Mechanical stability and differentially conserved physical–chemical properties of titin Ig‐domains

The mechanisms that determine mechanical stabilities of protein folds remain elusive. Our understanding of these mechanisms is vital to both bioengineering efforts and to the better understanding and eventual treatment of pathogenic mutations affecting mechanically important proteins such as titin. We present a new approach to analyze data from single‐molecule force spectroscopy for different domains of the giant muscle protein titin. The region of titin found in the I‐band of a sarcomere is composed of about 40 Ig‐domains and is exposed to force under normal physiological conditions and connects the free‐hanging ends of the myosin filaments to the Z‐disc. Recent single‐molecule force spectroscopy data show a mechanical hierarchy in the I‐band domains. Domains near the C‐terminus in this region unfold at forces two to three times greater than domains near the beginning of the I‐band. Though all of these Ig‐domains are thought to share a fold and topology common to members of the Ig‐like fold family, the sequences of neighboring domains vary greatly with an average sequence identity of only 25%. We examine in this study the relation of these unique mechanical stabilities of each I‐band Ig domain to specific, conserved physical–chemical properties of amino acid sequences in related Ig domains. We find that the sequences of each individual titin Ig domain are very highly conserved, with an average sequence identity of 79% across species that are divergent as humans, chickens, and zebra fish. This indicates that the mechanical properties of each domain are well conserved and tailored to its unique position in the titin molecule. We used the PCPMer software to determine the conservation of amino acid properties in titin Ig domains grouped by unfolding forces into “strong” and “weak” families. We found two motifs unique to each family that may have some role in determining the mechanical properties of these Ig domains. A detailed statistical analysis of properties of individual residues revealed several positions that displayed differentially conserved properties in strong and weak families. In contrast to previous studies, we find evidence that suggests that the mechanical stability of Ig domains is determined by several residues scattered across the β‐sandwich fold, and force sensitive residues are not only confined to the A′‐G region. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

A mechanical unfolding intermediate in an actin-crosslinking protein

Many F-actin crosslinking proteins consist of two actin-binding domains separated by a rod domain that can vary considerably in length and structure. In this study, we used single-molecule force spectroscopy to investigate the mechanics of the immunoglobulin (Ig) rod domains of filamin from Dictyostelium discoideum (ddFLN). We find that one of the six Ig domains unfolds at lower forces than do those of all other domains and exhibits a stable unfolding intermediate on its mechanical unfolding pathway. Amino acid inserts into various loops of this domain lead to contour length changes in the single-molecule unfolding pattern. These changes allowed us to map the stable core of approximately 60 amino acids that constitutes the unfolding intermediate. Fast refolding in combination with low unfolding forces suggest a potential in vivo role for this domain as a mechanically extensible element within the ddFLN rod.     

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 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).     

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.     

Cross-Species Mechanical Fingerprinting of Cardiac Myosin Binding Protein-C

Cardiac myosin binding protein-C (cMyBP-C) is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 8 Ig- and 3 fibronectin III (FNIII)-like domains along with a unique regulatory sequence referred to as the MyBP-C motif or M-domain. We previously used atomic force microscopy to investigate the mechanical properties of murine cMyBP-C expressed using a baculovirus/insect cell expression system. Here, we investigate whether the mechanical properties of cMyBP-C are conserved across species by using atomic force microscopy to manipulate recombinant human cMyBP-C and native cMyBP-C purified from bovine heart. Force versus extension data obtained in velocity-clamp experiments showed that the mechanical response of the human recombinant protein was remarkably similar to that of the bovine native cMyBP-C. Ig/Fn-like domain unfolding events occurred in a hierarchical fashion across a threefold range of forces starting at relatively low forces of ∼50 pN and ending with the unfolding of the highest stability domains at ∼180 pN. Force-extension traces were also frequently marked by the appearance of anomalous force drops suggestive of additional mechanical complexity such as structural coupling among domains. Both recombinant and native cMyBP-C exhibited a prominent segment ∼100 nm-long that could be stretched by forces <50 pN before the unfolding of Ig- and FN-like domains. Combined with our previous observations of mouse cMyBP-C, these results establish that although the response of cMyBP-C to mechanical load displays a complex pattern, it is highly conserved across species.     

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.     

