Simulated refolding of stretched titin immunoglobulin domains

Steered molecular dynamics (SMD) is used to investigate forced unfolding and spontaneous refolding of immunoglobulin I27, a domain of the muscle protein titin. Previous SMD simulations revealed the events leading to stretch-induced unfolding of I27, the rupture of hydrogen bonds bridging beta-strands A and B, and those bridging beta-strands A' and G, the latter rupture occurring at an extension of approximately 15 A and preceding the complete unfolding. Simulations are now used to study the refolding of partially unfolded I27 domains. The results reveal that stretched domains with ruptured interstrand hydrogen bonds shrink along the extension direction. Two types of refolding patterns are recognized: for separated beta-strands A' and G, in most simulations five of the six hydrogen bonds between A' and G stably reformed in 2 ns, whereas for separated beta-strands A and B hydrogen bonds seldom reformed in eight 2-ns simulations. The mechanical stability of the partially refolded intermediates has been tested by re-stretching.     

Relation of nebulin and connectin (titin) to dynamics of actin in nascent myofibrils of cultured skeletal muscle cells.

Cultured embryonic chicken skeletal muscle cells microinjected with rhodamine (rh)-labeled actin were stained with antibodies against nebulin and connectin (titin). In premyofibril areas, nebulin was observed as dotted structures, many of which were arranged in a linear fashion. These structures were associated with injected rh-actin. Among these linearly arranged dots of nebulin and rh-actin, numerous small nebulin dots without rh-actin incorporation were scattered. It is probable that the dots of nebulin and/or its associated protein(s) represent a preformed scaffold upon which actin monomers accumulate; exogenously introduced actin associates initially with small nebulin dots, which in turn coalesce to form rh-actin dots and are arranged linearly. In developing myofibrils, two patterns of nebulin distribution were found: "singlets" and "doublets." Recovery of rh-actin's fluorescence after photobleaching was slowest in the nonstriated dotted portions, followed by the striated myofibrillar portions with nebulin singlets and those with doublets, in that order. Thus, the distribution patterns of nebulin seem to be related to the accessibility/exchangeability of actin into nascent myofibrils. It is possible that early nebulin filaments exhibiting singlets are not tightly associated with actin filaments and that this loose association allows myofibrils to exchange nonadult isoforms of actin and other proteins into adult types. Connectin formed a striated pattern before the formation of rh-actin/nebulin striations. It appears that connectin does not have any significant role in the accessibility of actin into nascent myofibrils.     

Synchronous behavior of spontaneous oscillations of sarcomeres in skeletal myofibrils under isotonic conditions.

An isotonic control system for studying dynamic properties of single myofibrils was developed to evaluate the change of sarcomere lengths in glycerinated skeletal myofibrils under conditions of spontaneous oscillatory contraction (SPOC) in the presence of inorganic phosphate and a high ADP-to-ATP ratio. Sarcomere length oscillated spontaneously with a peak-to-peak amplitude of about 0.5 microns under isotonic conditions in which the external loads were maintained constant at values between 1.5 x 10(4) and 3.5 x 10(4) N/m2. The shortening and yielding of sarcomeres occurred in concert, in contrast to the previously reported conditions (isomeric or auxotonic) under which the myofibrillar tension is allowed to oscillate. This synchronous SPOC appears to be at a higher level of synchrony than in the organized state of SPOC previously observed under auxotonic conditions. The period of sarcomere length oscillation did not largely depend on external load. The active tension under SPOC conditions increased as the sarcomere length increased from 2.1 to 3.2 microns, although it was still smaller than the tension under normal Ca2+ contraction (which is on the order of 10(5) N/m2). The synchronous SPOC implies that there is a mechanism for transmitting information between sarcomeres such that the state of activation of sarcomeres is affected by the state of adjacent sarcomeres. We conclude that the change of myofibrillar tension is not responsible for the SPOC of each sarcomere but that it affects the level of synchrony of sarcomere oscillations.     

