Rabbit skeletal muscle myosin unfolded carboxyl‐terminus and its role in molecular assembly

Maéda, Kayo; Rösch, Andrea; Maéda, Yuichiro; Kalbitzer, Hans Robert; Wittinghofer, Alfred

doi:10.1016/0014-5793(91)80349-8

Cited by 25 publications

(22 citation statements)

References 21 publications

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“…Myosin as well as the chymotryptic myosin subfragment 1 (Sl), myosin rod and LMM were prepared as described [ 121. An LMM fragment comprising the carboxy-terminal 30-kDa portion of rabbit fast skeletal muscle LMM was expressed in Escherichio coli and purified as described [14]. The construct was a generous gift by Dr A. Wittinghofer (Max-Planck-Institute for Molecular Physiology, Dortmund, Germany).…”

Section: Generation Of Stable Proteolytic Fragments Of Myomesinmentioning

confidence: 99%

Purification and Biochemical Characterization of Myomesin, a Myosin‐Binding and Titin‐Binding Protein, from Bovine Skeletal Muscle

Obermann

Plessmann

Weber

et al. 1995

European Journal of Biochemistry

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We report a method for isolating homogeneous myomesin from mammalian skeletal muscle. The identity of the purified bovine protein was confirmed by its reactivity with myomesin-specific monoclonal antibodies and with polyclonal antibodies raised against peptides derived from the amino-terminal and carboxy-terminal ends of the sequence predicted by the human myomesin cDNA. All partial sequences obtained from bovine myomesin can be aligned along the human sequence predicted by its cloned cDNA. Electron microscopy of myomesin revealed short flexible rods with a molecular length of about SO nm. Circular dichroism spectra showed a high degree of structure as expected for a member of the immunoglobulin superfamily of proteins. Alignment of the sequences of the class I and I1 domains of myomesin with the sequences of domains of known three-dimensional structure provides a more detailed model of myomesin. In agreement with this view, the cleavage sites observed by limited proteolysis locate primarily between individual domains.In a solid-phase overlay assay myomesin specifically bound to the myosin rod and to light meromyosin (LMM), but not to the carboxy-terminal 30-kDa fragment of LMM. The myosin-binding site seemed to be confined to the amino-terminal 240 residues of the molecule. The cross-reactivity of myomesin with the phosphorylation-dependent monoclonal neurofilament antibody NE14 [Shaw, G. E., Debus, E. & Keywords: myomesin ; sarcomeric M band ; myosin-binding protein ; titin-associated protein.Vertebrate striated muscle sarcomeres contain in addition to thick and thin filaments a complex cytoskeleton (see [2] and references therein). This cytoskeleton is primarily based on the giant protein titin (also called connectin). In the M band region of the sarcomere, titin seems to be associated with M-protein and myomesin [3, 41. While M-protein was purified and biochemically characterized rather early [5, 61, myomesin was only identified by mAb 171. These antibodies helped to establish differential tissue distributions, and also indicated distinct developmental expression patterns [&lo]. Thus myomesin seemed to be present in all cardiac and skeletal muscle fibres examined, while M-protein was found only in fast skeletal and cardiac fibres.cDNA cloning revealed that both M-protein and myomesin are primarily comprised of repeating immunoglobulin cII-like and fibronectin-type-111-like domains [4, 111. The order of these domains is the same in both proteins and the overall sequence identity is about SO% [4]. This striking similarity implies that

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Section: Generation Of Stable Proteolytic Fragments Of Myomesinmentioning

confidence: 99%

Purification and Biochemical Characterization of Myomesin, a Myosin‐Binding and Titin‐Binding Protein, from Bovine Skeletal Muscle

Obermann

Plessmann

Weber

et al. 1995

European Journal of Biochemistry

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“…In contrast, LMM fragments greater than 75 kDa were insoluble (lane P of Figure 9b). The teprtr.Digests were sampled at the time points indicated and subjected to SDS/PAGE (%gl.Ln using myosin with partially deleted sequences provide further evidence that deletion of a short peptide at the C-terminus significantly increases its solubility in low-ionic-strength buffer [28], decreases aggregation [29] and increases by 50-fold the critical concentration for myosin rod assembly [30]. In sharp contrast, deleting a peptide of similar length at the N-terminus of the rod or LMM has only minor effects [28,30].…”

Section: Solubility Properties Of Lmm Fragmentsmentioning

confidence: 99%

Protease-susceptible sites and properties of fragments of aortic smooth-muscle myosin

