Titin Domains Research Articles

Single Molecule Oxidative Folding

More than a third of human proteins contain disulfide bonds, yet our understanding of how these are formed in vivo has remained rudimentary. Formation of disulfides takes place during oxidative folding and is catalyzed by the enzyme Protein Disulfide Isomerase (PDI). We have developed a single molecule approach to study this process in real-time, using force-clamp AFM. We investigated the oxidative folding of human titin domains, in the presence and absence of oxidase enzymes such as human PDI. Our approach enables, for the first time, independent kinetic measurements of folding and disulfide formation in individual proteins. Strikingly, we discovered a critical period for disulfide formation in the titin I1 domain. After protein folding was completed, disulfide formation could no longer be catalyzed by PDI. Thus, the competing kinetics of PDI-mediated oxidation and protein folding determines the outcome of oxidative folding. The involvement of glutathione in oxidative folding has long been under speculation. We show that glutathionylation can retard the kinetics of protein folding by more than 10-fold, revealing a possible novel role of this ubiquitous small molecule. In protein substrates with more than two cysteines, our data show that correct pairing is achieved through substrate folding rather than through the action of the oxidase enzyme. Therefore, the accuracy of cysteine pairing increases along the folding pathway of the substrate. These unprecedented observations shed new light on how disulfide formation relates to protein folding in vivo. Taken together, we are able to construct a complete kinetic model of oxidative folding, incorporating experimentally measured rates for the individual reaction steps. The technique presented here can be used to elucidate the pathway of oxidative folding for a wide variety of proteins, and can also be used to identify mechanisms that trigger misfolding and disease.

Small Heat Shock Proteins Associate under Stress Conditions with Elastic Titin Filaments and Provide Protection from Aggregation

The I-band portion of the giant muscle-protein titin is elastic but detailed knowledge of titin arrangement and interactions in this region is lacking. We determined whether or not select domains from this region can associate with one another, as observed for titin's “distal” immunoglobulin domain-like (Ig-) region. Using sedimentation-velocity centrifugation and size-exclusion chromatography of purified human titin fragments, we detected a monomeric state of the titin N2B-domain or the N2B-unique sequence contained therein. The constitutively expressed PEVK-domain and the Ig-only fragment I9-I12 also behaved as monomers in-vitro, whereas the N2A-segment showed both dimeric and (to a lesser degree) monomeric behavior. Yeast-2-hybrid and GST-pulldown interaction tests demonstrated that both the N2B and the N2A domain, but not the PEVK-fragment, bind to the small heat-shock proteins (sHSPs), alphaB-crystallin and HSP27. These interactions were confirmed on isolated human cardiac or rabbit psoas myofibrils incubated ex-vivo with either sHSP type. Stretching myofibrils to promote titin-domain unfolding increased the propensity of sHSPs to bind I-band titin. Protein unfolding in-vitro by 8M urea caused aggregation of the N2A-segment under acidic conditions (pH6.7), but not at pH7.2. Importantly, alphaB-crystallin prevented the aggregation of the N2A-segment at pH6.7 partially (molar ratio of N2A:alphaB-crystallin, 1:5) or fully (ratio, 1:10). In cultured neonatal rat cardiomyocytes, both sHSPs translocated from the cytosol to the sarcomeric Z-disk/I-band region on inhibition of the proteasome. In adult rat cardiomyocytes both sHSPs associated with I-band titin already under normal culture conditions, suggesting an age-related increase in chaperoning activity. We conclude that titin filaments may run through the elastic segment mainly as monomers; sHSPs associate with elastic titin domains under various stress conditions; and sHSPs are able to protect I-band titin regions from aggregation under adverse intracellular circumstances.

Computational investigation of the effect of thermal perturbation on the mechanical unfolding of titin I27

The emergence of single-molecule force measurement experiments has facilitated a better understanding of protein folding pathways and the thermodynamics involved. Computational methods such as steered molecular dynamics (SMD) simulations are helpful in providing atomistic level information on the unfolding pathways. Recent experimental studies have showed that combinations of single-molecule experiments with traditional methods such as chemical and/or thermal denaturation yield additional insights into the folding phenomenon. In this study, we report results from extensive computations (a total of about 60 SMD simulations with a total length of about 0.4 μs) that address the effect of thermal perturbation on the mechanical stability of the I27 domain of the protein titin. A wide range of temperatures (280-340 K) were considered for the pulling, which was done at both constant velocity and constant force using SMD simulations. Good agreement with experimental data, such as for the trends in changes in average force and the maximum force with respect to the temperature, was obtained. This study identifies two competing pathways for the mechanical unfolding of I27, and illustrates the significance of combining various techniques to examine protein folding.

