLC monoclonal antibody (7C10; Winkelman and Lowey, 1986) which was adsorbed to the nitrocellulose-coated coverslips at 100 µg/ml. Alternatively, equimolar concentrations of papain S1 and antibody (15 µM of each) were incubated at 0 °C for about 2 h, and applied at RT to a Superose 6 column (1 30 cm; FPLC) equilibrated with PBS, 0.1 mM ATP. The papain S1antibody complex was eluted in the void volume and applied to cleaned glass for the assay. All motilities were performed at 30 °C.

Electron Microscopy and Image Analysis

Low-dose (<1000 e/nm) images of filaments suspended in an unbroken sheet of negative stain (1% uranyl acetate) over holes in a carbon support film where recorded at nominal 60,000 magnification on a Philips CM12 electron microscope. The micrographs were digitized on a raster corresponding to about 0.75 nm in the filaments using an Eikonix model 1412 scanner interfaced to a Digital Equipment Corp. VAXstation 3100. Filament images used for further processing were selected on the basis of uniform S1 or MD binding, even staining, and the presence of straight or only gently curved filaments. Images of curved filaments were straightened by reinterpolation using a cubic spline function fitted to the filament axis (Egelman, 1986). Fourier transform data extending to about 2.7 nm axial and 2.0 nm radial resolution from about 20 images of each type were averaged semiautomatically (Morgan and DeRosier, 1992), aligned to a common origin, and used for three-dimensional helical reconstruction by standard methods (cf. DeRosier and Moore, 1970). ``Difference maps'' were calculated in Fourier space between each pair of reconstructed structures, and difference peaks that were statistically significant at greater than the 99.5% confidence level (dashed lines in Fig. 4, D-F) were identified by applying a Student's t test to the densities and their variances (Milligan and Flicker, 1987).


Figure 4: Density maps in projection of reconstructed filaments. Acto-MD (A), acto-S1A1 (B), and acto-S1A2 (C) in projection down the long strand n = 2 helices (``helical projections''). Acto-S1A1 and the statistically significant density difference (dashed lines) between it and acto-MD (D); acto-S1A2 and the difference between it and acto-MD (E); acto-S1A1 and the difference between it and acto-S1A2 (F). The greater length of the S1s is reflected in difference peaks 1 in D and E; the extra density close to actin (difference peak 2) is present in D and F but not in E. Closer to the helix axis, where reconstructions of helical structures are less reliable due to their limited radial resolution, other difference peaks occur at the positions of the outer actin domains in D and F, and probably represent residual scaling errors between the maps.




RESULTS

Preparation of MD

Proteolytic digestion of filamentous myosin has been routinely used to isolate S1 and the myosin rod. Papain cleaves at Leu-842 resulting in an S1 that contains both RLC and ELC, whereas chymotrypsin cleaves the heavy chain at Phe-814, leading to a somewhat smaller S1 with just the ELC (reviewed in Lowey, 1994). A slight difference in mobility of the heavy chain for the two S1 s is evident in SDS-polyacrylamide gel electrophoresis (Fig. 1, lanes 4 and 5). Until recently, it was not possible to prepare a more truncated form of S1, but the ability to strip the LCs from the heavy chain (lane 2) and reconstitute the myosin with just RLC (LC2, lane 3) provided a method for isolating the globular or motor domain (MD) of myosin (Lowey et al., 1993a). The heavy chain has a potential cleavage site for chymotrypsin at Phe-797 in the region where ELC normally binds. We assume this residue is the C terminus of MD, but end group analysis is needed to confirm the assignment. A small difference in mobility between MD (lane 6) and chymotryptic S1 (lane 5) is suggested by the gel pattern, but a difference in size of only 17 residues is difficult to detect. A slight displacement in elution volume was also seen between MD and chymotryptic S1 by gel permeation chromatography on Superose 12 (FPLC, data not shown). The preparation of MD is very homogeneous as judged by the single electrophoretic band on SDS gels, and a single symmetrical peak by gel filtration. The yield from 10 mg of myosin was 1 mg.


