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

Abstract

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

A Molecular Map of the Interactions between Titin and Myosin‐Binding Protein C

“…This also seems to he the case for the titin-binding and myosin-binding protein niyomesin (Obermann et al, 1995). We therefore suggest that a cooperative interaction in the ternary complex of titin, myosin and the MyBP-family members on specific regions of the thick filament will define their ultimately very strong and specific association to the myofibril.…”

“…To screen for the interaction of titin with MyBP-C, a number of titin multidomain fragments from the A-band and including controls from the I-band and M-line regions of titin was expressed in E. coli (Fig. 4A) and assayed for their MyBP-C binding ability in a solid-phase assay which previously proved most sensitive for related proteins (Obermann et al, 1995). The nomenclature of the A-band fragments on the titin domain pattern is illustrated in (Fig.…”

“…Recombinant fragments of titin or MyBP-C were diluted to 0.1 mglml in assay buffer similar to that described by Obermann et al (1995) (20mM imidazolelHC1, pH 7.0, 100 mM KCI, 5 mM MgCl,, 1 mM EGTA, 1 mM dithiothreitol, 0.05% Tween 20, 1 mg/ml BSA) without BSA and spotted onto wetted nitrocellulose membranes (Schleicher & Schiill BA-85) using a Scotslab dot-blotter. Protein density was between 2 pmol/mm2 (for multiple-domain constructs) and 5 pmol/ mm2 (for single-domain constructs).…”

Mapping of a Myosin-binding Domain and a Regulatory Phosphorylation Site in M-Protein, a Structural Protein of the Sarcomeric M Band

“…Myosin rod and LMM were prepared as described (Obermann et al, 1995). Purification of bovine M-protein was performed as reported (Vinkemeier et al, 1993).…”

Fast-folding α-helices as reversible strain absorbers in the muscle protein myomesin

“…Here, the 185 kDa protein myomesin (6) as well as other filamentous proteins form a large network constituting, together with metabolic enzymes and kinase domains, a well-organized compartment that has both structural and metabolic properties (7). Myomesin comprises 13 domains, with the first one (My1) being unique and the others (My2-My13) either of the immunoglobulin (Ig) or fibronectin type III fold (8). It is part of a complex network that involves interactions with myosin, titin, obscurin, and obscurin-like 1 (9,10).…”