Mechanical characterization of skeletal muscle myofibrils.

A new instrument, based on a technique described previously, is presented for studying mechanics of micron-scale preparations of two to three myofibrils or single myofibrils from muscle. Forces in the nanonewton to micronewton range are measurable with 0.5-ms time resolution. Programmed quick (200-microseconds) steps or ramp length changes are applied to contracting myofibrils to test their mechanical properties. Individual striations can be monitored during force production and shortening. The active isometric force, force-velocity relationship, and force transients after rapid length steps were obtained from bundles of two to three myofibrils from rabbit psoas muscle. Contrary to some earlier reports on myofibrillar mechanics, these properties are generally similar to expectations from studies on intact and skinned muscle fibers. Our experiments provide strong evidence that the mechanical properties of a fiber result from a simple summation of the myofibrillar force and shortening of independently contracting sarcomeres.     

Ligand-binding alters the mechanical stability of muscle protein titin domains

Abstract

Communicated by Ramaswamy H. Sarma     

Birth and death of protein domains: A simple model of evolution explains power law behavior

Background  

Power distributions appear in numerous biological, physical and other contexts, which appear to be fundamentally different. In biology, power laws have been claimed to describe the distributions of the connections of enzymes and metabolites in metabolic networks, the number of interactions partners of a given protein, the number of members in paralogous families, and other quantities. In network analysis, power laws imply evolution of the network with preferential attachment, i.e. a greater likelihood of nodes being added to pre-existing hubs. Exploration of different types of evolutionary models in an attempt to determine which of them lead to power law distributions has the potential of revealing non-trivial aspects of genome evolution.     

Nonlinear force-length relationship in the ADP-induced contraction of skeletal myofibrils

The regulatory mechanism of sarcomeric activity has not been fully clarified yet because of its complex and cooperative nature, which involves both Ca²⁺ and cross-bridge binding to the thin filament. To reveal the mechanism of regulation mediated by the cross-bridges, separately from the effect of Ca²⁺, we investigated the force-sarcomere length (SL) relationship in rabbit skeletal myofibrils (a single myofibril or a thin bundle) at SL > 2.2 μm in the absence of Ca²⁺ at various levels of activation by exogenous MgADP (4-20 mM) in the presence of 1 mM MgATP. The individual SLs were measured by phase-contrast microscopy to confirm the homogeneity of the striation pattern of sarcomeres during activation. We found that at partial activation with 4-8 mM MgADP, the developed force nonlinearly depended on the length of overlap between the thick and the thin filaments; that is, contrary to the maximal activation, the maximal active force was generated at shorter overlap. Besides, the active force became larger, whereas this nonlinearity tended to weaken, with either an increase in [MgADP] or the lateral osmotic compression of the myofilament lattice induced by the addition of a macromolecular compound, dextran T-500. The model analysis, which takes into account the [MgADP]-and the lattice-spacing-dependent probability of cross-bridge formation, was successfully applied to account for the force-SL relationship observed at partial activation. These results strongly suggest that the cross-bridge works as a cooperative activator, the function of which is highly sensitive to as little as ≤1 nm changes in the lattice spacing.     

Biosynthesis of titin in cultured skeletal muscle cells

Although significant progress has been made regarding the structure and function of titin, little data exist on the biosynthesis of this large protein in developing muscle. Using pulse-labeling with [35S]methionine and immunoprecipitation with an anti-titin mAb, we have examined the biosynthesis of titin in synchronized cultures of skeletal muscle cells derived from day 12 chicken embryos. We find that: (a) titin synthesis increases greater than 4-fold during the first week in culture and during this same time period, synthesis of muscle-specific myosin heavy chain increases greater than 12-fold; (b) newly synthesized titin has a t1/2 of approximately 70 h; (c) titin is resistant to extraction with Triton X-100 both during and immediately after its synthesis. These observations suggest that newly synthesized titin molecules are stable proteins that rapidly associate with the cytoskeleton of developing myotubes.     