Mechanical properties of cardiac titin's N2B-region by single-molecule atomic force spectroscopy

Titin is a giant protein responsible for passive-tension generation in muscle sarcomeres. Here, we used single-molecule AFM force spectroscopy to investigate the mechanical characteristics of a recombinant construct from the human cardiac-specific N2B-region, which harbors a 572-residue unique sequence flanked by two immunoglobulin (Ig) domains on either side. Force-extension curves of the N2B-construct revealed mean unfolding forces for the Ig-domains similar to those of a recombinant fragment from the distal Ig-region in titin (I91-98). The mean contour length of the N2B-unique sequence was 120 nm, but there was a bimodal distribution centered at approximately 95 nm (major peak) and 180 nm (minor peak). These values are lower than expected if the N2B-unique sequence were a permanently unfolded entropic spring, but are consistent with the approximately 100 nm maximum extension of that segment measured in isolated stretched cardiomyofibrils. A contour-length below 200 nm would be reasonable, however, if the N2B-unique sequence were stabilized by a disulphide bridge, as suggested by several disulphide connectivity prediction algorithms. Since the N2B-unique sequence can be phosphorylated by protein kinase A (PKA), which lowers titin-based stiffness, we studied whether addition of PKA (+ATP) affects the mechanical properties of the N2B-construct, but found no changes. The softening effect of PKA on N2B-titin may require specific conditions/factors present inside the cardiomyocytes.     

Differential mechanical stability of filamin A rod segments

Prompted by recent reports suggesting that interaction of filamin A (FLNa) with its binding partners is regulated by mechanical force, we examined mechanical properties of FLNa domains using magnetic tweezers. FLNa, an actin cross-linking protein, consists of two subunits that dimerize through a C-terminal self-association domain. Each subunit contains an N-terminal spectrin-related actin-binding domain followed by 24 immunoglobulinlike (Ig) repeats. The Ig repeats in the rod 1 segment (repeats 1–15) are arranged as a linear array, whereas rod 2 (repeats 16–23) is more compact due to interdomain interactions. In the rod 1 segment, repeats 9–15 augment F-actin binding to a much greater extent than do repeats 1–8. Here, we report that the three segments are unfolded at different forces under the same loading rate. Remarkably, we found that repeats 16–23 are susceptible to forces of ∼10 pN or even less, whereas the repeats in the rod 1 segment can withstand significantly higher forces. The differential force response of FLNa Ig domains has broad implications, since these domains not only support the tension of actin network but also interact with many transmembrane and signaling proteins, mostly in the rod 2 segment. In particular, our finding of unfolding of repeats 16–23 at ∼10 pN or less is consistent with the hypothesized force-sensing function of the rod 2 segment in FLNa.     

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.     

Single-molecule force spectroscopy reveals a stepwise unfolding of Caenorhabditis elegans giant protein kinase domains

Myofibril assembly and disassembly are complex processes that regulate overall muscle mass. Titin kinase has been implicated as an initiating catalyst in signaling pathways that ultimately result in myofibril growth. In titin, the kinase domain is in an ideal position to sense mechanical strain that occurs during muscle activity. The enzyme is negatively regulated by intramolecular interactions occurring between the kinase catalytic core and autoinhibitory/regulatory region. Molecular dynamics simulations suggest that human titin kinase acts as a force sensor. However, the precise mechanism(s) resulting in the conformational changes that relieve the kinase of this autoinhibition are unknown. Here we measured the mechanical properties of the kinase domain and flanking Ig/Fn domains of the Caenorhabditis elegans titin-like proteins twitchin and TTN-1 using single-molecule atomic force microscopy. Our results show that these kinase domains have significant mechanical resistance, unfolding at forces similar to those for Ig/Fn β-sandwich domains (30-150 pN). Further, our atomic force microscopy data is consistent with molecular dynamic simulations, which show that these kinases unfold in a stepwise fashion, first an unwinding of the autoinhibitory region, followed by a two-step unfolding of the catalytic core. These data support the hypothesis that titin kinase may function as an effective force sensor.     

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.     

