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PubMed1995

The folding and stability of titin immunoglobulin-like modules, with implications for the mechanism of elasticity.

Titin (first known as connectin) is a vast modular protein found in vertebrate striated muscle. It is thought to assist myofibrillogenesis and to provide a passive elastic restoring force that helps to keep the thick filaments properly centered in the sarcomere. We show that representative titin modules do indeed fold independently, and report their stabilities (i.e., delta G of unfolding and melting temperature) as measured by circular dichroism, fluorescence, and nuclear magnetic resonance spectroscopies. We find that there is a region-dependent variation in stability, although we find no evidence to support a proposed elastic mechanism based on a molten-globular-like equilibrium folding intermediate, nor do our calculations support any mechanism based on the configurational entropy of the molecule itself; instead we suggest a model based on hydrophobic hinge regions that would not be strongly dependent on the precise folding pattern of the chain.

Animals↗
PubMed1995

Microscopic analysis of the elastic properties of nebulin in skeletal myofibrils.

The elastic properties of nebulin were studied by measuring the elasticity of single skeletal myofibrils, from which the portion of the thin filament located at the I band had been selectively removed by treatment with plasma gelsolin under rigor conditions. In this myofibril model, a portion of each nebulin molecule at the I band was expected to be free of actin filaments and exposed. The length of the exposed portion of the nebulin molecule was controlled by performing the gelsolin treatment at various sarcomere lengths. The relation between the passive tension and extension of the exposed portion of the nebulin showed a convex curve starting from a slack length, apparently in a fashion similar to that of wool. The slack sarcomere length shifted depending on the length of the exposed portion of the nebulin, however, the relation being represented by a single master curve. The elastic modulus of nebulin was estimated to be two to three orders of magnitude smaller than that of an actin filament. Based on these results, we conclude that nebulin attaches to an actin filament in a side-by-side fashion and that it does not significantly contribute to the elastic modulus of thin filaments. The relation between the passive tension and extension of connectin (titin) was obtained for a myofibril from which thin filaments had been completely removed with gelsolin under contracting conditions; this showed a concave curve, consistent with the previous results obtained in single fibers.

Actins↗
PubMed1996

Nonuniform elasticity of titin in cardiac myocytes: a study using immunoelectron microscopy and cellular mechanics.

Titin (also known as connectin) is a muscle-specific giant protein found inside the sarcomere, spanning from the Z-line to the M-line. The I-band segment of titin is considered to function as a molecular spring that develops tension when sarcomeres are stretched (passive tension). Recent studies on skeletal muscle indicate that it is not the entire I-band segment of titin that behaves as a spring; some sections are inelastic and do not take part in the development of passive tension. To better understand the mechanism of passive tension development in the heart, where passive tension plays an essential role in the pumping function, we investigated titin's elastic segment in cardiac myocytes using structural and mechanical techniques. Single cardiac myocytes were stretched by various amounts and then immunolabeled and processed for electron microscopy in the stretched state. Monoclonal antibodies that recognize different titin epitopes were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. We found that only a small region of the I-band segment of titin is elastic; its contour length is estimated at approximately 75 nm, which is only approximately 40% of the total I-band segment of titin. Passive tension measurements indicated that the fundamental determinant of how much passive tension the heart develops is the strain of titin's elastic segment. Furthermore, we found evidence that in sarcomeres that are slack (length, approximately 1.85 microns) the elastic titin segment is highly folded on top of itself. Based on the data, we propose a two-stage mechanism of passive tension development in the heart, in which, between sarcomere lengths of approximately 1.85 microns and approximately 2.0 microns, titin's elastic segment straightens and, at lengths longer than approximately 2.0 microns, the molecular domains that make up titin's elastic segment unravel. Sarcomere shortening to lengths below slack (approximately 1.85 microns) also results in straightening of the elastic titin segment, giving rise to a force that opposes shortening and that tends to bring sarcomeres back to their slack length.

Animals↗
PubMed1997

Actin-titin interaction in cardiac myofibrils: probing a physiological role.

