Gerald Offer

Interaction of C-protein with myosin, myosin rod and light meromyosin*

C-protein, a component of vertebrate skeletal muscle myofibrils, is known to be located at specific positions along the thick myosin-containingc myofilaments. We have investigated its effects on the assembly of myosin into filaments in vitro and its interaction with low ionic strength aggregates of myosin rod and light meromyosin, the α-helical fragments of myosin. C-protein is not required for the formation of myosin filaments in vitro . Purified myosin, free of C-protein, can form long filaments with a demonstrable 14 nm longitudinal repeat. The presence of C-protein disrupts the regularity of the myosin filament structure, resulting in a reduced and more variable diameter and a loss of longitudinal order. However, C-protein does not appear to have any length-regulating role. Binding measurements at low ionic strength reveal a strong affinity of C-protein for myosin and also for rod and light meromyosin. The limiting stoichiometry of binding is about one mole C-protein per mole for myosin and somewhat less for rod and light meromyosin. The interaction of C-protein with rod and light meromyosin has also been investigated by electron microscopy. In the absence of C-protein, myosin rod forms large sheet-like paracrystals with a 14 nm longitudinal repeat. In the presence of C-protein the formation of these paracrystals is disrupted and in their place we find irregular narrow filaments. Formation of paracrystals of light meromyosin is not disrupted by C-protein. The C-protein forms a series of transverse stripes on the paracrystal with a longitudinal spacing identical to the principal repeat of about 40 nm which characterizes the light meromyosin assembly. We conclude that the C-protein in native thick filaments is probably bound to the shaft of the filament with a periodicity determined by the underlying myosin assembly.

The ultrastructural location of C-protein, X-protein and H-protein in rabbit muscle

SummaryPurified antibodies to the thick filament accessory proteins, C-protein, X-protein and H-protein, have been used to label fibres of three rabbit muscles, psoas (containing mainly fast white fibres), soleus (containing mainly slow red fibres) and plantaris (a muscle of mixed fibre type) and their location has been examined by electron microscopy.These accessory proteins are present on one or more of a set of eleven transverse stripes about 43 nm apart that have been observed previously in each half A-band. Each protein has a limited set of characteristic distributions. H-protein is present on stripe 3 (counting from the M-line) in the majority of psoas fibres but is absent in soleus and plantaris muscle. C-protein can occur (1) on stripes 4–11 (the commonest pattern seen in psoas); (2) on stripes 5–11 (in psoas and plantaris); (3) on stripe 3 together with stripes 5–11 (in plantaris); or (4) on none (in red fibres of all three muscles). X-protein can occur (1) on stripes 3–11 in the red fibres of all three muscles; (2) on stripe 4 only (in psoas and plantaris); (3) on stripes 3 and 4 (in psoas and plantaris) or (4) on none. Stripes labelled with anti-X are wider than those labelled with anti-C and consist of a doublet with an internal spacing of 16 nm. The patterns for the three accessory proteins, while overlapping, are in no case identical; this suggests the proteins do not simply substitute for one another.The precise axial positions of the anti-C labelled stripes differ from those of the anti-X stripes; the anti-X stripes lie about 8–9 nm further from the M-line than the corresponding anti-C stripes. This implies that the inner member of an X-protein doublet lies in a very similar position to a C-protein stripe. The anti-H labelled stripe seen in most psoas fibres lies 14 nm nearer the M-line than stripe 3 of the anti-X labelled array in psoas red fibres and is staggered from a continuation of the C-protein array by about 4 nm.The labelling patterns were constant within a fibre and suggest a very precise assembly mechanism. The number of classes of fibre, as defined by the accessory proteins present and their arrangement, exceeds the number of fibre types presently recognized.

Polarity of the myosin molecule

Abstract The direction of the heavy polypeptide chains of the myosin molecule has been established by finding where the acetylated N-termini are located. Three acetylated N-terminal peptides have been isolated from pronase digests of rabbit skeletal myosin. They are: 1. (a) N-acetyl-Ser-Ser-Asp-Ala-Asp; 2. (b) N-acetyl-Ser-Ser-Asp-Ala-Asp-Met-Ala; 3. (c) N-acetyl-Ser. The longer peptides (a) and (b) are derived from the two heavy polypeptide chains but N-acetyl-Ser is largely derived from the light chains and not from the heavy chains as had been supposed previously (Offer, 1965). The three acetyl peptides can be isolated from pronase digests of heavy meromyosin in higher yields than from myosin. The combined yield of the longer peptides (about 1.4 mol/mol) indicates that both heavy chains are acetylated and start with the same sequence. Since the acetylated sequences are present in heavy meromyosin but are absent in light meromyosin, we conclude that the N-termini of the two heavy chains are located in the two globular heads of the myosin molecule and the C-termini are at the opposite end of the molecule. Thus the highly α-helical rod-like tail of the myosin molecule is a parallel-chain structure rather than an antiparallel-chain structure. Extended N-terminal analysis of peptides produced by chymotryptic digestion has revealed that two slightly different sequences are present in the heavy chains of rabbit skeletal myosin: N-acetyl-Ser-Ser-Asp-Ala-Asp-Met-Ala-(Val, Phe) and N-acetyl-Ser-Ser-Asp-Ala-Asp-Met-Ala-(Ile, Phe). The ratio of the isoleucine-containing component to the valine-containing component is about 1.8:1. It is concluded that myosin from mixed back and leg muscles of the rabbit contains two types of heavy chain. This information together with published information on the light chain composition suggests that such myosin preparations contain two isoenzymic forms, each form having two heavy chains with the same N-terminal sequence and two pairs of identical light chains.

