Elastic Protein Of Muscle Research Articles

Human Cardiac Myosin Binding Protein C: Structural Flexibility within an Extended Modular Architecture

New insights into the modular organization and flexibility of the N-terminal half of human cardiac myosin binding protein C (cMyBP-C) and information on the association state of the full-length protein have been deduced from a combined small-angle X-ray scattering (SAXS) and NMR study. SAXS data show that the first five immunoglobulin domains of cMyBP-C, which include those implicated in interactions with both myosin and actin, remain monodisperse and monomeric in solution and have a highly extended yet distinctively ‘bent’ modular arrangement that is similar to the giant elastic muscle protein titin. Analyses of the NMR and SAXS data indicate that a proline/alanine-rich linker connecting the cardiac-specific N-terminal C0 domain to the C1 domain provides significant structural flexibility at the N-terminus of the human isoform, while the modular arrangement of domains C1–C2–C3–C4 is relatively fixed. Domain fragments from the C-terminal half of the protein have a propensity to self-associate in vitro, while full-length bacterially expressed cMyBP-C forms flexible extended dimers at micromolar protein concentrations. In summary, our studies reveal that human cMyBP-C combines a distinctive modular architecture with regions of flexibility and that the N-terminal half of the protein is sufficiently extended to span the range of interfilament distances sampled within the dynamic environment of heart muscle. These structural features of cMyBP-C could facilitate its putative role as a molecular switch between actin and myosin and may contribute to modulating the transverse pliancy of the C-zone of the A-band across muscle sarcomeres.

Rigid Conformation of an Immunoglobulin Domain Tandem Repeat in the A-band of the Elastic Muscle Protein Titin

Most of the structure of the giant muscle protein titin is formed by small modular domains. Many of them are predicted to be arranged in repeats with short linkers that may be key determinants of the peculiar elastic properties of titin. Here, we present the molecular structure of a tandem arrangement of two immunoglobulin-like domains, A168 and A169, located within the A-band segment of titin. The two domains are connected by a 17 residue long β-strand and form a common interface. Based on these data, we establish general principles to estimate the amount of conformational flexibility of tandem domain motifs in titin. An unusual bulge within the second domain, A169, is directly involved into binding to a sarcomeric ligand, MURF-1, thus suggesting a dual role of this tandem for both the mechanical properties of titin and for sarcomeric signaling.

Mechanically Induced Titin Kinase Activation Studied by Force-Probe Molecular Dynamics Simulations

The conversion of mechanical stress into a biochemical signal in a muscle cell requires a force sensor. Titin kinase, the catalytic domain of the elastic muscle protein titin, has been suggested as a candidate. Its activation requires major conformational changes resulting in the exposure of its active site. Here, force-probe molecular dynamics simulations were used to obtain insight into the tension-induced activation mechanism. We find evidence for a sequential mechanically induced opening of the catalytic site without complete domain unfolding. Our results suggest the rupture of two terminal β-sheets as the primary unfolding steps. The low force resistance of the C-terminal relative to the N-terminal β-sheet is attributed to their different geometry. A subsequent rearrangement of the autoinhibitory tail is seen to lead to the exposure of the active site, as is required for titin kinase activity. These results support the hypothesis of titin kinase as a force sensor.

Induction and Myofibrillar Targeting of CARP, and Suppression of the Nkx2.5 Pathway in the MDM Mouse with Impaired Titin-based Signaling

Muscular dystrophy with myositis ( mdm) is a recessive mouse mutation that is caused by a small deletion in the giant elastic muscle protein titin. Homozygous mdm/ mdm mice develop a progressive muscular dystrophy, leading to death at ∼2 months of age. We surveyed the transcriptomes of skeletal muscles from 24 day old homozygous mdm/ mdm and +/+ wild-type mice, an age when MDM animals have normal passive and active tensions and sarcomeric structure. Of the 12,488 genes surveyed (U74 affymetrix array), 75 genes were twofold to 30-fold differentially expressed, including CARP (cardiac ankyrin repeat protein), ankrd2/Arpp (a CARP-like protein) and MLP (muscle LIM protein), all of which associate with the titin filament system. The four genes most strongly affected (eightfold to 30-fold change) were all members of the CARP-regulated Nkx-2.5-dependent signal pathway, and CARP mRNA level was 30-fold elevated in MDM skeletal muscle tissues. The CARP protein overexpressed in MDM became associated with the I-band region of the sarcomere. The mdm mutation excises the C-terminal portion of titin's N2A region, abolishing its interaction with p94/calpain-3 protease. Thus, the composition of the titin N2A protein complex is altered in MDM by incorporation of CARP and loss of p94/calpain-3. These changes were absent from the following control tissues (1) cardiac muscles from homozygous mdm/ mdm animals, (2) skeletal and cardiac muscle from heterozygous mdm/+ animals, and (3) dystrophic muscles from MDX mice. Thus, the altered composition of the titin N2A complex is specific for the titin-based skeletal muscular dystrophy in MDM.

