Segments from myosin rods

J. iklol. Biol. (1970) 47, 605-609

Segments from Myosin Rods The rod portion

of myosin

forms bipolar

“segment”

aggregates

when precipitated

with divalent cations, This in vitro structure establishes a minimum length for the rod, and indicates one mode of molecular packing which may be related to the bare zone of the myosin filament. The distinctive appearance of the myosin filament reflects the unusual form of the molecule. Myosin consists of an z-helical, coiled-coil rod region about 1400 A long, terminating in two enzymic globular portions (Slayter & Lowey, 1967). The core of the filament is built by the rod regions, and the globular units (contributing to the “cross-bridges”) are regularly arrayed at the surface where they interact with actin (Huxley 8z Brown, 1967). Although the symmetry of the cross-bridge arrangement in the polar portion of the filament is known, the exact packing relations of the molecules in the filament are not yet established. Myosin filaments produced in vitro resemble the native filaments. The assembly is initiated by the aggregation of molecules which point in opposite directions, forming a “bare-zone” at the center of the filament. Growth proceeds at each end by the addition of molecules pointing in the same direction. The bipolarity of the filament provides the directional specificity recluired by the sliding filament mechanism of contraction (Huxley, 1963). Analysis of filament organization is aided by the availability of well-defined subfragments of myosin which may be prepared by proteolytic enzymes. Trypsin produces LMlVlt, the fully helical tail portion of myosin, and HMM the remainder of the molecule (Gergely, 1953; Szent-GyGrgyi, 1953). The HMiV S-2 may be obtained by an insoluble derivative of trypsin (Lowey, Goldstein, Cohen & Luck, 1967) or by papain digestion (Lowey, Slayter, Weeds & Baker, 1969)$ Aggregates of LMM and HMM S-2 display periodicities of 145 A or 430 A which are characteristic of the cross-bridge arrangement in the polar portion of the thick filament (Lowey et al., 1967; Szent-Gyorgyi, Cohen & Philpott, 1960). It has recently been shown that papain digestion of precipitated myosin cleaves the entire rod portion of myosin from the globular subunits (Lowey et al., 1969). The assembly of this new rod subfragment, which contains the interaction properties of intact myosin, is a key to understanding the organization of the filament. The simple, rod-shaped a-helical muscle proteins, tropomyosin (Caspar, Cohen & Longley, 1969) and paramyosin (Kendrick-Jones, Cohen, Szent-Gyorgyi & Longley, 1969), form a number of ordered states when precipitated with divalent cations. Here we report the formation of “segment” aggregates of the rod and LMM by precipitation with oalcium and magnesium. Our results establish a minimum length for the rod portion of myosin and indicate one mode of packing of the rod which may be related to the bare zone of the myosin filament. Myosin from rabbit and chicken breast muscle was prepared as previously described (Holtzer & Lowey, 1959). Pecten myosin was purified according to Barany $ Abbreviations used: LMM, light meromyosin; HMM rod portion of HMM. $ See Fig. 1 for diagram illustrating nomenclature. 005

heavy

meromyosin;

HMM

S-2, helical

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C. COHEN

ET

AL.

