Unfolding of titin immunoglobulin domains by steered molecular dynamics simulation.

Titin, a 1-microm-long protein found in striated muscle myofibrils, possesses unique elastic and extensibility properties in its I-band region, which is largely composed of a PEVK region (70% proline, glutamic acid, valine, and lysine residue) and seven-strand beta-sandwich immunoglobulin-like (Ig) domains. The behavior of titin as a multistage entropic spring has been shown in atomic force microscope and optical tweezer experiments to partially depend on the reversible unfolding of individual Ig domains. We performed steered molecular dynamics simulations to stretch single titin Ig domains in solution with pulling speeds of 0.5 and 1.0 A/ps. Resulting force-extension profiles exhibit a single dominant peak for each Ig domain unfolding, consistent with the experimentally observed sequential, as opposed to concerted, unfolding of Ig domains under external stretching forces. This force peak can be attributed to an initial burst of backbone hydrogen bonds, which takes place between antiparallel beta-strands A and B and between parallel beta-strands A' and G. Additional features of the simulations, including the position of the force peak and relative unfolding resistance of different Ig domains, can be related to experimental observations.     

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.     

Molecular evolution of immunoglobulin and fibronectin domains in titin and related muscle proteins.

The family of regulatory and structural muscle proteins, which includes the giant kinases titin, twitchin and projectin, has sequences composed predominantly of serially linked immunoglobulin I set (Ig) and fibronectin type III (FN3) domains. This paper explores the evolutionary relationships between 16 members of this family. In titin, groups of Ig and FN3 domains are arranged in a regularly repeating pattern of seven and 11 domains. The 11-domain super-repeat has its origins in the seven-domain super-repeat and a model for the duplications which gave rise to this super-repeat is proposed. A super-repeat composed solely of immunoglobulin domains is found in the skeletal muscle isoform of titin. Twitchin and projectin, which are presumed to be orthologs, have undergone significant insertion/deletion of domains since their divergence. The common ancestry of myomesin, skelemin and M-protein is shown. The relationship between myosin binding proteins (MyBPs) C and H is confirmed, and MyBP-H is proposed to have given rise to MyBP-C by the acquisition of some titin domains.     

Mechanical fatigue in repetitively stretched single molecules of titin

Relaxed striated muscle cells exhibit mechanical fatigue when exposed to repeated stretch and release cycles. To understand the molecular basis of such mechanical fatigue, single molecules of the giant filamentous protein titin, which is the main determinant of sarcomeric elasticity, were repetitively stretched and released while their force response was characterized with optical tweezers. During repeated stretch-release cycles titin becomes mechanically worn out in a process we call molecular fatigue. The process is characterized by a progressive shift of the stretch-force curve toward increasing end-to-end lengths, indicating that repeated mechanical cycles increase titin's effective contour length. Molecular fatigue occurs only in a restricted force range (0-25 pN) during the initial part of the stretch half-cycle, whereas the rest of the force response is repeated from one mechanical cycle to the other. Protein-folding models fail to explain molecular fatigue on the basis of an incomplete refolding of titin's globular domains. Rather, the process apparently derives from the formation of labile nonspecific bonds cross-linking various sites along a pre-unfolded titin segment. Because titin's molecular fatigue occurs in a physiologically relevant force range, the process may play an important role in dynamically adjusting muscle's response to the recent history of mechanical perturbations.     

The hydrophilic domain of small ankyrin-1 interacts with the two N-terminal immunoglobulin domains of titin

Little is known about the mechanisms that organize the internal membrane systems in eukaryotic cells. We are addressing this question in striated muscle, which contains two novel systems of internal membranes, the transverse tubules and the sarcoplasmic reticulum (SR). Small ankyrin-1 (sAnk1) is an approximately 17-kDa transmembrane protein of the SR that concentrates around the Z-disks and M-lines of each sarcomere. We used the yeast two-hybrid assay to determine whether sAnk1 interacts with titin, a giant myofibrillar protein that organizes the sarcomere. We found that the hydrophilic cytoplasmic domain of sAnk1 interacted with the two most N-terminal Ig domains of titin, ZIg1 and ZIg2, which are present at the Z-line in situ. Both ZIg1 and ZIg2 were required for binding activity. sAnk1 did not interact with other sequences of titin that span the Z-disk or with Ig domains of titin near the M-line. Titin ZIg1/2 also bound T-cap/telethonin, a 19-kDa protein of the Z-line. We show that titin ZIg1/2 could form a three-way complex with sAnk1 and T-cap. Our results indicate that titin ZIg1/2 can bind sAnk1 in muscle homogenates and suggest a role for these proteins in organizing the SR around the contractile apparatus at the Z-line.     

