Steered molecular dynamics studies of titin I1 domain unfolding

The cardiac muscle protein titin, responsible for developing passive elasticity and extensibility of muscle, possesses about 40 immunoglobulin-like (Ig) domains in its I-band region. Atomic force microscopy (AFM) and steered molecular dynamics (SMD) have been successfully combined to investigate the reversible unfolding of individual Ig domains. However, previous SMD studies of titin I-band modules have been restricted to I27, the only structurally known Ig domain from the distal region of the titin I-band. In this paper we report SMD simulations unfolding I1, the first structurally available Ig domain from the proximal region of the titin I-band. The simulations are carried out with a view toward upcoming atomic force microscopy experiments. Both constant velocity and constant force stretching have been employed to model mechanical unfolding of oxidized I1, which has a disulfide bond bridging beta-strands C and E, as well as reduced I1, in which the disulfide bridge is absent. The simulations reveal that I1 is protected against external stress mainly through six interstrand hydrogen bonds between its A and B beta-strands. The disulfide bond enhances the mechanical stability of oxidized I1 domains by restricting the rupture of backbone hydrogen bonds between the A'- and G-strands. The disulfide bond also limits the maximum extension of I1 to approximately 220 A. Comparison of the unfolding pathways of I1 and I27 are provided and implications to AFM experiments are discussed.     

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.     

Stretching molecular springs: elasticity of titin filaments in vertebrate striated muscle

Titin, the giant protein of striated muscle, provides a continuous link between the Z-disk and the M-line of a sarcomere. The elastic I-band section of titin comprises two main structural elements, stretches of immunoglobulin-like domains and a unique sequence, the PEVK segment. Both elements contribute to the extensibility and passive force development of nonactivated muscle. Extensibility of the titin segments in skeletal muscle has been determined by immunofluorescence/immunoelectron microscopy of sarcomeres stained with sequence-assigned titin antibodies. The force developed upon stretch of titin has been measured on isolated molecules or recombinant titin fragments with the help of optical tweezers and the atomic force microscope. Force has also been measured in single isolated myofibrils. The force-extension relation of titin could be readily fitted with models of biopolymer elasticity. For physiologically relevant extensions, the elasticity of the titin segments was largely explainable by an entropic-spring mechanism. The modelling explains why during stretch of titin, the Ig-domain regions (with folded modules) extend before the PEVK domain. In cardiac muscle, I-band titin is expressed in different isoforms, termed N2-A and N2-B. The N2-A isoform resembles that of skeletal muscle, whereas N2-B titin is shorter and is distinguished by cardiac-specific Ig-motifs and nonmodular sequences within the central I-band section. Examination of N2-B titin extensibility revealed that this isoform extends by recruiting three distinct elastic elements: poly-Ig regions and the PEVK domain at lower stretch and, in addition, a unique 572-residue sequence insertion at higher physiological stretch. Extension of all three elements allows cardiac titin to stretch fully reversibly at physiological sarcomere lengths, without the need to unfold individual Ig domains. However, unfolding of a very small number of Ig domains remains a possibility.     

Mechanics and structure of titin oligomers explored with atomic force microscopy

Titin is a giant polypeptide that spans half of the striated muscle sarcomere and generates passive force upon stretch. To explore the elastic response and structure of single molecules and oligomers of titin, we carried out molecular force spectroscopy and atomic force microscopy (AFM) on purified full-length skeletal-muscle titin. From the force data, apparent persistence lengths as long as approximately 1.5 nm were obtained for the single, unfolded titin molecule. Furthermore, data suggest that titin molecules may globally associate into oligomers which mechanically behave as independent wormlike chains (WLCs). Consistent with this, AFM of surface-adsorbed titin molecules revealed the presence of oligomers. Although oligomers may form globally via head-to-head association of titin, the constituent molecules otherwise appear independent from each other along their contour. Based on the global association but local independence of titin molecules, we discuss a mechanical model of the sarcomere in which titin molecules with different contour lengths, corresponding to different isoforms, are held in a lattice. The net force response of aligned titin molecules is determined by the persistence length of the tandemly arranged, different WLC components of the individual molecules, the ratio of their overall contour lengths, and by domain unfolding events. Biased domain unfolding in mechanically selected constituent molecules may serve as a compensatory mechanism for contour- and persistence-length differences. Variation in the ratio and contour length of the component chains may provide mechanisms for the fine-tuning of the sarcomeric passive force response.     