Q Barany (1966) with extensive washing to remove paramyosin. Rods and HMM S-2 from myosin were obtained by papain digestion (Lowey et al., 1969) ; LMYM by tryptic digestion (Lowey & Cohen, 1962). The proteins at concentrations of about 2 mg/ml. were dialyzed against O-05 MTris-HCl, pH 8.2 ; KSCN at 0.05 M was added to solubilize the rod and certain LMM preparations. Previous studies with paramyosin have indicated that this reagent prevents aggregation, and often monomeric preparations may be obtained. Precipitation was effected by addition of 0.05 M-CaCl, or MgCl,. The material was negatively stained with 1% uranyl acetate and examined in the Philips 200 microsoope. The rods from chicken myosin precipitate with CaCl, in the presence of KSCN in the form of remarkably ordered segments (Plate I (a) and (b) ). These often appear as flat ribbons up to two microns long and about 1800 A in width. Structures of two general types may be seen: one type of aggregate is clearly bipolar (has dihedral symmetry). This kind of segment shows a tightly packed fringe about 145 A in width at either end, and extending from this a second far less densely packed fringe about 100 A in width. Another type of segment, which is less frequently observed, appears polar (Plate II (a)). This form is more variable in appearance than the bipolar aggregates, and widths of 1600 to 1800 A have been measured. One end shows a relatively narrow, tightly-packed fringe about 130 A in width, enhanced by a dense line of stain. The other end often shows a relatively large, loosely packed region about 300 to 500 A in width. Chicken myosin alone forms filaments but not segments with divalent cations and KSCN. Incorporation of the myosin into segments may be achieved by mixing rods and myosin at a ratio of about 5/l by weight. The segments formed with calcium and KSCN show an unusual fringe appearance : at the end of the outer fringe, negativelystained material may be observed, which we interpret as the globular regions of myosin (Plate I(c)). Segment formation depends both on the nature of the subfragment, and on the animal species and organ from which the myosin was obtained. Unlike rods from chicken myosin, rabbit rods do not form well-ordered segments. Varying the solvent conditions by use of other divalent cations and dispersing reagents did not affect this result. Chicken LMM usually precipitated as fibrous sheets with a 430-A period (Plate II(b)). However, when chicken LMM was prepared by very limited tryptic digestion, a small proportion of the material formed a fibrous long-spacing aggregate with a 760 A repeat (Plate III(a)). Although rabbit LMM generally formed poorly ordered segments, on occasion, well-ordered segments about 900 A long have been observed (Plate III(b)). The only preparation of LMM we have yet found, which consistently formed a large proportion of well-organized segments, comes from the molluscan myosin of smooth or striated adductor muscle of Pecten irradians. These aggregates are about 700 A in length and are often attached to fibers showing a 145 A axial repeat (Plate II(c)). While the LMM subfragment of the rod can in oertain cases form segments, this property does not seem to be shared by the more soluble HMM S-2 subfragment: HMM S-2 from both chicken and rabbit myosin precipitated as amorphous aggregates in the presence of divalent cations. These results provide information on the molecular structure and interactions of myosin and its subfragments. Myosin is a polar molecule with the two-chain coiledcoil rod connected by a pair of single chain links to the globular head regions (Fig. l(a)). It is evident that the bipolar segment is composed of two oppositely oriented arrays

PLATE I. Segments HCl, (a) (b) center (c) All

from myosin rods. The protein is dispersed in 0.05 WI-KSCN-0.05 pH 8.2, and precipitated with 0.05 M-C&I,. Ribbon-like segment (outer fringe obscured by stain). “double” fringe. The staining pattern Representative shorter segments showing of the segments consists of three light bands about 100 A apart. Segments formed from mixing rods and intact myosin. preparations negatively stained with 1% many1 acetate. (a) x 108,000; (b)

x-Tris-

at

the

and

(c)

x105,000. [facing p. 606

64

(b)

(4

PLATE II. (a) “Polar” segments from rods. These forms are found in preparations containing the bipolar segments shown in Plate I. Segments showing an unusual stain incorporation are also reproduced (see text). (b) Chicken light meromyosin dispersed in 0.06 M-KSCN-0.05 m-Tris-HCI, pH 8.2, and precipitated with 0.05 M-CaCl,. The axial period of the aggregates is 430 d. (c) Segments from light meromyosin prepared from a tryptic digest of P&en smooth muscle myosin. Formed by precipitation with 0.05 M-MgCl,. All preparations negatively stained with 1% uranyl acetate. x 105,000.

(a)

(b) PLATE III. Tactoids and segments from chicken and rabbit LMM prepared by limited proteolytic digestion. The chicken and rabbit myosins were digested for 2 min and 5 min, respectively, according to the procedure described in Lowey & Cohen (1962). The proteins were precipitated under the conditions described in Plate I. (a) The preparations of chicken LMM showed primarily tactoids with a 430 .& period (upper); occasionally a fibrous long-spacing aggregate wais observed with a repeat of about 760 A and a molecular overlap of 100 A (lower). (b) Preparations of rabbit LMM showed tactoids with a 145 A period and occasional segments about 900 A long. All preparations negatively stained with 1% uranyl acetate. x 105,000.