Figure 1: SDS-polyacrylamide gel electrophoresis (12.5%) of myosin and its subfragments. Myosin (lane 1), myosin depleted of light chains (lane 2), heavy chain reconstituted with RLC (LC2, lane 3), papain S1 (lane 4), chymotryptic S1 (lane 5), and motor domain (lane 6). Note the slight increase in mobility of the subfragment heavy chain as it is truncated and the absence of light chains in the motor domain.



Characterization of MD by Electron Microscopy

Actin filaments were decorated with MD in order to obtain a low resolution picture of the MD structure. For comparison, actin filaments were also decorated with the two light chain isoforms, S1A1 and S1A2, the difference between them residing in the N-terminal extension of the A1 light chain (Lowey, 1994). Visual inspection of the negatively stained images in Fig. 2reveals the characteristic arrowhead appearance of the complexes. The MD-decorated actin filament looks significantly narrower, however, and the individual truncated heads are more distinct than for S1-decorated filaments. Surface views of reconstructed densities of these filaments show that all the head fragments bind similarly to actin, but the S1 s are longer by virtue of the ELC- (A1 or A2) binding domain (Fig. 3). There is also extra density near the actin interface in the case of S1A1-decorated actin (panel B), which is not present in the surface view of MD-decorated actin (panel A). These differences are more clearly seen in density maps formed by projecting the density of the reconstructed filaments onto a plane perpendicular to the helix axis (Fig. 4). The maps show that S1A1 and S1A2 have additional mass at their C termini relative to MD (arrows marked 1 in panels D and E), which is consistent with the presence of the ELC-binding site. Furthermore, the extra density near the actin surface seen in S1A1 (arrow marked 2 in panel D), is also seen, but to a lesser extent, when comparing S1A1 and S1A2 (panel F). This extra density has been seen previously in three-dimensional maps of decorated filaments obtained by cryo-electron microscopy and was ascribed to the N-terminal domain of the A1 light chain (Milligan et al., 1990).


Figure 2: Electron microscopy of F-actin decorated with myosin head fragments. Electron micrographs of negatively stained acto-MD (A and B), acto-S1A1 (C), and acto-S1A2 (D), recorded under high dose (A) or low dose conditions (B-D). The different width of the arrowhead complexes in B compared to C and D is best seen by viewing the filaments along their lengths at a glancing angle. The high dose image (A) emphasizes the clear separation of individual MD units bound to adjacent actin monomers. Bar, 50 nm.




Figure 3: Surface views of reconstructed density maps. Acto-MD (A), acto-S1A1 (B), and acto-S1A2 (C). The three myosin head fragments bind in a similar way to actin, but the S1s are longer (region 1) and acto-S1A1 also has extra density (region 2) close to the actin interface which is absent from acto-MD. Region 2 is not as dense in acto-S1A2 (see Fig. 4C) and does not appear as a statistically significant difference peak when acto-S1A2 and acto-MD are compared (see Fig. 4E).



Functional Properties of MD

The actin-activated MgATPase rate for MD was the same as for S1 prepared by papain or chymotrypsin, within the experimental error (Fig. 5). The average V and K for the subfragments was about 20 s and 60 µM, respectively. These kinetic constants are well within the range of values usually found for S1 (Margossian and Lowey, 1973; Wagner et al., 1979). Removal of the light chain binding region does not appear to affect the steady state ATPase activity of MD; whether transient rates are affected remains to be determined.


Figure 5: Actin-activated ATPase activity of myosin subfragments. The activity was normalized to the value, 14.5 s obtained for chymotryptic S1 at 100 µM actin. Each rate is the average of three time points at any given actin concentration, and rates were determined for several independent preparations. V extrapolated from double-reciprocal plots was 30 s for chymotryptic S1 (triangles), 22 s for papain S1 (squares), and 18 s for motor domain (circles). Note that the myosin from which motor domain was prepared had been exposed to high concentrations of NHCl, conditions which lead to a loss of about 20% activity (Lowey et al., 1993a).