Individual Globular Domains and Domain Unfolding Visualized in Overstretched Titin Molecules with Atomic Force Microscopy

Titin is a giant elastomeric protein responsible for the generation of passive muscle force. Mechanical force unfolds titin’s globular domains, but the exact structure of the overstretched titin molecule is not known. Here we analyzed, by using high-resolution atomic force microscopy, the structure of titin molecules overstretched with receding meniscus. The axial contour of the molecules was interrupted by topographical gaps with a mean width of 27.7 nm that corresponds well to the length of an unfolded globular (immunoglobulin and fibronectin) domain. The wide gap-width distribution suggests, however, that additional mechanisms such as partial domain unfolding and the unfolding of neighboring domain multimers may also be present. In the folded regions we resolved globules with an average spacing of 5.9 nm, which is consistent with a titin chain composed globular domains with extended interdomain linker regions. Topographical analysis allowed us to allocate the most distal unfolded titin region to the kinase domain, suggesting that this domain systematically unfolds when the molecule is exposed to overstretching forces. The observations support the prediction that upon the action of stretching forces the N-terminal ß-sheet of the titin kinase unfolds, thus exposing the enzyme’s ATP-binding site and hence contributing to the molecule’s mechanosensory function.     

Mechanics and structure of titin oligomers explored with atomic force microscopy

Titin is a giant polypeptide that spans half of the striated muscle sarcomere and generates passive force upon stretch. To explore the elastic response and structure of single molecules and oligomers of titin, we carried out molecular force spectroscopy and atomic force microscopy (AFM) on purified full-length skeletal-muscle titin. From the force data, apparent persistence lengths as long as ∼1.5 nm were obtained for the single, unfolded titin molecule. Furthermore, data suggest that titin molecules may globally associate into oligomers which mechanically behave as independent wormlike chains (WLCs). Consistent with this, AFM of surface-adsorbed titin molecules revealed the presence of oligomers. Although oligomers may form globally via head-to-head association of titin, the constituent molecules otherwise appear independent from each other along their contour. Based on the global association but local independence of titin molecules, we discuss a mechanical model of the sarcomere in which titin molecules with different contour lengths, corresponding to different isoforms, are held in a lattice. The net force response of aligned titin molecules is determined by the persistence length of the tandemly arranged, different WLC components of the individual molecules, the ratio of their overall contour lengths, and by domain unfolding events. Biased domain unfolding in mechanically selected constituent molecules may serve as a compensatory mechanism for contour- and persistence-length differences. Variation in the ratio and contour length of the component chains may provide mechanisms for the fine-tuning of the sarcomeric passive force response.     

Human myocytes are protected from titin aggregation-induced stiffening by small heat shock proteins

In myocytes, small heat shock proteins (sHSPs) are preferentially translocated under stress to the sarcomeres. The functional implications of this translocation are poorly understood. We show here that HSP27 and αB-crystallin associated with immunoglobulin-like (Ig) domain-containing regions, but not the disordered PEVK domain (titin region rich in proline, glutamate, valine, and lysine), of the titin springs. In sarcomeres, sHSP binding to titin was actin filament independent and promoted by factors that increased titin Ig unfolding, including sarcomere stretch and the expression of stiff titin isoforms. Titin spring elements behaved predominantly as monomers in vitro. However, unfolded Ig segments aggregated, preferentially under acidic conditions, and αB-crystallin prevented this aggregation. Disordered regions did not aggregate. Promoting titin Ig unfolding in cardiomyocytes caused elevated stiffness under acidic stress, but HSP27 or αB-crystallin suppressed this stiffening. In diseased human muscle and heart, both sHSPs associated with the titin springs, in contrast to the cytosolic/Z-disk localization seen in healthy muscle/heart. We conclude that aggregation of unfolded titin Ig domains stiffens myocytes and that sHSPs translocate to these domains to prevent this aggregation.     

Towards a Molecular Understanding of the Elasticity of Titin

Vertebrate striated muscle behaves elastically when stretched and this property is thought to reside primarily within the giant filamentous protein, titin (connectin). The elastic portion of titin comprises two distinct structural motifs, immunoglobulin (Ig) domains and the PEVK titin, which is a novel motif family rich in proline, glutamate, valine and lysine residues. The respective contributions of the titin Ig and the PEVK sequences to the elastic properties of the molecule have been unknown so far. We have measured both the passive tension in single, isolated myofibrils from cardiac and skeletal muscle and the stretch-induced translational movement of I-band titin antibody epitopes following immunofluorescent labelling of sites adjacent to the PEVK and Ig domain regions. We found that with myofibril stretch, I-band titin does not extend homogeneously. The Ig domain region lengthened predominantly during small stretch, but such lengthening did not result in measurable passive tension and might be explained by straightening, rather than by unfolding, of the Ig repeats. At moderate to extreme stretch, the main extensible region was found to be the PEVK segment whose unravelling was correlated with a steady passive tension increase. In turn, PEVK domain transition from a linearly extended to a folded state appears to be principally responsible for the elasticity of muscle fibers. Thus, the length of the PEVK sequence may determine the tissue-specificity of muscle stiffness, whereas the expression of different Ig domain motif lengths may set the characteristic slack sarcomere length of a muscle type.     