Unfolding domains of recombinant fusion alpha alpha-tropomyosin.

The thermal unfolding of the coiled-coil alpha-helix of recombinant alpha alpha-tropomyosin from rat striated muscle containing an additional 80-residue peptide of influenza virus NS1 protein at the N-terminus (fusion-tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion-tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 degrees C involving the middle of the molecule; (2) a major transition at 46 degrees C involving no more than 36% of the helix from the C-terminus; (3) a major transition at 56 degrees C involving about 46% of the helix from the N-terminus; and (4) a transition from the nonhelical fusion domain at about 70 degrees C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 degrees C or 70 degrees C transition. The very stable fusion unfolding domain of fusion-tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a cross-link to stabilize the adjacent N-terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C-terminal domain at 46 degrees C from the stabilized N-terminal unfolding domain at 56 degrees C. Thus, strong localized interchain interactions in coiled-coil molecules can increase the stability of neighboring domains.     

Cross-bridge duty cycle in isometric contraction of skeletal myofibrils

During interaction of actin with myosin, cross-bridges impart mechanical impulses to thin filaments resulting in rotations of actin monomers. Impulses are delivered on the average every tc seconds. A cross-bridge spends a fraction of this time (ts) strongly attached to actin, during which it generates force. The "duty cycle" (DC), defined as the fraction of the total cross-bridge cycle that myosin spends attached to actin in a force generating state (ts/ tc), is small for cross-bridges acting against zero load, like freely shortening muscle, and increases as the load rises. Here we report, for the first time, an attempt to measure DC of a single cross-bridge in muscle. A single actin molecule in a half-sarcomere was labeled with fluorescent phalloidin. Its orientation was measured by monitoring intensity of the polarized TIRF images. Actin changed orientation when a cross-bridge bound to it. During isometric contraction, but not during rigor, actin orientation oscillated between two values, corresponding to the actin-bound and actin-free state of the cross-bridge. The average ts and tc were 3.4 and 6 s, respectively. These results suggest that, in isometrically working muscle, cross-bridges spend about half of the cycle time attached to actin. The fact that 1/ tc was much smaller than the ATPase rate suggests that the bulk of the energy of ATP hydrolysis is used for purposes other than performance of mechanical work.     

Extra actin filaments at the periphery of skeletal muscle myofibrils.

Myofibrils isolated from a variety of vertebrate muscle fibers have a set of peripheral filaments associated with the periphery of the Z line free to move away from the surface of the myofibril. Decoration with myosin subfragment 1 shows that these are actin filaments.     

Stability and folding rates of domains spanning the large A-band super-repeat of titin

Titin is a very large (>3 MDa) protein found in striated muscle where it is believed to participate in myogenesis and passive tension. A prominent feature in the A-band portion of titin is the presence of an 11-domain super-repeat of immunoglobulin superfamily and fibronectin-type-III-like domains. Seven overlapping constructs from human cardiac titin, each consisting of two or three domains and together spanning the entire 11-domain super-repeat, have been expressed in Escherichia coli. Fluorescence unfolding experiments and circular dichroism spectroscopy have been used to measure folding stabilities for each of the constructs and to assign unfolding rates for each super-repeat domain. Immunoglobulin superfamily domains were found to fold correctly only in the presence of their C-terminal fibronectin type II domain, suggesting close and possibly rigid association between these units. The domain stabilities, which range from 8.6 to 42 kJ mol(-1) under physiological conditions, correlate with previously reported mechanical forces required to unfold titin domains. Individual domains vary greatly in their rates of unfolding, with a range of unfolding rate constants between 2.6 x 10(-6) and 1.2 s(-1). This variation in folding behavior is likely to be an important determinant in ensuring independent folding of domains in multi-domain proteins such as titin.     

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.     