The high stiffness of relaxed cardiac myofibrils is explainable mainly by the expression of a short-length titin (connectin), the giant elastic protein of the vertebrate myofibrillar cytoskeleton. However, additional molecular features could account for this high stiffness, such as interaction between titin and actin, which has previously been reported in vitro. To probe this finding for a possible physiological significance, isolated myofibrils from rat heart were subjected to selective removal of actin filaments by a calcium-independent gelsolin fragment, and the "passive" stiffness of the specimens was recorded. Upon actin extraction, stiffness decreased by nearly 60%, and to a similar degree after high-salt extraction of thick filaments. Thus actin-titin association indeed contributes to the stiffness of resting cardiac muscle. To identify possible sites of association, we employed a combination of different techniques. Immunofluorescence microscopy revealed that actin extraction increased the extensibility of the previously stiff Z-disc-flanking titin region. Actin-titin interaction within this region was confirmed in in vitro cosedimentation assays, in which multimodule recombinant titin fragments were tested for their ability to interact with F-actin. By contrast, such assays showed no actin-titin-binding propensity for sarcomeric regions outside the Z-disc comb. Accordingly, the results of mechanical measurements demonstrated that competition with native titin by recombinant titin fragments from Z-disc-remote, I-band or A-band regions did not affect passive myofibril stiffness. These results indicate that it is actin-titin association near the Z-disc, but not along the remainder of the sarcomere, that helps to anchor the titin molecule at its N-terminus and maintain a high stiffness of the relaxed cardiac myofibril.

Actins↗
PubMed1997

Titin elasticity and mechanism of passive force development in rat cardiac myocytes probed by thin-filament extraction.

Titin (also known as connectin) is a giant filamentous protein whose elastic properties greatly contribute to the passive force in muscle. In the sarcomere, the elastic I-band segment of titin may interact with the thin filaments, possibly affecting the molecule's elastic behavior. Indeed, several studies have indicated that interactions between titin and actin occur in vitro and may occur in the sarcomere as well. To explore the properties of titin alone, one must first eliminate the modulating effect of the thin filaments by selectively removing them. In the present work, thin filaments were selectively removed from the cardiac myocyte by using a gelsolin fragment. Partial extraction left behind approximately 100-nm-long thin filaments protruding from the Z-line, whereas the rest of the I-band became devoid of thin filaments, exposing titin. By applying a much more extensive gelsolin treatment, we also removed the remaining short thin filaments near the Z-line. After extraction, the extensibility of titin was studied by using immunoelectron microscopy, and the passive force-sarcomere length relation was determined by using mechanical techniques. Titin's regional extensibility was not detectably affected by partial thin-filament extraction. Passive force, on the other hand, was reduced at sarcomere lengths longer than approximately 2.1 microm, with a 33 +/- 9% reduction at 2.6 microm. After a complete extraction, the slack sarcomere length was reduced to approximately 1.7 microm. The segment of titin near the Z-line, which is otherwise inextensible, collapsed toward the Z-line in sarcomeres shorter than approximately 2.0 microm, but it was extended in sarcomeres longer than approximately 2.3 microm. Passive force became elevated at sarcomere lengths between approximately 1.7 and approximately 2.1 microm, but was reduced at sarcomere lengths of >2.3 microm. These changes can be accounted for by modeling titin as two wormlike chains in series, one of which increases its contour length by recruitment of the titin segment near the Z-line into the elastic pool.

Amino Acid Sequence↗
PubMed1998

The mechanical stability of immunoglobulin and fibronectin III domains in the muscle protein titin measured by atomic force microscopy.

The domains of the giant muscle protein titin (connectin) provide interaction sites for other sarcomeric proteins and fulfill mechanical functions. In this paper we compare the unfolding forces of defined regions of different titin isoforms by single-molecule force spectroscopy. Constructs comprising six to eight immunoglobulin (Ig) domains located in the mechanically active I-band part of titin are compared to those containing fibronectin III (Fn3) and Ig domains from the A-band part. The high spatial resolution of the atomic force microscope allows us to detect differences in length as low as a few amino acids. Thus constructs of different lengths may be used as molecular rulers for structural comparisons with other modular proteins. The unfolding forces range between 150 and 300 pN and differ systematically between the constructs. Fn3 domains in titin exhibit 20% lower unfolding forces than Ig domains. Fn3 domains from tenascin, however, unfold at forces only half those of titin Fn3 domains. This indicates that the tightly folded titin domains are designed to maintain their structural integrity, even under the influence of stretching forces. Hence, at physiological forces, unfolding is unlikely unless the forces are applied for a long time (longer than minutes).

Biomechanical Phenomena↗
PubMed2002

Titin organisation and the 3D architecture of the vertebrate-striated muscle I-band.