LETTERS

+i 145+-e I< >

TO

THE

607

EDITOR

1450R-.----4 <

(b)

>Fm 1. (a) Schematic diagram of the myosin molecule. (b) Two-dimensional representation of possible packing of myosin rods in the bipolar segment. The average length of the segment (excluding the outer, less dense fringe) w&s 1589 f 44 A from ~1total of 61 mectsurements. The length of the inner fringe was 143 & 5 A from 117 measurements. All readings were made directly from the electron microscope plates using a Nikon optical comparator. The magnification of each series of plates was determined using tropomyosin Mg-tactoids with e 395A axial repeat (&spar et al., 1969) es a calibration standard.

of polar rods related by perpendicular dyads (Fig. l(b)). The determination of the molecular length of the rod depends on the interpretation of both fringes seen in these segments. The inner dense fringe may be accounted for by assuming that the head end of the rod is displaced from the tail end in the bipolar arrays (Pig. l(b)). The minimum length of the rod would then be 1450 A. Alternatively, there may be some differentiation along either end of the rod, so that a region of about 145 A incorporates stain. In this case,with head-to-tail register between oppositely directed rods, the rod would be about 1600 d iu length. It is possible that the outer, less dense fringe consists of the single links which connect the head region to the rod (Slayter & Lowey, 1967). The very low density of this region may indicate that papain cleavage in myosin yields a small proportion of rods which contains these connecting links. The experiments on the incorporation of myosin into the rod segments seems to support this interpretation: the globular head units of myosin appear to be located at the end of the outer fringe (Plate I(c)). Various other lines of evidence tend to support the shorter value for the rod length. Measurements of the contour lengths of isolated rods from electron micrographs of shadow-cast molecules give an average value of 1360 A (Lowey et al., 1969). Moreover, the molecular weight of a two chain a-helical coiled-coil 1450 A long would be 220,000, which corresponds well with molecular weights determined by sedimentation equilibrium ultraoentrifugation (Lowey et al., 1969). We should stress, however, that the absolute determination of the length of the rod will require the defmitive identification of both ends of the molecule. The length of the polar segments we have observed would suggest a rod 1600 to 1700 A long, if these segments are, in fact, built of molecules all pointing in the same direction and in register. Inspection of a number of these apparently polar segments has indicated, however, that they may be artifacts of staining (Plate II(a)). Their general lack of fine structure, the piling up of stain at one edge of the segment only, and their occasional resemblance to the bipolar form prevent us from attaching too much significance to their structure at this stage of the study. It is noteworthy that the parts of the rod from chicken myosin do not aggregate as segments: the LMM forms periodic fibers with divalent cations, and the HMN S-2 does not form ordered states. These results are in marked contrast to observations

608

C. COHEN

ET

AL.

on collagen, which forms highly ordered polar segments (Schmitt, Gross & Highberger, 1953). When the collagen molecule is split by proteolytic enzymes, the separated fragments can be re-associated to form partial polar segments which show the same fine structure as those from native collagen (Kuehn, Rauterberg, Zimmerman & Tkocz, 1968). Our results show that the myosin rods preferentially form bipolar segments, where the HMM S-2 portion of the rods of one array packs well with the LMM regions of the oppositely oriented array. The packing in the bipolar rod segment (Pig. l(b)) suggests that there is sufficient overlap so that LMM alone could form segments about 1300 d in length. However, neither the LMM (nor the HMM S-2) from chicken myosin associate in this way. Moreover, segments formed by LMIM molecules from other species do not show this bonding arrangement. It appears, therefore, that stable association in bipolar rod segments may require head-to-tail interactions between both parts of the rod. The bipolar rod segments have dihedral symmetry which characterizes the myosin filaments in muscle, and the in vitro myosin aggregates. It is possible that the molecular packing in the segments corresponds to certain dimer relations occurring in the bare zone of the native myosin filament. The distance between the globular regions marking the boundaries of the bare zone, i.e. the so-called pseudo-H-zone, has been reported to be from 1500 to 2000 A (Huxley, 1963). The length of the bipolar segment is about 1800 d. This distance would correspond to the maximum length possible for the bare zone if the initiating dimer were that of the bipolar segment. It is important to note that the in vitro bipolar segments grow by addition of molecules in a lateral direction only. To build the polar portion of the myosin filament, additional interactions must occur to generate the helical packing of molecules at either end of the bare zone. The results presented here lead also to some inferences about the packing of the myosin molecules in the polar portion of the native filaments. Since the “bridges” at the surface of the myosin filament maintain an exact 430 A repeat in resting muscle (I-Iuxley & Brown, 1967), the whole rod region must be regularly packed. We have established a minimum length for the rod, which is not an integral multiple of 430 d. In this fibrous protein system, as in collagen (Hodge & Petruska, 1962) and paramyosin (Kendrick-Jones et al., 1969), there is no end-to-end interaction between molecules, and thus no integral relation between the molecular length and the axial periodicity. Although the exact orientation of the molecular axes relative to the fiber axis has not been established in these systems, the myosin rods must be tilted relative to the axis in order to position the globular enzymic units on the surface of the thick filament. The information provided here on the molecular length and association properties of myosin, together with knowledge of the symmetry of the cross-bridge arrangement (Huxley & Brown, 1967) and the structure of the M-line region (Pepe, 1967; Knappeis & Carlsen, 1968) furnish considerable constraints for building a realistic model of the myosin filament. This work was supported by Public Health Service grant AM-02633 to one of us (C. C.) by Public Health Service grant AM-04762 and Public Health Service Research Career Program award K3-AM-10630 to another (S. L.), and by Public Health Service grant GM-14675 and National Science Foundation grant GB5368 to a further author (A. G. S-G.). This study was aided by a grant from the Muscular Dystrophy Association of America, Inc.

