Optical Diffraction Studies on the Structure of

Transcription

1 J. Biochem., 72, (1972) Optical Diffraction Studies on the Structure of Troponin-Tropomyosin-Actin Paracrystals* Iwao OHTSUKI and Takeyuki WAKABAYASHI Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo Received for publication, December 28, In order to clarify the structure of paracrystals of actin (Hanson, 1967) and two types of paracrystals made of troponin-tropomyosinactin complex, electron micrographs of negatively stained specimens of these paracrystals were studied by optical diffraction and optical filtering methods. 2. The actin paracrystal consists of parallel arrays of actin filaments. The filaments align in register along the filament axis, being in contact with each other at the portions between the crossover points of double strands. 3. The first type of paracrystal made of troponin-tropomyosinactin complex shows essentially the same arrangement of actin filaments as that in the actin paracrystal. 4. The second type of troponin-tropomyosinactin paracrystal shows clear regular transverse striations of about 380 A period, which seem to be made of troponin mole cules. The period corresponds to the length of seven actin molecules. The actin subunits of neighboring filaments are not in register. 5. The structure of tropomn-tropomyosinactin filament was discussed. It has been shown that the action of Ca ion in regulating the contractile process is exerted only in the presence of a protein component, named native tropomyosin (1 ). This compo nent, a complex of tropomyosin and troponin (2, 3), was demonstrated to be localized in the thin filament (4). Immunoelectron micro scopy has further revealed that troponin dis tributes itself along the thin filament at regular * This work was supported in part by the research grants of the U.S. Public Health Service, AM-04810, Muscular Dystrophy Association of America, Inc., Ministry of Health and Welfare, Japan, No. 216, the Iatrochemical Foundation and Toray Science Foundation. intervals of 380 A (5 ). This periodic localiza tion of troponin has been shown to be based on the end-to-end distribution of tropomyosin with the same periodicity along the thin fila ment (6). These findings suggest that the thin filament is an ordered aggregate of tropo nin-tropomyosinactin (7 ). The present investigation was undertaken to study the structural relation of the troponin tropomyosin system to the actin filament, the double-stranded structure of which has already been visualized under the electron microscope (8). Paracrystals of troponin-tropomyosin actin complex were made in the presence of Mg ion at neutral ph (9) and the electron micrographs of negatively stained specimens Vol. 72, No. 2,

2 OHTSUKI and T. WAKABAYASHI of the resulting paracrystals were examined by the optical diffraction and optical filtering methods (10, 11). EXPERIMENTAL PROCEDURE Preparation of Muscle Proteins-Skeletal muscle of rabbit was usually used for prepar. ing proteins. Actin was usually extracted from acetone powder prepared by the method of Ebashi and Maruyama (12 ), and then purified by Mommaerts' procedure (13). When acetone powder prepared according to the method of Straub (14) was used, the crude water ex tract was treated with trypsin [EC ] (1/200, by weight) for 10 min at 20 Ž and then purified by Mommaerts' procedure( 13 ). Actin prepared from chicken skeletal muscle was also used in some experiments. Native tropo myosin was prepared by the method of Ebashi and Ebashi (I ), except that the protein was precipitated by 32.5 g of ammonium sulfate per 100 ml instead of 37 g. Native tropomyosin thus prepared showed a higher troponin-tropo myosin ratio than the usual preparation. Troponin and tropomyosin were prepared by the method described in the previous papers (3, 15), Preparation of Paracrystals-Paracrystals were formed by dialyzing the protein solution against mm MgC12 buffered with Tris maleate (ph 6.8) at 2 Ž. The protein solution contained 1.0 mg/ml of G-actin, or 1.0 mg/ml of G-actin and O mg/ml of troponin tropomyosin complex. Electron Microscopy - Paracrystals were negatively stained with uranyl acetate solution on carbon-coated collodion film and observed in an electron microscope (JEOL, JEM-7) with an accelerating voltage of 80 kv, a beam current of 15 ƒêa and an objective aperture of 30 p diameter. Optical Diffraction and Optical Filtering- The optical diffraction pattern of the paracrystals on electron micrographs was observed using an optical diffractometer (10 ). Magni fications of electron micrographs were from x 30,000 to x 50,000. The region of paracrystal structure on the film was selected and the rest of the film was masked. A parallel beam from a He-Ne-gas laser (NEC, GLG 2009) was projected on the film and the diffraction pattern was observed at the focal plane of the lens (focal length 120 cm) behind the film. The spacings of the diffraction patterns were meas ured on a profile projector (Nikon. 6 C). Optical filtering (11) was carried out as follows. The mask made of an opaque plate with holes permitting the selected beams to pass through, was put at the diffraction plane. The lens (focal length 50 cm) behind the dif fraction plane produced the filtered image, which was reduced to about half of the origi nal scale of the electron micrograph. RESULTS Aggregates of actin formed in the presence of Mg ion are electron microscopically shown to consist of bundles of actin filaments of vari ous thickness. Paracrystal structure is found in the portion of the bundle where actin fila ments align side by side on the flat supporting film. Figure 1A shows an electron micrograph of such an actin paracrystal. Actin filaments align parallel with each other and a beaded structure is observed in each filament. Gross transverse periodicity of 355 A is found along the parallel array of filaments and the distance between neighboring filaments is 60 A. In Fig. 1C the optical diffraction pattern of Fig. 1A is shown. The longitudinal spac ings of these layer lines given in Table I cor- TABLE I. Spacings of the diffraction pattern of actin paracrystal in Fig. 1C. f Number of layer line corresponds to that indicated at the right side of the diffraction pattern in Fig. 1C. J. Biochem.