Can a myosin molecule bind to two actin filaments

It is suggested that in striated muscles the two heads of one myosin molecule are able to interact with different actin filaments. This would provide a simple explanation for the appearance and arrangement of cross-bridges in insect flight muscle in rigor.

H-protein and X-protein: Two new components of the thick filaments of vertebrate skeletal muscle

With a view to obtaining a more complete view of the composition and structure of the thick filaments of vertebrate skeletal muscle, we have isolated and characterized two new myofibrillar components, H-protein and X-protein. These were purified by hydroxyapatite column chromatography of an impure C-protein preparation itself made from impure myosin extracted from rabbit back and leg muscles. H-protein is the protein responsible for band H on sodium dodecyl sulphate/polyacrylamide gel electrophoresis of crude myosin. X-protein, although present in such preparations in significant quantities, was not detected previously since it is difficult to resolve from C-protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Physical-chemical parameters have been determined for the new proteins and compared with those of C-protein. The apparent chain weight of H-protein estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis is 69,000, whereas that of X-protein (152,000) is only slightly greater than that of C-protein (140,000). The molecular weights of H- and X-proteins determined by sedimentation equilibrium centrifugation show that the molecules contain only a single polypeptide chain. The circular dichroism spectra indicate that the proteins have low alpha-helical contents. Both proteins, particularly H-protein, have a high proline content. Although X-protein is of similar chain weight to C-protein, the two show distinct differences in other properties. The sedimentation coefficient of X-protein is markedly lower than that of C-protein, suggesting X-protein is a more asymmetrical molecule. The amino acid compositions, although broadly similar, also show clear differences. Antibodies to H-protein, X-protein and C-protein have been raised in goats and shown not to cross-react.

Skip residues correlate with bends in the myosin tail

Sharp bends have previously been observed in the tail of the skeletal myosin molecule at well-defined positions 44, 75 and 135 nm from the head-tail junction, and in vertebrate smooth myosin at two positions about 45 and 96 nm from this junction. The amino acid sequence of the heavy chain does not straightforwardly account for such bending on the original model of the tail in which an invariant proline residue is present at the head-tail junction and the repeating seven amino acid pattern of hydrophobic residues lies entirely in the tail. Recently, a revised model has been proposed by Rimm et al. in which the first seven to eight heptads lie in the heads. It is shown here that with this model the observed bends in the tail of skeletal myosin coincide with three of the four additional (skip) residues that interrupt the heptad repeat. It is concluded that the skip residues, by causing localized instability of the coiled-coil, are responsible for the bends. Smooth myosin lacks the second of these skip residues explaining the absence of a bend at 75 nm.

Interaction of monomeric and polymeric actin with myosin subfragment 1

Abstract The abilities of F-actin and G-actin to activate the ATPase of subfragment 1 of myosin have been compared at 15 °C in a medium of low ionic strength in which polymerization of G-actin does not occur. F-actin gives a very much larger activation than G-actin, but the activation by G-actin is still appreciable. At 25 °C the difference between G-actin and F-actin is accentuated. At higher ionic strengths the rate of hydrolysis of ATP by subfragment 1 and G-actin increases with time, due to polymerization of G-actin. Photo-oxidized G-actin, which is incapable of polymerization, has a similar effect to unmodified G-actin. The activation by G-actin is destroyed by treatment with urea or guanidinium chloride, and other proteins do not activate. Experiments demonstrate that the activation by G-actin is not due to an indirect effect on the ionic composition of the medium. It is concluded that the activation by G-actin is due to protein-protein interaction, and that the structure of G-actin is unlikely to differ very greatly from an F-actin subunit.