LETTERS

TO THE

EDITOR

609

We thank Miss Marjorie Kasac and Mr Paul Norton for electron microscopy and Dr B. Uzman, director of the laboratory of tissue ultrastructure, for use of electron microscope facilities. We are grateful to Dr D. L. D. Caspar for discussion. The Children’s Cancer Research Foundation and Harvard Medical School Boston, Massachusetts, U.S.A.

CAROLYN COHEN SUSAN LOWEY RICHAXD G. HARRISON

Department of Biology Brandeis University Waltham, Massachusetts,

JOIXN ?&NDRICK-JONES ANDREW G. SZENT-GYORGYI

Received

16 September

U.S.A. 1969

REFERENCES Barany, M. & Barany, K. (1966). Biochem. 2. 345, 37. Caspar, D. L. D., Cohen, C. & Longley, W. (1969). J. Mol. BioZ. 41, 87. Gergely, 5. (1953). J. Biol. Chem. 200, 543. Hodge, A. J. & Petruska, J. A. (1962). In Electron Micro.sc~y, ed. by S. S. Breese, Jr., vol. 1, paper Q&-l. New York: Academic Press. Holtzer, A. & Lowey, S. (1959). J. Amer. Chem. Sot. 81, 1370. Huxley, H. E. (1963). J. Mol. BioZ. 7, 281. Huxley, H. E. & Brown, W. (1967). J. Mol. BioZ. 30, 383. Kendriok-Jones, J., Cohen, C., Szent-Gyijrgyi, A. G. & Longley, W. (1969). Science, 163, 1196. Knappeis, G. 8. & Carlsen, F. (1968). J. Cell BioZ. 38, 202. Kuehn, K., Rauterberg, J., Zimmerman, B. & Tkocz, C. (1968). In Syrqnoaium on Fibrous Proteins 1967, ed. by W. G. Crewther, p. 181. Australia: Butterworth. Lowey, S. & Cohen, C. (1962). J. Mol. Biol. 4, 293. Lowey, S., Goldstein, L., Cohen, C. & Luck, S. M. (1967). J. Mol. Biol. 23, 287. Lowey, S., Slayter, H. S., Weeds, A. G. & Baker, H. (1969). J. Mol. BioZ. 42, 1. Pepe, F. A. (1967). J. Mol. Biol. 27, 203. Schmitt, F. O., Gross, J. & Highberger, J. H. (1953). Proc. Nat. Acad. Sci., Wash. 39, 459. Slayter, H. S. & Lowey, S. (1967). Proc. Nat. Acud. Sk., Wash. 58, 1611. Szent-Gyiirgyi, A. G. (1953). Arch. Biochem. Biophys. 42, 305. Szent-Gyorgyi, A. G., Cohen, C. & Philpott, D. E. (1960). J. Mol. Biol. 2, 133.