Beta-Actinin, Cap Z, connectin and titin: what's in a name?

beta-Actinin, Cap Z, connectin and titin: what's in a name?

Responsive hybrid hydrogels with volume transitions modulated by a titin immunoglobulin module.

The I28 immunoglobulin (Ig)-like module of human cardiac titin, an elastic muscle protein, was used to cross-link acrylamide (AAm) copolymers into hybrid hydrogels. Cross-linking was accomplished through metal coordination bonding between terminal histidine tags (His tags) of the I28 module and metal-chelating nitrilotriacetic acid (NTA)-containing side chains on the copolymer. In solution, the beta-sheet structure of the I28 module unfolded with a transition midpoint of about 58 degrees C as the temperature was elevated. Hydrogels cross-linked with the I28 module demonstrated positive temperature responsiveness; they swelled to 3 times their initial volume at temperatures above the melting temperature of the cross-links. Positive temperature responsiveness is unusual for synthetic hydrogels. The I28 hybrid hydrogels demonstrate that cross-linking synthetic polymers with natural, well-characterized protein modules is a practical strategy for creating new materials with unique environmental responsiveness predictably determined by the mechanical properties of the protein cross-links. These new materials may be useful for controlled chemical delivery.

Connectin/titin, giant elastic protein of muscle.

The structure and function of the giant elastic protein connectin/titin are described on the basis of recent investigations. The 3000 kDa protein links the Z line to the myosin filament in striated muscle sarcomeres. The NH2-terminal region of connectin filament is involved in the Z line binding, and the COOH-terminal region is bound onto the myosin filament with an overlap between the counter-connectin filaments at the M line. The PEVK region in the I band is shown to be mainly responsible for passive tension generation. The longitudinal continuity of myosin-, actin-free sarcomeres is explained by the linkage of freed connectin filaments extending from both sides of the Z lines in a sarcomere. The role of connectin in myofibrillar differentiation and the biodiversity of connectin-related proteins in the animal kingdom are briefly reviewed.

Improvements in atomic force microscopy protocols for imaging fibrous proteins

Myosin is one of the main structural components of biological systems and due to its unique shape is easily recognized. This makes it an ideal subject for the development of preparation techniques for atomic force microscopy (AFM) of other fibrous proteins. Myosin, prepared using a glycerol/mica deposition technique, has been imaged using contact mode as well as the newly developed tapping mode AFM in liquid. Improved resolution using the tapping mode under propanol has allowed us to resolve the ∼7 nm half‐repeat of the coiled‐coil α‐helix of the myosin tail. Myosin has also been imaged under aqueous conditions. We have also imaged the elastic muscle protein, titin, prepared in a similar fashion. Titin is easily deformed, making contact mode imaging in aqueous conditions a destructive process. However, by using tapping mode AFM in liquid it has been possible to image titin under aqueous conditions without damage to the molecules at a resolution equivalent to contact mode in propanol. Using tapping mode AF...

A partial connectin cDNA encoding a novel type of RSP motifs isolated from chicken embryonic skeletal muscle.

A partial cDNA encoding 811 amino acids of connectin (titin), a giant elastic protein of muscle (3,000 kDa), was cloned from a chicken embryonic skeletal muscle cDNA library using antibodies to muscle connectin. The encoded product was the C terminal segment of connectin. The predicted sequences consisted of 5 type II motifs (immunoglobulin C2 type) separated by 5 interdomain insertions. One interdomain insertion had significant homology (RSP) to KSP repeats found in human cardiac C-terminal connectin and another had a high sequence homology to porin (67.7%; 31 amino acids).

Connectin filaments in stretched skinned fibers of frog skeletal muscle.

Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.

Connectin, an elastic protein of muscle. Identification of "titin" with connectin.

When whole muscle fibers or myofibrils of rabbit and chicken skeletal muscles are directly solubilized in hot SDS solution, a very high molecular protein called titin can be isolated by gel filtration (Wang et al. 1979). Connectin, an elastic protein of muscle (Maruyama et al. 1977), can be isolated by a similar method from thoroughly extracted muscle residues. Studies of electrophoretic mobility on 2-3% polyacrylamide gel electrophoresis, amino acid composition, and localization in myofibrils determined by the indirect immunofluorescence technique showed that titin and connectin are identical. Connectin was found to be unstable in SDS solution on storage for a few days at room temperature; the doublet band of connectin on SDS gel electrophoresis became diffuse and eventually disappeared. Connectin was concentrated around the A-I junction region of a myofibril, although it was present in an entire sarcomere except for the Z lines. On removal of myosin, the A-I junction was still fluorescent, when treated with fluorescent antibody against connectin. In the KI-extracted myofibril, materials accumulated on both sides of the Z lines were strongly stained, and there were fluorescent filaments between the neighboring Z lines, but the Z lines were not stained at all.

Connectin, an elastic protein of muscle. Effects of proteolytic enzymes in situ.

The effects of various proteolytic enzymes on the high molecular weight protein (connectin) present in a direct sodium dodecyl sulfate extract of myofibrils from chicken breast muscle were studied in detail. To keep the high molecular weight proteins intact, myofibrils had to be prepared in the presence of EGTA. Trypsin, chymotrypsin, papain, and nagarse readily hydrolyzed connectin (doublet band of titin) and the band 3 protein (N2-line protein). Pepsin did not attack connectin, but digested the band 3 protein and myosin. Calcium-activated neutral proteinase hydrolyzed the band 3 protein, leaving connectin intact. On the other hand, serine protease digested connectin but not the band 3 protein.

Connectin, an elastic protein of muscle. A connectin-like protein from the plasmodium Physarum polycephalum.

A completely sodium dodecyl sulfate-insoluble protein was obtained from plasmodia of Physarum polycephalum. The amino acid composition of this protein was very similar to that of connectin, an elastic protein of muscle. When the protein suspension was treated with 6 M guanidine-HCl in the presence of 0.1 M 2-mercaptoethanol, entanglements of very thin filaments, less than 5 nm in width, were observed under an electron microscope. Since the network structure is almost the same as that of isolated muscle connectin, this plasmodium protein can be regarded as forming an elastic net structure. Using an antiserum against the insoluble protein, it appeared to be located at the peripheral region of the plasmodium. This elastic net structure may act as a cytoskeleton at the cytoplasmic surface of the plasmodium cell membrane.

Carp connectin: Its contents in white and dark muscles.

The white and dark muscles of carp have been studied by a simple method which was devised for the isolation and determination of connectin, an elastic protein of muscle. The connectin contents in the white and dark muscles were estimated to be approximately 4.4% and 2.8% of the total muscle protein, respectively. When compared with the white muscle, however, the dark one was found to be rich in muscle stroma which was mainly composed of connectin and collagen. Connectin preparations of both muscles were almost free from connective tissue proteins, collagen and elastin, and exhibited a close similarity in their amino acid compositions.

Fine structure of connectin nets in cardiac myofibrils.

A net-like structure extending through Z lines of myofibrils in frog cardiac muscle was clearly demonstrated under an electron microscope after the removal of myosin and actin. The diameter of the very thin filaments forming the nets was approximately 2 nm; this width was thinner than that of the actin filament. The net structure is ascribed to connectin, an elastic protein of muscle.

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Pure myofibrils were isolated from bovine heart by sucrose layer ultracentrifugation. Cardiac myofibrils thus prepared contained more protein as insoluble stroma than skeletal muscle. The insoluble stroma largely consisted of connectin, an elastic protein of muscle. The connectin content in cardiac myofibrils was about 18% of the total myofibrillar protein and was three times that in skeletal myofibrils. In view of the role of connectin as an elastic component of muscle, the abundance of connectin in cardiac myofibrils may be responsible for keeping myofibrils short at rest. This would account for the more effective tension generation in cardiac muscle on passive stretching due to blood inflow (Stirling's law).