The giant muscle protein titin (connectin) is known to serve as a cytoskeletal element in muscle sarcomeres. It elastically restrains lengthening sarcomeres, it aids the integrity and central positioning of the A-band in the sarcomere and it may act as a template upon which some sarcomeric components are laid down during myogenesis. A puzzle has been how titin molecules, arranged systematically within the hexagonal A-band lattice of myosin filaments, can redistribute through the I-band to their anchoring sites in the tetragonal Z-band lattice. Recent work by Liversage and colleagues has suggested that there are six titin molecules per half myosin filament. Since there are two actin filaments per half myosin filament in a half sarcomere, this means that there are three titin molecules interacting with each Z-band unit cell containing one actin filament in the same sarcomere and one of opposite polarity from the next sarcomere. Liversage et al. suggested that the three titins might be distributed with two on an actin filament of one polarity and one on the filament of opposite polarity. Here, we build on this suggestion and discuss the transition of titin from the A-band to the Z-band. We show that there are good structural and mechanical reasons why titin might be organised as Liversage et al., suggested and we discuss the possible relationships between A-band arrangements in successive sarcomeres along a myofibril.

Animals↗
PubMed1995

Dissecting titin into its structural motifs: identification of an alpha-helix motif near the titin N-terminus.

Titin, also known as connectin, is a giant modular protein specifically found in vertebrate striated muscle. Since the huge size of titin does not allow a direct structure determination, we have started a long-term project to characterize the protein by cutting it into smaller domains or structural units. The major part of the titin sequence is assembled by modules approximately 100 amino acids long that belong to two major protein superfamilies. Most of these modules are linked together by stretches of variable length with unique sequence. No direct structural characterization has been achieved so far for any of these linkers. We present here a study of a stretch located in the titin N-terminus and part of a linker between two modules. Our attention was drawn toward this region because it shows 100% probability to form a coiled coil when analyzed by a prediction program. A synthetic 38 amino acid peptide spanning such a sequence was studied in aqueous solution by circular dichroism, nuclear magnetic resonance, and analytical ultracentrifugation at various pH, salt, and peptide concentrations. Under all conditions, it shows a strong tendency to form alpha-helical structures. In the presence of salt, this conformation is associated with the formation of helical bundles below pH 5. Above pH 5, any aggregate breaks, and the titin peptide is a monomeric helix in equilibrium with its random coil conformation. We discuss the factors which stabilize the helical conformation and the possible role of this stretch in vivo.

Amino Acid Sequence↗
PubMed1998

Structural basis for activation of the titin kinase domain during myofibrillogenesis.

The giant muscle protein titin (connectin) is essential in the temporal and spatial control of the assembly of the highly ordered sarcomeres (contractile units) of striated muscle. Here we present the crystal structure of titin's only catalytic domain, an autoregulated serine kinase (titin kinase). The structure shows how the active site is inhibited by a tyrosine of the kinase domain. We describe a dual mechanism of activation of titin kinase that consists of phosphorylation of this tyrosine and binding of calcium/calmodulin to the regulatory tail. The serine kinase domain of titin is the first known non-arginine-aspartate kinase to be activated by phosphorylation. The phosphorylated tyrosine is not located in the activation segment, as in other kinases, but in the P + 1 loop, indicating that this tyrosine is a binding partner of the titin kinase substrate. Titin kinase phosphorylates the muscle protein telethonin in early differentiating myocytes, indicating that this kinase may act in myofibrillogenesis.

Amino Acid Sequence↗
PubMedDate not supplied

A physiological role for titin and nebulin in skeletal muscle.

Production of active force in skeletal muscle results from the interaction of myosin-containing thick filaments with actin-containing thin filaments. These muscles are also passively elastic, producing forces that resist stretch independently of ATP splitting or of interaction between the filaments. The mechanism of this passive elasticity is unknown; suggestions include gap filaments in the region between thick and thin filaments in muscles stretched beyond filament overlap, or intermediate filaments which connect successive Z-disks. Recently, the two exceptionally large proteins titin (also called connectin) and nebulin (originally called band 3) have been implicated in passive elasticity (for review see refs 7, 8). Here, we show that after these proteins are degraded by low doses of ionizing radiation, the ability of single skinned muscle cells to generate both passive tension in response to stretch and active tension in response to calcium is greatly reduced. These effects are accompanied by axial misalignment of thick filaments. Titin and/or nebulin apparently provide axial continuity for the production of resting tension on stretch and also tend to keep the thick filaments centred within the sarcomere during force generation.