3 TROPONIN-TROPOMYOSIN-ACTIN PARACRYSTALS 371 respond to those of the helix containing approximately thirteen subunits in the structural repeat of 355A. The measurement in four preparations of the ratio of spacings of the first to sixth layer line gives an average value of 5.93±0.05. The contribution of the diffrac tion caused by the arrays of filaments, with the spacing of 60 A perpendicular, accentuates the layer line reflections of the first and sixth orders. Optical filtering was applied to this electron micrograph (Fig. 1B). The mask was put at the diffraction plane and among diffracted beams those with spacings presented in Table I were allowed to pass through. The filtered image thus obtained in Fig. 113 shows clearly the arrangement of globular subunits of the actin filaments in the paracrystal structure. Actin subunits are arranged in a helical way in each filament. The filaments align in re gister and are in contact with each other in the midst of the crossover points of the double strands, where the subunits of adjacent fila ments seem to interdigitate ( 16, 17). This results in narrower separation of the filaments than that expected from the size of the subunit of 55 A diameter (8 ). The corresponding subunits of the neighboring filaments are con tained in the same plane perpendicular to the axis of filament (16, 17). Troponin-tropomyosinactin complex formed two types of paracrystal structures under the same condition as that for the preparation of actin paracrystals. One type is similar in appearance to the actin paracrystal (Fig. 2), and the other is the one showing distinct cross striations of 380A period along the bundle of filaments (Fig. 3). Figure 2A shows an electron micrograph of the first type of troponin-tropomyosinactin paracrystal, which consists of parallel arrays of filaments with the gross periodicity of 360 A. The distance between adjacent filaments is 80 A. This type of paracrystal was formed when native tropomyosin or reconstituted tro ponintropomyosin complex was used. The layer line spacings in the diffraction pattern shows essentially the same characteristics as those of actin paracrystal (Fig. 2C, Table II). The average spacing ratio of the first to sixth layer line in five preparations is 5.97± TABLE II. Spacings of the diffraction pattern of native tropomyosinactin paracrystal in Fig. 2C. t Number of layer line corresponds to that indicated at the right side of the diffraction pattern in Fig. 2C. In the optically filtered image, globular sub units are also arranged essentially in the same way as those in the actin paracrystal (Fig. 2B). In Fig. 2C, diffuse reflections around the meridional region near the center can be seen. However, the spacing could not be measured precisely because of the interference with the reflections caused by the mask. The second type of troponin-tropomyosin actin paracrystal is shown in Fig. 3A. This type of paracrystal could be observed only when native tropomyosin in which the amount of troponin is several times that of tropomyosin was used. The regular transverse striations of about 380 A intervals along the bundle of filaments are remarkable. On the optical dif fraction pattern (Fig. 3C), diffraction of 382 A spacing and its second order near the me ridian are clearly seen. When these reflections are masked, the striations do not appear in the filtered image, leaving a zigzag appearance of the filaments (Fig. 3B-ii). Off meridional diffractions derived from the helical structure of the filament is rather obscure in this type of paracrystal, and only one layer line of around 59 A spacing appears, which roughly corresponds to the strong sixth layer line in other types of paracrystals. By masking this layer line diffraction, the zigzag pattern along each filament disappears from the filtered image (Fig. 3B-iii), indicating that the intervals of zigzag pattern along the filament approx- Vol. 72, No. 2, 1972

4 372 I. OHTSUKI and T. WAKABAYASHI J. Biochem.