Different molecular mechanics displayed by titin's constitutively and differentially expressed tandem Ig segments

Titin is a giant elastic protein responsible for passive force generated by the stretched striated-muscle sarcomere. Passive force develops in titin's extensible region which consists of the PEVK segment in series with tandemly arranged immunoglobulin (Ig)-like domains. Here we studied the mechanics of tandem Ig segments from the differentially spliced (I65-70) and constitutive (I91-98) regions by using an atomic force microscope specialized for stretching single molecules. The mechanical stability of I65-70 domains was found to be different from that of I91-98 domains. In the range of stretch rates studied (0.05-1.00 microm/s) lower average domain unfolding forces for I65-70 were associated with a weaker stretch-rate dependence of the unfolding force, suggesting that the differences in the mechanical stabilities of the segments derive from differences in the zero force unfolding rate (K(0)(u)) and the characteristic distance (location of the barrier) along the unfolding reaction coordinate (DeltaX(u)). No effect of calcium was found on unfolding forces and persistence length of unfolded domains. To explore the structural basis of the differences in mechanical stabilities of the two fragment types, we compared the amino acid sequence of I65-70 domains with that of I91-98 domains and by using homology modeling analyzed how sequence variations may affect folding free energies. Simulations suggest that differences in domain stability are unlikely to be caused by variation in the number of hydrogen bonds between the force-bearing beta-strands at the domain's N- and C-termini. Rather, they may be due to differences in hydrophobic contacts and strand orientations.     

Secondary and tertiary structure elasticity of titin Z1Z2 and a titin chain model

The giant protein titin, which is responsible for passive elasticity in muscle fibers, is built from approximately 300 regular immunoglobulin-like (Ig) domains and FN-III repeats. While the soft elasticity derived from its entropic regions, as well as the stiff mechanical resistance derived from the unfolding of the secondary structure elements of Ig- and FN-III domains have been studied extensively, less is known about the mechanical elasticity stemming from the orientation of neighboring domains relative to each other. Here we address the dynamics and energetics of interdomain arrangement of two adjacent Ig-domains of titin, Z1, and Z2, using molecular dynamics (MD) simulations. The simulations reveal conformational flexibility, due to the domain-domain geometry, that lends an intermediate force elasticity to titin. We employ adaptive biasing force MD simulations to calculate the energy required to bend the Z1Z2 tandem open to identify energetically feasible interdomain arrangements of the Z1 and Z2 domains. The finding is cast into a stochastic model for Z1Z2 interdomain elasticity that is generalized to a multiple domain chain replicating many Z1Z2-like units and representing a long titin segment. The elastic properties of this chain suggest that titin derives so-called tertiary structure elasticity from bending and twisting of its domains. Finally, we employ steered molecular dynamics simulations to stretch individual Z1 and Z2 domains and characterize the so-called secondary structure elasticity of the two domains. Our study suggests that titin's overall elastic response at weak force stems from a soft entropic spring behavior (not described here), from tertiary structure elasticity with an elastic spring constant of approximately 0.001-1 pN/A and, at strong forces, from secondary structure elasticity.     

Titin Domains Progressively Unfolded by Force Are Homogenously Distributed along the Molecule

Titin is a giant filamentous protein of the muscle sarcomere in which stretch induces the unfolding of its globular domains. However, the mechanisms of how domains are progressively selected for unfolding and which domains eventually unfold have for long been elusive. Based on force-clamp optical tweezers experiments we report here that, in a paradoxical violation of mechanically driven activation kinetics, neither the global domain unfolding rate, nor the folded-state lifetime distributions of full-length titin are sensitive to force. This paradox is reconciled by a gradient of mechanical stability so that domains are gradually selected for unfolding as the magnitude of the force field increases. Atomic force microscopic screening of extended titin molecules revealed that the unfolded domains are distributed homogenously along the entire length of titin, and this homogeneity is maintained with increasing overstretch. Although the unfolding of domains with progressively increasing mechanical stability makes titin a variable viscosity damper, the spatially randomized variation of domain stability ensures that the induced structural changes are not localized but are distributed along the molecule''s length. Titin may thereby provide complex safety mechanims for protecting the sarcomere against structural disintegration under excessive mechanical conditions.     