Folding-unfolding of immunoglobulin domains in titin: a simple two-state model

The folding-unfolding reaction rate process in the giant protein titin is studied within a simple two-state model. The molecule is assumed to be stretched by an external force which modulates the potential barrier associated with the folded state. A two-state model for this process is assumed (i.e., the immunoglobulin domains are considered to be either folded or unfolded, with no intermediate states at all). Simple calculations yield a relation between the force and the pulling speed that agrees fairly well with data from experiments and Monte Carlo simulations performed recently. Moreover, in a regime involving ultrafast pulling, the results show that the detailed form of the potential barrier is irrelevant, a conclusion that agrees with the current theoretical work on molecular dynamics.     

Alteration of actin-tropomyosin interaction in 2,4-pentanedione-treated rabbit skeletal myofibrils

In previous work, we (El-Saleh, S., Theiret, R., Johnson, P., and Potter, J. D. (1984) J. Biol. Chem. 259, 11014-11021) presented evidence that Ca2+ activation of skeletal myofilaments depends on a specific actin domain. We showed that rabbit skeletal thin filaments reconstituted with actin modified at Lys-237 activate heavy meromyosin X Mg2+-ATPase activity independently of the Ca2+ ion concentration. The modification, which apparently blocks the inhibitory effects of troponin-tropomyosin (Tn X Tm), on acto-heavy meromyosin X Mg2+-ATPase activity, consisted of conversion of Lys-237 to an enamine by reaction of purified actin with 2,4-pentanedione (PD). In experiments reported here, we have treated myofibrils with PD with the idea of altering actin in its native state within the myofilament lattice. Preparations of native and Tn X Tm free ("desensitized") myofibrils were incubated with PD (100 mol/mol of actin lysine) under rigorous conditions (10 mM 4-morpholinepropanesulfonic acid, pH 7.0, 2.0 nM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.4 mM dithiothreitol, and 0.15 mM NaN3). Actin isolated from PD X myofibrils contained 0.5 mol of enamine/mol. In the presence of Ca2+, the Mg2+-ATPase activity of PD-treated myofibrils was 110-120% of the maximum Ca2+-stimulated Mg2+-ATPase activity of untreated control myofibrils. In low free Ca2+ (pCa greater than 8), the Mg2+-ATPase activity of the PD-treated myofibrils was not suppressed and remained at 100-106% of the maximum activity of the control myofibrils. Ca2+ sensitivity of the PD-treated myofibrils was restored following treatment with hydroxylamine, which hydrolyzes enamine's products. Preparations of desensitized myofibrils reconstituted with PD-modified or unmodified Tn X Tm demonstrated the same Ca2+-sensitive ATPase activities. On the other hand, preparations reconstituted with unmodified or PD-modified Tn X Tm and PD-modified desensitized myofibrils were insensitive to Ca2+ ion concentration. The Mg2+-ATPase activity of preparations of myosin treated with PD was not activated by modified or unmodified actin. Our results indicate that is is possible to produce an active state(s) of the myofibrils in the absence and presence of Ca2+ by specific alteration of the actin X Tm interaction following modification of myofibrillar actin most likely at Lys-237.     

Adaptive behavior of titin isoforms from skeletal and cardiac muscles of ground squirrels (Citellus undulatus) during hibernation

By the use of modified SDS electrophoresis in agarose-strengthened 2% polyacrylamide gels, the adaptive behavior of titin isoforms in skeletal and cardiac muscles of ground squirrels (Citellus undulatus) during hibernation was studied. The presence of two titin isoforms (short and long) with molecular weights approximately 3700 and approximately 3800 kDa in m. soleus, approximately 3400 and approximately 3600 kDa in m. psoas, approximately 3000 and approximately 3400 kDa in the left ventricle of myocardium was found. It was found that the content of the short titin isoform in the above muscles of hibernating and arousing ground squirrels is considerably lower than that of the long titin isoform. The preservation of the long titin isoform in skeletal and cardiac muscles of hibernating and arousing ground squirrels can be regarded as an evolutionarily determined adaptive mechanism favoring the survival of animal under extreme conditions without pathological consequences.