5 TROPONIN-TROPOMYOSIN-ACTIN PARACRYSTALS 373 Vol. 72, No. 2, 1972

6 374 I. OHTSUKI and T. WAKABAYASHI J. Biochem.

7 TROPONIN-TROPOMYOSIN-ACTIN PARACRYSTALS 375 TABLE III. Relation between the period formed by troponin and actin subunit size. No. 1 in this table is the same sample as shown in Fig. 3. Paracrystals were formed under the conditions described in the legend for Fig. 3. Each value is the average of five measurements.? It is postulated that the 59A layer line corresponds to the sixth layer line of the helix, with thirteen subunits per six turns. Size of actin subunit (column 3)=59A layer line spacing (column 2)X(6/13)X2. Size of actin subunit (column 7)=axial interval of zigzag pattern (column 6)x(6113) ~2. imately corresponds to the spacing of the 59 A layer line. The spacings determined from three optical diffraction patterns of this type of paracrystals are given in Table III. These values indicate that the period of 380 A corresponds just to the length of a chain of seven actin subunits (column 4 in Table III). The measurements on the filtered image also gave the same results (column 8 in Table III). DISCUSSION It has been found that actin has ability to form a paracrystal structure in the presence of divalent cations (9). The present investiga tion shows that the troponin-tropomyosinactin complex formed two types of paracrystals under the same condition. Optical diffraction Fig. 1. Actin paracrystal. A. Electron micrograph of negatively stained actin paracrystal. Magnification X430,000; B : Optically filtered image of A ; C : Optical diffraction pattern of A. Layer line numbers are indicated at the right side of the figure. Paracrystals were made by dialyzing actin against 25 mm MgC12 and 25 mm Trismaleate (ph 6.8). Fig. 2. Paracrystal of native tropomyosinactin complex. A : Electron micrograph of negatively stained paracrystal. Magnification x430,000; B : Optically filtered image of A ; C : Optical dif fraction pattern of A. Layer line numbers are indicated at the right side of the figure. Paracrystals were made in 25 mm MgC12 and 20 mm Trismaleate (ph 6.8). Fig. 3. Paracrystal of native tropomyosinactin complex. A : Electron micrograph of negatively stained paracrystal. Magnification X170,000. B : Optically filtered image of A. (i) Diffracted beams of both meridional and off meridional region were allowed to pass through ; (ii) Off-meri dional reflections were allowed to pass through and meridional reflections were masked ; (iii) Meri dional reflections were allowed to pass through and off meridional one was masked ; C : Optical diffraction pattern of A. Paracrystals were made by dialyzing the protein against 50 mm MgC12 and 20 mm Trismaleate (ph 6.8). Vol. 72, No. 2, 1972

8 I 376 I. OHTSUKI and T. WAKABAYASHL studies of the electron micrographs of the specimens clarified the structure of these para crvstals to some extent. The first type of troponintropomyosin actin paracrystal (Fig. 2A) has apparently the same structure as that of actin paracrystal (Fig. 1A) (Essentially the same structure was also observed in the paracrystals made of thin filaments separated in a relaxing medium ; I. Ohtsuki, unpublished). Optical filtering shows that actin subunits have almost the same arrangement as that in actin paracrystal (Fig. 1B, Fig. 2B). The model of this type of paracrystal is presented in Fig. 4B. The structure seems to be determined mainly by the interaction of actin subunits of neighboring filaments. Tropomyosin, consequently troponin, should attach to the actin filament in such a way that these protein molecules do not in tensely affect the interaction of actin subunits of neighboring filaments in the paracrystal. Hence, grooves along double strands of actin are the most plausible region for the endto end aggregates of fibrous tropomyosin mole cules (7, 8). Troponin molecules between the neighboring filaments may not be related to each other (Fig. 4B). This is indicated by the obscure reflections around the meridional region of the diffraction pattern (Fig. 2C). The second type of paracrystal of troponin tropomyosinactin complex (Fig. 3) could be observed only when native tropomyosin in which the content of troponin is several times that of tropomyosin was used. The process of formation of characteristic striations with 380 A intervals would be as follows. The native complex of troponintropomyosin dis tributes along the actin filament with 380 A period. Excess free troponin molecules then form aggregates with the troponin molecule which has been attached to the actin filament through tropomyosin and thus connects one filament with other filaments side by side. The parallel arrangement of actin filaments is not strictly preserved, as shown in optically filtered image (Fig. 3B). The model of this paracrystal is presented in Fig. 4C. The exact relation between the perioc formed by the troponintropomyosin systere and actin in the filament was obtained by Fig. 4. Models of troponintropomyosinactin para crystals. (A) Troponintropomyosinactin filament. Globular actin subunits of 55A diameter, form double stranded structure (8). End-to-end filaments of tropomyosin are supposed to lie in both grooves of actin double strands (3, 7). Troponin or subunits of troponin (shown in the fugure as the black glo bular shapes) attach to tropomyosin filament with the intervals corresponding to the length of a chain of seven actin molecules (380A). (B) Troponin tropomyosinactin paracrystal (Fig. 2). In this type of paracrystal, the filaments are arranged in parallel. Actin filaments align in register, whereas troponin molecules of adjacent filaments are not specifically related to each other. (C) Troponintropomyosin actin paracrystal (Fig. 3). In this type of para crystal, the location of troponin of neighboring fila ments is in register, whereas actin filaments are not in register. The formation of troponin aggregates on troponin attached to the filament intensifies the cross striations of 380A period. using the second type of paracrystal (Table III). The result indicates that the period cor responds to the length of a chain of seven actin molecules in the filament. This finding, i.e., that the length of an integral number of actin molecules corresponds to the period of end-to-end distribution of tropomyosin along the filament, suggests that the interaction of tropomyosin and actin is based on a strict T. Biochem.