Molecular basis of passive stress relaxation in human soleus fibers: assessment of the role of immunoglobulin-like domain unfolding

Titin (also known as connectin) is the main determinant of physiological levels of passive muscle force. This force is generated by the extensible I-band region of the molecule, which is constructed of the PEVK domain and tandem-immunoglobulin segments comprising serially linked immunoglobulin (Ig)-like domains. It is unresolved whether under physiological conditions Ig domains remain folded and act as "spacers" that set the sarcomere length at which the PEVK extends or whether they contribute to titin's extensibility by unfolding. Here we focused on whether Ig unfolding plays a prominent role in stress relaxation (decay of force at constant length after stretch) using mechanical and immunolabeling studies on relaxed human soleus muscle fibers and Monte Carlo simulations. Simulation experiments using Ig-domain unfolding parameters obtained in earlier single-molecule atomic force microscopy experiments recover the phenomenology of stress relaxation and predict large-scale unfolding in titin during an extended period (> approximately 20 min) of relaxation. By contrast, immunolabeling experiments failed to demonstrate large-scale unfolding. Thus, under physiological conditions in relaxed human soleus fibers, Ig domains are more stable than predicted by atomic force microscopy experiments. Ig-domain unfolding did not become more pronounced after gelsolin treatment, suggesting that the thin filament is unlikely to significantly contribute to the mechanical stability of the domains. We conclude that in human soleus fibers, Ig unfolding cannot solely explain stress relaxation.     

The origin of passive force enhancement in skeletal muscle

The aim of the present study was to test whether titin is a calcium-dependent spring and whether it is the source of the passive force enhancement observed in muscle and single fiber preparations. We measured passive force enhancement in troponin C (TnC)-depleted myofibrils in which active force production was completely eliminated. The TnC-depleted construct allowed for the investigation of the effect of calcium concentration on passive force, without the confounding effects of actin-myosin cross-bridge formation and active force production. Passive forces in TnC-depleted myofibrils (n = 6) were 35.0 +/- 2.9 nN/ microm(2) when stretched to an average sarcomere length of 3.4 microm in a solution with low calcium concentration (pCa 8.0). Passive forces in the same myofibrils increased by 25% to 30% when stretches were performed in a solution with high calcium concentration (pCa 3.5). Since it is well accepted that titin is the primary source for passive force in rabbit psoas myofibrils and since the increase in passive force in TnC-depleted myofibrils was abolished after trypsin treatment, our results suggest that increasing calcium concentration is associated with increased titin stiffness. However, this calcium-induced titin stiffness accounted for only approximately 25% of the passive force enhancement observed in intact myofibrils. Therefore, approximately 75% of the normally occurring passive force enhancement remains unexplained. The findings of the present study suggest that passive force enhancement is partly caused by a calcium-induced increase in titin stiffness but also requires cross-bridge formation and/or active force production for full manifestation.     

Modeling AFM-induced PEVK extension and the reversible unfolding of Ig/FNIII domains in single and multiple titin molecules

Molecular elasticity is associated with a select number of polypeptides and proteins, such as titin, Lustrin A, silk fibroin, and spider silk dragline protein. In the case of titin, the globular (Ig) and non-globular (PEVK) regions act as extensible springs under stretch; however, their unfolding behavior and force extension characteristics are different. Using our time-dependent macroscopic method for simulating AFM-induced titin Ig domain unfolding and refolding, we simulate the extension and relaxation of hypothetical titin chains containing Ig domains and a PEVK region. Two different models are explored: 1) a series-linked WLC expression that treats the PEVK region as a distinct entropic spring, and 2) a summation of N single WLC expressions that simulates the extension and release of a discrete number of parallel titin chains containing constant or variable amounts of PEVK. In addition to these simulations, we also modeled the extension of a hypothetical PEVK domain using a linear Hooke's spring model to account for "enthalpic" contributions to PEVK elasticity. We find that the modified WLC simulations feature chain length compensation, Ig domain unfolding/refolding, and force-extension behavior that more closely approximate AFM, laser tweezer, and immunolocalization experimental data. In addition, our simulations reveal the following: 1) PEVK extension overlaps with the onset of Ig domain unfolding, and 2) variations in PEVK content within a titin chain ensemble lead to elastic diversity within that ensemble.     