Despite a constant ATPase activity, the subfragments showed marked differences in their ability to move actin filaments in an in vitro motility assay (Fig. 6). After purification by gel filtration, papain S1 showed the fastest movement, 2.6 µm/s; chymotryptic S1 was 1.5 µm/s; and MD was 0.5 µm/s. The motility depended to some extent on the surface of the coverslip: the best movement was observed on cleaned glass coverslips; fewer actin filaments moved on nitrocellulose treated surfaces, although the rates of the filaments that did move were not substantially different. In order to avoid unfavorable interactions between S1 and the substratum, we first adsorbed the nitrocellulose-coated coverslip with a monoclonal antibody (7C10) specific for the N-terminal region of the RLC (Winkelmann and Lowey, 1986). The advantages of using monoclonal antibodies in the motility assay for skeletal (Lowey et al., 1993a) and smooth muscle myosin (Trybus et al., 1994; Trybus, 1994) have been described previously. Attaching papain S1 by the antibody (7C10) interaction increased the velocity to 4 µm/s; the same rate was obtained when S1 and antibody were mixed in solution prior to adhesion to the coverslip (Fig. 6).


Figure 6: In vitro motility assay of myosin subfragments. The subfragments: papain S1, chymotryptic S1, and motor domain were applied directly to cleaned glass coverslips. Each velocity value represents the average of a minimum of 40 filaments, and two independent preparations (indicated by bars of different shadings). In the case of (S1+Ab), papain S1 was either attached to the coverslip via an antibody specific for the N terminus of RLC (7C10), which was first adsorbed to a nitrocellulose-coated coverslip (filled bar), or papain S1 was mixed with antibody in solution and the complex after gel filtration was applied to the coverslip (striped bar). All velocities are expressed as the mean ± standard deviation.



Unfortunately, this type of attachment cannot be used for chymotryptic S1 or MD, since antibodies binding closer to the active sites will have an inhibitory effect on S1 movement (data not shown). A certain degree of uncertainty in motility values is unavoidable when attaching head fragments to a surface, be it by an antibody or by an engineered tag. However, it has been our experience, and that of others (Stewart et al. 1993)), that if a protein supports movement, the rate of movement will not change by more than 2-fold independent of the mode of attachment to the substratum.


DISCUSSION

A proteolytic fragment (MD) encompassing the catalytic and actin binding sites of S1, but devoid of the light chain binding region, was prepared from ELC-deficient myosin. In principle, MD could be prepared by simply digesting isolated myosin heavy chain free of all LCs, but in practice the yield of MD is poor, probably due to the tendency of free heavy chains to aggregate (Lowey et al., 1993a). Alternatively, it has been shown that ELC can be removed from chymotryptic S1 by ion-exchange chromatography at 37 °C (Sivaramakrishnan and Burke, 1982), or by immunoadsorption in the presence of 4.6 M NHCl (Wagner and Giniger, 1981). However, it is difficult to eliminate all traces of intact S1 by these procedures, and, moreover, the isolated S1 heavy chain is relatively unstable (Wagner and Stone, 1983). By using ELC-deficient myosin as the starting material, it has been possible to overcome these difficulties and prepare a homogeneous MD with good enzymatic activity and stability over time.

A recombinant fragment consisting of the motor domain has been expressed in the cellular slime mold Dictyostelium discoideum (Itakura et al., 1993). The C terminus of this truncated head, Asp-781 (numbered according to the adult chicken myosin sequence, Maita et al., 1991), is only 16 residues shorter than chicken motor domain. But the difference in enzymatic properties between the two motor domains is striking: whereas the activity of chicken MD is similar to that of S1 prepared by papain or chymotrypsin, Dictyostelium MD showed a >10-fold higher actin-activated MgATPase than Dictyostelium S1. The latter was expressed with both LCs and the heavy chain terminated at Pro-840 (Itakura et al., 1993).