Specific titin and myomesin domains stimulate myoblast proliferation

Myofibrillar proteins titin and myomesin stimulated myoblast proliferation as determined by MTT-test and labelled thymidine incorporation in the DNA. Specific Fn type III and Ig-like domains of these proteins were able to exert mitogenic effects as well. Proliferative effect of Fn type III domains was highly sensitive to inhibition of Ca²⁺/calmodulin dependent protein kinase, whereas the effect of Ig-like domains showed greater sensitivity to the inhibition of adenylyl cyclase – cAMP – PKA pathway. IGF-1 autocrine signalling inhibition partially suppressed mitogenic effects revealed by both domain types.     

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).     

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

Abstract

Communicated by Ramaswamy H. Sarma     

Independent refolding of domains in the pancreatic serine proteinases

Ser-neotrypsinogen and Val-neotrypsinogen are two-chain modifications of bovine trypsinogen produced on limited proteolysis with trypsin. Ser-neotrypsinogen has Lys131-Ser132 cleaved in the connecting peptide (the autolysis loop) linking the amino- and carboxyl-terminal domains. Val-neotrypsinogen has Arg105-Val106 cleaved which is located within the amino-terminal domain. The mixed disulfide derivative of Ser-neotrypsinogen was successfully refolded. A functional molecule was regenerated from the polypeptide fragments with the correct molecular weight of neotrypsinogen in an overall yield of 7%. Val-Neotrypsinogen could not be refolded. The first-order rate constants for the regeneration of Ser-neotrypsinogen were determined from the formation of active enzyme molecules as a function of time and from the regain of the correct molecular weight. Both kinetic values were the same indicating that refolding of the polypeptide chains first forms globular domain structures. The two domains then associate and the disulfide bonds between the domains and the correct geometry of the active site residues are formed last. The same kinetic results were also found in refolding Thr-neochymotrypsinogen (Duda, C. T., and Light, A. (1982) J. Biol. Chem. 257, 9866-9871) where peptide bond cleavage also occurred in the connecting peptide. These observations support the hypothesis that the pathway of folding of serine proteinases proceeds with the independent refolding of domains.     

Unfolding of titin domains explains the viscoelastic behavior of skeletal myofibrils

The elastic section of the giant muscle protein titin contains many immunoglobulin-like domains, which have been shown by single-molecule mechanical studies to unfold and refold upon stretch-release. Here we asked whether the mechanical properties of Ig domains and/or other titin regions could be responsible for the viscoelasticity of nonactivated skeletal-muscle sarcomeres, particularly for stress relaxation and force hysteresis. We show that isolated psoas myofibrils respond to a stretch-hold protocol with a characteristic force decay that becomes more pronounced following stretch to above 2.6-microm sarcomere length. The force decay was readily reproducible by a Monte Carlo simulation taking into account both the kinetics of Ig-domain unfolding and the worm-like-chain model of entropic elasticity used to describe titin's elastic behavior. The modeling indicated that the force decay is explainable by the unfolding of only a very small number of Ig domains per titin molecule. The simulation also predicted that a unique sequence in titin, the PEVK domain, may undergo minor structural changes during sarcomere extension. Myofibrils subjected to 1-Hz cycles of stretch-release exhibited distinct hysteresis that persisted during repetitive measurements. Quick stretch-release protocols, in which variable pauses were introduced after the release, revealed a two-exponential time course of hysteresis recovery. The rate constants of recovery compared well with the refolding rates of Ig-like or fibronectin-like domains measured by single-protein mechanical analysis. These findings suggest that in the sarcomere, titin's Ig-domain regions may act as entropic springs capable of adjusting their contour length in response to a stretch.     

A kinetic molecular model of the reversible unfolding and refolding of titin under force extension.

Molecular elasticity is a physicomechanical property that is associated with a select number of polypeptides and proteins, such as the giant muscle protein, titin, and the extracellular matrix protein, tenascin. Both proteins have been the subject of atomic force microscopy (AFM), laser tweezer, and other in vitro methods for examining the effects of force extension on the globular (FNIII/Ig-like) domains that comprise each protein. In this report we present a time-dependent method for simulating AFM force extension and its effect on FNIII/Ig domain unfolding and refolding. This method treats the unfolding and refolding process as a standard three-state protein folding model (U right arrow over left arrow T right arrow over left arrow F, where U is the unfolded state, T is the transition or intermediate state, and F is the fully folded state), and integrates this approach within the wormlike chain (WLC) concept. We simulated the effect of AFM tip extension on a hypothetical titin molecule comprised of 30 globular domains (Ig or FNIII) and 25% Pro-Glu-Val-Lys (PEVK) content, and analyzed the unfolding and refolding processes as a function of AFM tip extension, extension rate, and variation in PEVK content. In general, we find that the use of a three-state protein-folding kinetic-based model and the implicit inclusion of PEVK domains can accurately reproduce the experimental force-extension curves observed for both titin and tenascin proteins. Furthermore, our simulation data indicate that PEVK domains exhibit extensibility behavior, assist in the unfolding and refolding of FNIII/Ig domains in the titin molecule, and act as a force "buffer" for the FNIII/Ig domains, particularly at low and moderate extension forces.     