Chemistry on a single protein, vascular cell adhesion molecule-1, during forced unfolding

Proteins of many types experience tensile forces in their normal function, and vascular cell adhesion molecule-1 (VCAM-1) is typical in this. VCAM has seven Ig domains, and each has a disulfide bond (-S-S-) buried in its core that covalently stabilizes about half of each domain against unfolding. VCAM is extended here by single molecule atomic force microscopy in the presence or absence of reducing agents. In the absence of reducing agent, a sawtooth pattern of forced unfolding reveals an average period and total length consistent with disulfide locations in VCAM. With increasing reducing agent, accessible disulfides are specifically reduced (to SH); the average period for unfolding increases up to saturation together with additional metrics of unfolding. Steered molecular dynamics simulations of unfolding indeed show that the core disulfide bond is solvent-exposed in the very earliest stages of protein extension. Michaelis-Menten kinetics emerge with reduction catalyzed by force (tau(reduction) approximately 10(-4) s). The results establish single molecule reduction, one bond at a time, and show that mechanical forces can play a key role in modulating the redox state of cell adhesion proteins that are invariably stressed in cell adhesion.     

The effect of core destabilization on the mechanical resistance of I27

It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27)(5)*) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27)(5)* in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 x 10(-3) s(-1)). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 x 10(-2) s(-1)), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.     

Viscoelastic study of the mechanical unfolding of a protein by AFM

We have applied a dynamic force modulation technique to the mechanical unfolding of a homopolymer of immunoglobulin (Ig) domains from titin, (C47S C63S I27)5, [(I27)5] to determine the viscoelastic response of single protein molecules as a function of extension. Both the stiffness and the friction of the homopolymer system show a sudden decrease when a protein domain unfolds. The decrease in measured friction suggests that the system is dominated by the internal friction of the (I27)5 molecule and not solvent friction. In the stiffness-extension spectrum we detected an abrupt feature before each unfolding event, the amplitude of which decreased with each consecutive unfolding event. We propose that these features are a clear indication of the formation of the known unfolding intermediate of I27, which has been observed previously in constant velocity unfolding experiments. This simple force modulation AFM technique promises to be a very useful addition to constant velocity experiments providing detailed viscoelastic characterization of single molecules under extension.     

The protein import motor of mitochondria: a targeted molecular ratchet driving unfolding and translocation

Unfolding and import of preproteins into mitochondria are facilitated by a molecular motor in which heat shock protein 70 (Hsp70) in the matrix plays an essential role. Here we present two different experimental approaches to analyze mechanisms underlying this function of Hsp70. First, preproteins containing stretches of glutamic acid (polyE) or glycine (polyG) repeats in front of folded domains were imported into mitochondria. This occurred although Hsp70 cannot pull on these stretches to unfold the folded domains, since it does not bind to polyE and polyG. Secondly, preproteins containing titin immunoglobulin (Ig)-like domains were imported into mitochondria, despite the fact that forces of >200 pN are required to mechanically unfold these domains. Since molecular motors generate forces of approximately 5 pN, Hsp70 could not promote unfolding of the Ig-like domains by mechanical pulling. Our observations suggest that Hsp70 acts as an element of a Brownian ratchet, which mediates unfolding and translocation of preproteins across the mitochondrial membranes.     

Molecular mechanics of cardiac titin's PEVK and N2B spring elements.

Titin is a giant elastic protein that is responsible for the majority of passive force generated by the myocardium. Titin's force is derived from its extensible I-band region, which, in the cardiac isoform, comprises three main extensible elements: tandem Ig segments, the PEVK domain, and the N2B unique sequence (N2B-Us). Using atomic force microscopy, we characterized the single molecule force-extension curves of the PEVK and N2B-Us spring elements, which together are responsible for physiological levels of passive force in moderately to highly stretched myocardium. Stretch-release force-extension curves of both the PEVK domain and N2B-Us displayed little hysteresis: the stretch and release data nearly overlapped. The force-extension curves closely followed worm-like chain behavior. Histograms of persistence length (measure of chain bending rigidity) indicated that the single molecule persistence lengths are approximately 1.4 and approximately 0.65 nm for the PEVK domain and N2B-Us, respectively. Using these mechanical characteristics and those determined earlier for the tandem Ig segment (assuming folded Ig domains), we modeled the cardiac titin extensible region in the sarcomere and calculated the extension of the various spring elements and the forces generated by titin, both as a function of sarcomere length. In the physiological sarcomere length range, predicted values and those obtained experimentally were indistinguishable.     

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.     

Mechanical unfolding of cardiac myosin binding protein-C by atomic force microscopy

Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.