An increase in enzymatic activity with increasing truncation is reminiscent of the mechanochemical protein, kinesin, and related members of this superfamily of microtubule-dependent motor proteins (Stewart et al., 1993; Chandra et al., 1993). Expressed Drosophila kinesin with as few as 340 N-terminal residues had a microtubule-stimulated ATPase rate that was 60-fold higher than the rate for bovine kinesin (Huang and Hackney, 1994). If the high ATPase rate for Dictyostelium MD is due to truncation, it is not clear why chicken MD of similar length does not have an elevated ATPase activity. Perhaps the region of the heavy chain linking the MD to the light chain-binding domain is folded differently in these two widely divergent species, and cleavage in this critical linker region affects the active site of one myosin more than the other. A comparison of the x-ray structure of the truncated Dictyostelium heavy chain (MD) with the three-dimensional structure of chicken S1 has revealed remarkable conservation of structure, except for differences in the C terminus of the heavy chain following the reactive thiols (Rayment et al., 1995). No firm conclusions can be drawn at the present time, however, since the Dictyostelium structure with its bound nucleotide and truncated heavy chain is too different from chicken S1 for a direct comparison.

The motor domains from chicken myosin and Dictyostelium myosin both support gliding of actin filaments by an in vitro motility assay, but the velocity is much slower than observed for myosin. Dictyostelium MD moved actin at 0.12 µm/s compared to 1-2 µm/s for Dictyostelium myosin (25 °C, Itakura et al., 1993). Chicken MD moved actin filaments at a rate of 0.55 µm/s compared to 7-8 µm/s for chicken myosin (30 °C). Therefore, there is good agreement between the two species if one only considers the relative rates of MD:myosin. There are difficulties, however, when comparing the velocities of MD:S1. Dictyostelium S1 was able to support actin gliding when bound directly to nitrocellulose (0.13 µm/s, Manstein et al., 1989), but Dictyostelium MD could not support movement until it was attached to the glass surface via a biotin-avidin system (Itakura et al., 1993). This fusion MD was now able to promote sliding motion, but the velocity was the same as found earlier for Dictyostelium S1. Addition of a biotin-avidin attachment site to the S1 did not increase its velocity which remained at 0.13 µm/s.

In contrast to the Dictyostelium system, chicken S1 prepared by papain, which contains both the RLC and the ELC, moved actin at an appreciably faster rate (2.6 µm/s) than chicken MD (0.5 µm/s). Chymotryptic S1, which lacks the RLC, moved actin at an intermediate velocity. The rate for papain S1 could be increased to 4 µm/s when the S1 was attached to the surface by means of an antibody directed against the N-terminal region of the RLC (Winkelman and Lowey, 1986). It seems reasonable to assume, therefore, that the movement of chicken MD would not increase beyond 1 µm/s even if an attachment site were to be designed. This rate for MD is remarkably similar to the rate of actin movement found for myosin heavy chain free of all light chains (Lowey et al., 1993a).

We conclude that the velocities for the chicken subfragments are in qualitative agreement with the earlier results for chicken myosins deficient in one or both light chains. It would seem that independent of whether one removes light chain, and thereby exposes the heavy chain to solvent, or whether one removes the light chain-binding domain by proteolysis, the velocity of actin movement is roughly proportional to the length of the neck region. A third approach in the future will be to delete regions of the myosin light chain-binding domain by molecular biology (Trybus, 1994). For the present, the results are at least consistent with the hypothesis that the size of the working stroke is closely coupled to the length of the neck domain of the myosin molecule (Vibert and Cohen, 1988; Rayment et al., 1993).


FOOTNOTES

*
This work was supported by Grant AR17350 from the National Institutes of Health (to S. L.), National Science Foundation Grants IBN9119570 (to S. L.) and MCB9004746 (to P. V.), and the Muscular Dystrophy Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110.

To whom correspondence should be addressed. Tel.: 1-617-736-2464; Fax: 1-617-736-2405.

The abbreviations used are: S1, subfragment-1; RLC (or LC2), regulatory light chain; ELC (or LC1 and LC3 isoforms), essential light chain; MD, motor domain; S1A1 and S1A2, subfragment-1 containing the A1 (LC1) or A2 (LC3) light chain isoforms; DTT, dithiothreitol; FPLC, fast performance liquid chromatography; RT, room temperature; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank David Morgan for assistance with computer programs.


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