9 TROPONIN-TROPOMYOSIN-ACTIN PARACRYSTALS 377 stoichiometric relationship. In this connection,.a question is raised whether one tropomyosin molecule binds to one of two strands of an.actin filament or to both strands. Although an indication favoring the former possibility has been presented by Moore et al., (17), final conclusion must await further information. The structural repeat of actin filament in the paracrystal structure has the length of the chain of six and one-half subunits. Then the period formed by troponintropomyosin is longer by one-half subunit than the structural repeat of actin. This means, as shown in Fig. 4A, that the position of troponin rotates at a constant angle to the axis of the filament one by one. It is quite reasonable to consider that the thin filament would have essentially the same :structure as the above (Fig. 4A), as it has been presumed in a previous paper (7). Note added in proof : As to the ratio of the troponin period to the size of actin unit essen tially the same conclusion has also been reached by J.A. Spudich, H.E. Huxley, and J.T. Finch (J. Mol. Biol., in press). We wish to express our sincere thanks to Prof. S. Ebashi for his constant advice and encouragement. We are also indebted to Dr. M. Yanagida for his 1lelpful advice. REFERENCES 1. S. Ebashi and F. Ebashi, J. Biochem., 55, 604 (1964). 2. S. Ebashi and A. Kodama, J. Biochem., 58, 107 (1965). 3. S. Ebashi, A. Kodama, and F. Ebashi, J. Bio chem., 64, 465 (1968). 4. M. Endo, Y. Nonomura, T. Masaki, 1. Ohtsuki, and S. Ebashi, J. Biochem., 60, 605 (1966) Ohtsuki, T. Masaki, Y. Nonomura, and S. Ebashi, J. Biochem., 61, 817 (1967). 6. Y. Nonomura, W. Drabikowski, and S. Ebashi, J. Biochem., 64, 419 (1968). 7. S. Ebashi, M. Endo, and 1. Ohtsuki, Quart. Rev. Biophys., 2, 351 (1969). 8. J. Hanson and J. Lowy, J. Mot. Biol., 6, 46 (1963). 9. J. Hanson, "Symposium on Muscle," ed. by E. Ernst and F.B. Straub, Akademiai Kiado, Budapest, p. 99 (1968). 10. A. Kiug and J.E. Berger, J. Mot. Biol., 10, 565 (1964). 11. A. Klug and D.J. DeRosier, Nature, 212, 29 (1966). 12. S. Ebashi and K. Maruyama, J. Biochem., 58, 20 (1965). 13. W.F.H.M. Mommaerts, J. Biol. Chem., 198, 445 (1952). 14. F.B. Straub, Studies Inst. Med. Chem. Univ. Szeged, 2, 3 (1942). 15. S. Ebashi, T. Wakabayashi, and F. Ebashi, J. Biochem., 69, 441 (1971). 16. E.J. O'Brien, P.M. Bennett, and J. Hanson, Phil. Trans. Roy. Soc. B., 261, 196 (1971). 17. P.B. Moore, H.E. Huxley, and D.J. DeRosier. J. Mot. Biol., 50, 279 (1970). Vol. 72, No. 2, 1972