Is titin a ‘winding filament’? A new twist on muscle contraction

Recent studies have demonstrated a role for the elastic protein titin in active muscle, but the mechanisms by which titin plays this role remain to be elucidated. In active muscle, Ca(2+)-binding has been shown to increase titin stiffness, but the observed increase is too small to explain the increased stiffness of parallel elastic elements upon muscle activation. We propose a 'winding filament' mechanism for titin's role in active muscle. First, we hypothesize that Ca(2+)-dependent binding of titin's N2A region to thin filaments increases titin stiffness by preventing low-force straightening of proximal immunoglobulin domains that occurs during passive stretch. This mechanism explains the difference in length dependence of force between skeletal myofibrils and cardiac myocytes. Second, we hypothesize that cross-bridges serve not only as motors that pull thin filaments towards the M-line, but also as rotors that wind titin on the thin filaments, storing elastic potential energy in PEVK during force development and active stretch. Energy stored during force development can be recovered during active shortening. The winding filament hypothesis accounts for force enhancement during stretch and force depression during shortening, and provides testable predictions that will encourage new directions for research on mechanisms of muscle contraction.     

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).     

Mechanical design of the first proximal Ig domain of human cardiac titin revealed by single molecule force spectroscopy

The elastic I-band part of muscle protein titin contains two tandem immunoglobulin (Ig) domain regions of distinct mechanical properties. Until recently, the only known structure was that of the I27 module of the distal region, whose mechanical properties have been reported in detail. Recently, the structure of the first proximal domain, I1, has been resolved at 2.1A. In addition to the characteristic beta-sandwich structure of all titin Ig domains, the crystal structure of I1 showed an internal disulfide bridge that was proposed to modulate its mechanical extensibility in vivo. Here, we use single molecule force spectroscopy and protein engineering to examine the mechanical architecture of this domain. In contrast to the predictions made from the X-ray crystal structure, we find that the formation of a disulfide bridge in I1 is a relatively rare event in solution, even under oxidative conditions. Furthermore, our studies of the mechanical stability of I1 modules engineered with point mutations reveal significant differences between the mechanical unfolding of the I1 and I27 modules. Our study illustrates the varying mechanical architectures of the titin Ig modules.     

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.     

Mechanics on myocardium deficient in the N2B region of titin: the cardiac-unique spring element improves efficiency of the cardiac cycle

Titin (also known as connectin) is an intrasarcomeric muscle protein that functions as a molecular spring and generates passive tension upon muscle stretch. The N2B element is a cardiac-specific spring element within titin's extensible region. Our goal was to study the contribution of the N2B element to the mechanical properties of titin, particularly its hypothesized role in limiting energy loss during repeated stretch (diastole)-shortening (systole) cycles of the heart. We studied energy loss by measuring hysteresis from the area between the stretch and release passive force-sarcomere length curves and used both wild-type (WT) mice and N2B knockout (KO) mice in which the N2B element has been deleted. A range of protocols was used, including those that mimic physiological loading conditions. KO mice showed significant increases in hysteresis. Most prominently, in tissue that had been preconditioned with a physiological stretch-release protocol, hysteresis increased significantly from 320 ± 46 pJ/mm²/sarcomere in WT to 650 ± 94 pJ/mm²/sarcomere in N2B KO myocardium. These results are supported by experiments in which oxidative stress was used to mechanically inactivate portions of the N2B-Us of WT titin through cysteine cross-linking. Studies on muscle from which the thin filaments had been extracted (using the actin severing protein gelsolin) showed that the difference in hysteresis between WT and KO tissue cannot be explained by filament sliding-based viscosity. Instead the results suggest that hysteresis arises from within titin and most likely involves unfolding of immunoglobulin-like domains. These studies support that the mechanical function of the N2B element of titin includes reducing hysteresis and increasing the efficiency of the heart.     

Thermal effects in stretching of Go-like models of titin and secondary structures

The effect of temperature on mechanical unfolding of proteins is studied using a Go-like model with a realistic contact map and Lennard-Jones contact interactions. The behavior of the I27 domain of titin and its serial repeats is contrasted to that of simple secondary structures. In all cases, thermal fluctuations accelerate the unraveling process, decreasing the unfolding force nearly linearly at low temperatures. However, differences in bonding geometry lead to different sensitivity to temperature and different changes in the unfolding pattern. Due to its special native-state geometry, titin is much more thermally and elastically stable than the secondary structures. At low temperatures, serial repeats of titin show a parallel unfolding of all domains to an intermediate state, followed by serial unfolding of the domains. At high temperatures, all domains unfold simultaneously, and the unfolding distance decreases monotonically with the contact order, that is, the sequence distance between the amino acids that form the native contact.