Animals↗
PubMed1997

Elasticity and unfolding of single molecules of the giant muscle protein titin.

The giant muscle protein titin, also called connectin, is responsible for the elasticity of relaxed striated muscle, as well as acting as the molecular scaffold for thick-filament formation. The titin molecule consists largely of tandem domains of the immunoglobulin and fibronectin-III types, together with specialized binding regions and a putative elastic region, the PEVK domain. We have done mechanical experiments on single molecules of titin to determine their visco-elastic properties, using an optical-tweezers technique. On a fast (0.1s) timescale titin is elastic and force-extension data can be fitted with standard random-coil polymer models, showing that there are two main sources of elasticity: one deriving from the entropy of straightening the molecule; the other consistent with extension of the polypeptide chain in the PEVK region. On a slower timescale and above a certain force threshold, the molecule displays stress-relaxation, which occurs in rapid steps of a few piconewtons, corresponding to yielding of internal structures by about 20 nm. This stress-relaxation probably derives from unfolding of immunoglobulin and fibronectin domains.

Connectin↗
PubMed1993

Effects of different enzymic treatments on the release of titin fragments from rabbit skeletal myofibrils. Purification of an 800 kDa titin polypeptide.

In myofibrils, titin (also called connectin) molecules span from Z line to M line and constitute a third filament system containing an elastic domain in the I band. This giant protein is particularly sensitive to proteolysis in situ. Treatment of rabbit skeletal myofibrils with exogenous proteinases induces a release of titin fragments, which are detected in the soluble myofibrillar fraction. The cleavage of titin occurs at specific points localized at the proximity of Z line and could lead to a concomitant release of alpha-actinin.

Animals↗
PubMed1997

Differential titin isoform expression in human skeletal muscle.

Mammalian skeletal muscle expresses at least two isoforms of the cytoskeletal protein titin (connectin; MW approximately 3000 kDa). These isoforms are associated with different passive force curves, and thus may affect physical performance. To study the distribution of titin and its possible influence on performance in humans, muscle biopsies were obtained from 15 males (mean +/- SE; age = 25.4 +/- 2.9 years, height = 177.7 +/- 1.8 cm, weight = 76.5 +/- 2.2 kg). Two biopsies were obtained on separate occasions from both the right and left vastus lateralis, and one biopsy each from the lateral head of the right gastrocnemius and the right soleus, with all biopsies handled identically. Fibre type analyses were performed via mATPase histochemistry. Expression of titin and myosin heavy chain isoforms were determined by SDS-PAGE. Titin bands in the resulting gels were highly repeatable and were verified by migration patterns, as well as Western blot analysis. Two groups of subjects were identified: group 1 (n = 10) expressed only one titin isoform (titin-1) in all biopsies, and group 2 (n = 5) expressed two titin isoforms (titin-1 and titin-2) in all biopsies. No significant differences (P > 0.05) between groups were observed for percentage fibre types, percentage fibre type areas, fibre type cross-sectional areas, and percentage myosin heavy chain expression when comparing individual muscles, sampling times or bilateral comparisons. This is the first report of differential titin isoform expression in healthy, mature human skeletal muscle, but it is not clear why this occurs or what influence this may have on performance.

Adult↗
PubMed2003

Calcium-dependent molecular spring elements in the giant protein titin.

Titin (also known as connectin) is a giant protein with a wide range of cellular functions, including providing muscle cells with elasticity. Its physiological extension is largely derived from the PEVK segment, rich in proline (P), glutamate (E), valine (V), and lysine (K) residues. We studied recombinant PEVK molecules containing the two conserved elements: approximately 28-residue PEVK repeats and E-rich motifs. Single molecule experiments revealed that calcium-induced conformational changes reduce the bending rigidity of the PEVK fragments, and site-directed mutagenesis identified four glutamate residues in the E-rich motif that was studied (exon 129), as critical for this process. Experiments with muscle fibers showed that titin-based tension is calcium responsive. We propose that the PEVK segment contains E-rich motifs that render titin a calcium-dependent molecular spring that adapts to the physiological state of the cell.

Amino Acid Motifs↗
PubMed1998

Titin extensibility in situ: entropic elasticity of permanently folded and permanently unfolded molecular segments.

Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293-296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres (approximately 2.0 micron) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from approximately 2.0 to 2.7 micron, the "contracted" tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond approximately 2.7 micron, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112-1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force-SL relation that is not achievable with a single elastic segment.