Origins of immunoglobulin heavy chain domains

Summary Using computer programs that analyze the evolutionary history and probability of relationship of protein sequences, we have investigated the gene duplication events that led to the present configuration of immunoglobulin C regions, with particular attention to the origins of the homology regions (domains) of the heavy chains. We conclude that all of the sequenced heavy chains share a common ancestor consisting of four domains and that the two shorter heavy chains, alpha and gamma, have independently lost most of the second domain. These conclusions allow us to align corresponding regions of these sequences for the purpose of deriving evolutionary trees. Three independent internal gene duplications are postulated to explain the observed pattern of relationships among the four domains: first a duplication of the ancestral single domain C region, followed by independent duplications of the resulting first and last domains. In these studies there was no evidence of crossing-over and recombination between ancestral chains of different classes; however, certain types of recombinations would not be detectable from the available sequence data.     

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.     

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.     

Altered mechanical properties of titin immunoglobulin domain 27 in the presence of calcium

Titin (connectin) based passive force regulation has been an important physiological mechanism to adjust to varying muscle stretch conditions. Upon stretch, titin behaves as a spring capable of modulating its elastic response in accordance with changes in muscle biochemistry. One such mechanism has been the calcium-dependent stiffening of titin domains that renders the spring inherently more resistant to stretch. This transient titin-calcium interaction may serve a protective function in muscle, which could preclude costly unfolding of select domains when muscles elongate to great lengths. To test this idea, fluorescence spectroscopy was performed revealing a change in the microenvironment of the investigated immunoglobulin domain 27 (I27) of titin with calcium. Additionally, an atomic force microscope was used to evaluate the calcium-dependent regulation of passive force by stretching eight linked titin I27 domains until they unfolded. When stretching in the presence of calcium, the I27 homopolymer chain became stabilized, displaying three novel properties: (1) higher stretching forces were needed to unfold the domains, (2) the stiffness, measured as a persistence length (PL), increased and (3) the peak-to-peak distance between adjacent I27 domains increased. Furthermore, a peak order dependence became apparent for both force and PL, reflecting the importance of characterizing the dynamic unfolding history of a polymer with this approach. Together, this novel titin Ig-calcium interaction may serve to stabilize the I27 domain permitting titin to tune passive force within stretched muscle in a calcium-dependent manner.     

Salt-induced refolding in different domains of partially folded bovine serum albumin

In our earlier communication on urea denaturation of bovine serum albumin (BSA), we showed significant unfolding of domain III along with domain I prior to intermediate formation around 4.6-5.2 M urea based on the binding results of domain specific ligands:chloroform, bilirubin and diazepam for domains I, II and III, respectively. Here, we present our results on the salt-induced refolding of the two partially folded states of BSA obtained at 4.5 M urea and at pH 3.5, respectively. Both these states were characterized by significant unfolding of both domains I and III as indicated by decreased binding of chloroform and diazepam, respectively. Salt-induced stabilization of partially folded states of BSA was accompanied by nearly complete refolding of both domains I and III as the binding isotherms of chloroform and diazepam obtained in presence of approximately 1.0 M KCl were nearly identical to that obtained with native BSA at pH 7.4. From these observations, it can be concluded that the anion binding sites on serum albumin are not only confined to domain III (C-terminal region) but few sites are also present on domain I (or N-terminal region) of the protein.     

Unfolding and refolding of the constant fragment of the immunoglobulin light chain

The kinetics of reversible unfolding and refolding by guanidine hydrochloride of the constant fragment of the immunoglobulin light chain are described. The kinetic measurements were made at pH 7.5 and 25 °C using tryptophyl fluorescence and farultraviolet circular dichroism.The kinetics of unfolding of the constant fragment showed two phases in the conformational transition zone and a single phase above the transition zone. A double-jump experiment confirmed the presence of two forms of the unfolded molecule. These results were thoroughly explained on the basis of the three-species mechanism, U₁

U₂

N, where U₁ and U₂ are the slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. The equilibrium constant for the process of U₂ to U₁ was estimated to be about 10 and was independent of the conditions of denaturation. These findings were consistent with the view that the U₁

U₂ reaction is proline isomerization. The rates of interconversion between N and U₂ changed greatly with the concentration of guanidine hydrochloride. On the other hand, the refolding kinetics below the transition zone showed behavior unexpected from the three-species mechanism. Whereas the apparent rate constant of the slow phase of refolding was independent of the refolding conditions, its amplitude decreased markedly with the decrease in the final concentration of guanidine hydrochloride. On the basis of this and other results, formation of an intermediate during refolding was ascertained and the refolding kinetics were consistently explained in terms of a more general mechanism involving a kinetic intermediate probably containing non-native proline isomers. The intermediate seemed to have a folded conformation similar to native protein. Comparison of the refolding kinetics of the constant fragment with those of other domains of the immunoglobulin molecule suggested that Pro143 is responsible for the appearance of the slow phase.