King¹,

Jiang

Huang³

et al. 1995

Biochemical Journal

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We have examined the protease susceptibility of aortic myosin, the thermal unfolding profiles of myosin rod and light meromyosin (LMM) and the solubility properties of the LMM fragments. Two major protease-susceptible sites were found, located at the head-rod junction and the heavy meromyosin (HMM)-LMM junction. Both tryptic and chymotryptic digestion of aortic myosin rod produced the LMM (80-85 kDa) and short subfragment 2 (S-2) (40-45 kDa) segments, which were similar to those of gizzard myosin rod and differed from the short LMM (70 kDa) and long S-2 (58 kDa) segments produced from skeletal-muscle rod. The thermal unfolding profile of aortic myosin rods exhibited three helix-unfolding transitions, at 47.5, 51 and 54 degrees C, similar to those of gizzard rods yet different from those of skeletal-muscle rods. There was a dramatic difference in the solubility of aortic LMM fragments of various molecular mass, as for gizzard smooth-muscle LMM and rabbit skeletal-muscle LMM. LMM fragments of molecular mass 77 kDa or more were completely insoluble in low-ionic-strength buffer, whereas LMM fragments of molecular mass 73 kDa or less were completely soluble in low-ionic-strength buffer. Proteolytic digestion patterns of LMM showed two additional protease-susceptible sites located 13 and 30 kDa from the ends of the LMM molecule. This suggests the existence of flexible regions within the LMM molecule, which may be responsible for the folded form of aortic myosin.

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“…For example, mutations in the COOH terminus of Caenorhabditis elegans unc-54 MHC disrupts myosin filament assembly (Dibb et al, 1985) while fusion proteins containing Acanthamoeba myosin II tail sequences with deletions in the last 100 amino acids fail to assemble (Sinard et al, 1990). However, phosphorylation or deletion of part of the nonhelical COOH-terminal tail piece of Acanthamoeba myosin II has little apparent effect on filament formation (Atkinson et al, 1989;Sathyamoorthy et al, 1990;Ganguly et al, 1990) whereas removal of the 17 COOH-terminal amino acids from rabbit skeletal muscle myosin rod disturbs filament assembly especially at high pH values (Maeda et al, 1991).…”

mentioning

confidence: 99%

Role of the COOH-terminal nonhelical tailpiece in the assembly of a vertebrate nonmuscle myosin rod.

Hodge

Cross

Kendrick‐Jones

1992

The Journal of Cell Biology

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Abstract. A short nonhelical sequence at the COOHterminus of vertebrate nonmuscle myosin has been shown to enhance myosin filament assembly. We have analyzed the role of this sequence in chicken intestinal epithelial brush border myosin, using protein engineering/site-directed mutagenesis. Clones encoding the rod region of this myosin were isolated and sequenced. They were truncated at various restriction sites and expressed in Escherichia coli, yielding a series of mutant myosin rods with or without the COOH-terminal tailpiece and with serial deletions from their NH2-termini. Deletion of the 35 residue COOH-terminal nonhelical tailpiece was sufficient to increase the critical concentration for myosin rod assembly by 50-fold (at 150 mM NaC1, pH 7.5), whereas NH2-terminal deletions had only minor effects. The only exception was the longest NH2-terminal deletion, which reduced the rod to 119 amino acids and rendered it assembly incompetent. The COOH-terminal tailpiece could be reduced by 15 amino acids and it still efficiently promoted assembly. We also found that the tailpiece promoted assembly of both filaments and segments; assemblies which have different molecular overlaps. Rod fragments carrying the COOH-terminal tailpiece did not promote the assembly of COOH-terminally deleted material when the two were mixed together. The tailpiece sequence thus has profound effects on assembly, yet it is apparently unstructured and can be bisected without affecting its function. Taken together these observations suggest that the nonhelical tailpiece may act sterically to block an otherwise dominant but unproductive molecular interaction in the self assembly process and does not, as has been previously thought, bind to a specific target site(s) on a neighboring molecule.

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Rabbit skeletal muscle myosin unfolded carboxyl‐terminus and its role in molecular assembly

Cited by 25 publications

References 21 publications

Purification and Biochemical Characterization of Myomesin, a Myosin‐Binding and Titin‐Binding Protein, from Bovine Skeletal Muscle

Purification and Biochemical Characterization of Myomesin, a Myosin‐Binding and Titin‐Binding Protein, from Bovine Skeletal Muscle

Protease-susceptible sites and properties of fragments of aortic smooth-muscle myosin

Role of the COOH-terminal nonhelical tailpiece in the assembly of a vertebrate nonmuscle myosin rod.

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