[14] Preparation of C-Protein, H-Protein, X-Protein, and phosphofructokinase

Publisher Summary This chapter presents method that allows to purify C-protein, F-protein (phosphofructokinase), H-protein, X-protein, and myosin in a single procedure. In procedure, muscle mince is extracted with a high concentration of salt. The thick filament is largely depolymerized under these conditions, and most of its components are extracted in molecular form along with the soluble proteins of the muscle cell. The extract is diluted, whereupon myosin precipitates in the form of aggregates of thick filaments to which the other components bind. Phosphofructokinase and traces of actin may then be removed from the preparation by ammonium sulfate fractionation. Myosin (still contaminated with B-protein) is separated from the other compo nents by chromatography on DEAE-Sephadex. C-protein, H-protein, and X-protein may then be separated on a hydroxyapatite column. The advantage of this form of ion-exchange chromatography is that it is possible to maintain a high ionic strength (provided by KC1) throughout the fractionation and thus to prevent the association of the proteins that occurs at low ionic strength.

Location of C-protein, H-protein and X-protein in rabbit skeletal muscle fibre types

SummaryThe locations of C-protein, H-protein and X-protein in rabbit psoas, plantaris and soleus muscles have been investigated with fluorescently tagged specific antibodies. Two systems have been examined: isolated myofibrils allowed the locations of these proteins within the sarcomere to be determined, while cryosections allowed a comparison of the amounts of these proteins between different types of fibre in the three muscles.Using antibody-labelled cryosections, we find that the amounts of each of these proteins depends closely on the fibre type. In all the muscles studied, C-protein is present in the largest amounts in fast white and fast intermediate fibres and is absent from slow red fibres, while X-protein is absent from fast white fibres and is present in the largest amounts in fast and slow red fibres. In psoas muscle, H-protein is present in the largest amounts in fast white fibres and is absent in fast and slow red fibres. In plantaris muscle, however, H-protein is absent from fast white fibres but occurs in some slow red fibres.All psoas myofibrils label with anti-C and anti-H and a minority label with anti-X. In each case the pattern of labelling is a zone in each half of the A-band. Measured across the middle of the A-band, the zones for H-protein are much closer together than for C-protein; the centre-to-centre spacings are 0.35 µm for anti-H and 0.64 µm for anti-C. The fluorescent zones for X-protein are slightly but significantly closer (0.52 µm) than those for C-protein. All soleus myofibrils label with anti-X but the centre-to-centre spacing was greater (0.67 µm). With plantaris myofibrils, where labelling occurs with anti-C or anti-H, the spacings resemble those in psoas myofibrils, but with anti-X the spacing resembles that in soleus myofibrils.The spacing of the fluorescent zones in an A-band, whether produced by anti-C, anti-X or anti-H does not vary with sarcomere length. We conclude that X-protein and H-protein, like C-protein, are thick filament components.With both fibres and myofibrils, there is no simple relationship between the amount of X-protein and the amount of C-protein. Many fast intermediate fibres in psoas and plantaris muscle label as strongly with anti-C as do fast white fibres but also label as strongly with anti-X as do fast and slow red fibres. Similarly, some psoas and many plantaris myofibrils label strongly with both antibodies. We conclude that C-protein and X-protein can coexist on thick filaments but do not compete for the same sites.

Arrangement of cross-bridges in insect flight muscle in rigor

Abstract We have previously proposed that in insect flight muscle in rigor, the cross-bridges are formed by the attachment of one head of a myosin molecule to one actin filament and the other head to a neighbouring filament (Offer & Elliott, 1978). We have now analysed this type of model in more detail and considered the geometrical conditions that have to be fulfilled for such two-filament interaction to be possible. We find that two labelling patterns are possible. In both, only 2 7 of the actin subunits are labelled. In one type there is an unlabelled subunit between two labelled subunits of the long-pitched strands of the actin filament; in the other type, a similar grouping occurs in one long-pitched strand, while in the other strand there is a gap of two unlabelled subunits. We have shown that if the myosin molecules on the insect thick filament are arranged on a right-handed, four-stranded helix (Wray, 1979 a,b ), it is possible to find conditions where only two-filament interaction and no single-filament interaction would be expected. In this case, approximately 9 16 of the myosin heads attach, while the remainder are unattached. With other thick filament symmetries a mixture of single-filament and two-filament interactions would be expected. The two-filament interaction type of model can, with a suitable choice of actin-myosin binding geometry, explain the appearance of transverse and longitudinal sections of insect flight muscle in rigor (Reedy, 1968). The Fourier transforms of many variations of the two-filament interaction model have been calculated. We find several detailed models, differing principally in the slew angle of attachment of myosin heads and in the position of troponin, which are able to account satisfactorily for the observed X-ray diffraction patterns of this muscle in rigor (Miller & Tregear, 1972; Holmes et al. , 1980).