Chemical Phenomena↗
PubMed1997

An N-terminal fragment of titin coupled to green fluorescent protein localizes to the Z-bands in living muscle cells: overexpression leads to myofibril disassembly.

Cultures of nonmuscle cells, skeletal myotubes, and cardiomyocytes were transfected with a fusion construct (Z1.1GFP) consisting of a 1.1-kb cDNA (Z1.1) fragment from the Z-band region of titin linked to the cDNA for green fluorescent protein (GFP). The Z1.1 cDNA encodes only 362 amino acids of the approximately 2000 amino acids that make up the Z-band region of titin; nevertheless, the Z1.1GFP fusion protein targets the alpha-actinin-rich Z-bands of contracting myofibrils in vivo. This fluorescent fusion protein also localizes in the nascent and premyofibrils at the edges of spreading cardiomyocytes. Similarly, in transfected nonmuscle cells, the Z1.1GFP fusion protein localizes to the alpha-actinin-containing dense bodies of the stress fibers in vivo. A dominant negative phenotype was also observed in living cells expressing high levels of this Z1.1GFP fusion protein, with myofibril disassembly occurring as titin-GFP fragments accumulated. These data indicate that the Z-band region of titin plays an important role in maintaining and organizing the structure of the myofibril. The Z1.1 cDNA was derived from a chicken cardiac lambda gt11 expression library, screened with a zeugmatin antibody. Recent work has suggested that zeugmatin is actually part of the N-terminal region of the 81-kb titin cDNA. A reverse transcriptase polymerase chain reaction using a primer from the distal end (5' end) of the Z1.1 zeugmatin cDNA and a primer from the nearest known proximal (3' end) chicken titin (also called connectin) cDNA resulted in a predicted 0.3-kb polymerase chain reaction product linking the two known chicken titin cDNAs to each other. The linking region had a 79% identity at the amino acid level to human cardiac titin. This result and a Southern blot analysis of chicken genomic DNA hybridized with Z1.1 add further support to our original suggestion that zeugmatin is a proteolytic fragment from the N-terminal region of titin.

Amino Acid Sequence↗
PubMed1998

Molecular structure of the sarcomeric Z-disk: two types of titin interactions lead to an asymmetrical sorting of alpha-actinin.

The sarcomeric Z-disk, the anchoring plane of thin (actin) filaments, links titin (also called connectin) and actin filaments from opposing sarcomere halves in a lattice connected by alpha-actinin. We demonstrate by protein interaction analysis that two types of titin interactions are involved in the assembly of alpha-actinin into the Z-disk. Titin interacts via a single binding site with the two central spectrin-like repeats of the outermost pair of alpha-actinin molecules. In the central Z-disk, titin can interact with multiple alpha-actinin molecules via their C-terminal domains. These interactions allow the assembly of a ternary complex of titin, actin and alpha-actinin in vitro, and are expected to constrain the path of titin in the Z-disk. In thick skeletal muscle Z-disks, titin filaments cross over the Z-disk centre by approximately 30 nm, suggesting that their alpha-actinin-binding sites overlap in an antiparallel fashion. The combination of our biochemical and ultrastructural data now allows a molecular model of the sarcomeric Z-disk, where overlapping titin filaments and their interactions with the alpha-actinin rod and C-terminal domain can account for the essential ultrastructural features.

Actin Cytoskeleton↗
PubMed1986

A comparative study of high molecular weight proteins in various types of muscle across the animal kingdom.

A wide range of phyla have been surveyed by SDS-PAGE for the new large proteins of the myofibril. Connectin (or titin) appears to be widely distributed. It is seen as a band of constant intensity and mobility in vertebrate striated muscle, but is absent from smooth muscle. It appears in more variable amounts, in a form of constant but greater mobility in many invertebrates: worms, molluscs (adductor but not gastropod feet), insects, a myriapod, and even in human blood platelets. Nebulin shares the same distribution in vertebrate muscles except for its notable absence in all heart muscle examined. It too is found in many invertebrates, not always with titin. It has been found in a worm, molluscs (adductor and gastropod feet), insects, crustaceans and an echinoderm. The mobility of nebulin varies within the vertebrates and more so between invertebrates (where, as with titin, it is greater). The isoforms of filamin in skeletal, cardiac, and smooth muscles of vertebrates are recorded. C-protein in rabbit muscles has four isoforms: white, alpha-red (X-protein), beta-red, and cardiac.

Animals↗