Postmortem titin proteolysis is influenced by sarcomere length in bovine muscle1

The calpain protease system, in particular, μ-calpain is involved in the disassembly of specific myofibrillar proteins, resulting in tenderization of meat postmortem. Given the size, complexity, and integral nature of titin to the structure of the sarcomere, it is plausible that the length of a sarcomere may alter the susceptibility of various domains of titin to cleavage by the calpains. Therefore, we hypothesized titin degradation differs in a sarcomere-length-dependent manner in beef. After slaughter, beef carcasses were split and sides were either suspended by the Achilles tendon (normal suspension, NS) or by the aitchbone (hip suspension, HS). Immediately after suspension, samples were dissected from the LM, psoas major (PM), and semitendinosus (STN) muscles to serve as 0-d controls. After 24 h, 4 steaks were removed from each muscle and randomly assigned to 1-, 4-, 7-, or 10-d aging treatments. After the assigned aging period, myofibrils were purified for determination of sarcomere length. Warner-Bratzler shear force analysis was also performed to evaluate differences in tenderness. Muscle proteins were solubilized and subjected to SDS-VAGE (vertical agarose gel electrophoresis) to evaluate titin degradation. Sarcomere lengths differed (P < 0.0001) between contralateral muscles of NS and HS carcasses. Quantification of SDS-VAGE gels revealed less (P < 0.05) intact titin in the PM muscle of NS carcasses at each aging period compared with the PM of HS carcasses. No significant differences (P > 0.05) were detected in the disappearance of intact titin among suspension methods in the LM or STN. These data demonstrate that suspension method alters proteolysis of titin and suggest an increase in sarcomere length may contribute to the susceptibility of titin to postmortem proteolysis in beef.

Characterization of the in vitro expressed autoimmune rippling muscle disease immunogenic domain of human titin encoded by TTN exons 248–249

Autoimmune rippling muscle disease (ARMD) is an autoimmune neuromuscular disease associated with myasthenia gravis (MG). Past studies in our laboratory recognized a very high molecular weight skeletal muscle protein antigen identified by ARMD patient antisera as the titin isoform. These past studies used antisera from ARMD and MG patients as probes to screen a human skeletal muscle cDNA library and several pBluescript clones revealed supporting expression of immunoreactive peptides. This study characterizes the products of subcloning the titin immunoreactive domain into pGEX-3X and the subsequent fusion protein. Sequence analysis of the fusion gene indicates the cloned titin domain (GenBank ID: EU428784) is in frame and is derived from a sequence of N2-A spanning the exons 248–250 an area that encodes the fibronectin III domain. PCR and EcoR1 restriction mapping studies have demonstrated that the inserted cDNA is of a size that is predicted by bioinformatics analysis of the subclone. Expression of the fusion protein result in the isolation of a polypeptide of 52 kDa consistent with the predicted inferred amino acid sequence. Immunoblot experiments of the fusion protein, using rippling muscle/myasthenia gravis antisera, demonstrate that only the titin domain is immunoreactive.

Force Generation: ATP-Powered Proteasomes Pull the Rope

Recently, single-molecule force spectroscopy techniques have provided unprecedented opportunities to apply and to quantify forces that guide protein (un-)folding. A new study provides fascinating insights into the sophisticated mechanism by which an ATP-fueled proteolytic machine generates mechanical forces to unfold and translocate multidomain substrates.

Dependence of Proteasome Processing Rate on Substrate Unfolding

Protein degradation by eukaryotic proteasomes is a multi-step process involving substrate recognition, ATP-dependent unfolding, translocation into the proteolytic core particle, and finally proteolysis. To date, most investigations of proteasome function have focused on the first and the last steps in this process. Here we examine the relationship between the stability of a folded protein domain and its degradation rate. Test proteins were targeted to the proteasome independently of ubiquitination by directly tethering them to the protease. Degradation kinetics were compared for test protein pairs whose stability was altered by either point mutation or ligand binding, but were otherwise identical. In both intact cells and in reactions using purified proteasomes and substrates, increased substrate stability led to an increase in substrate turnover time. The steady-state time for degradation ranged from ∼5 min (dihydrofolate reductase) to 40 min (I27 domain of titin). ATP turnover was 110/min./proteasome, and was not markedly changed by substrate. Proteasomes engage tightly folded substrates in multiple iterative rounds of ATP hydrolysis, a process that can be rate-limiting for degradation.

Conformation-regulated mechanosensory control via titin domains in cardiac muscle.

The giant filamentous protein titin is ideally positioned in the muscle sarcomere to sense mechanical stimuli and transform them into biochemical signals, such as those triggering cardiac hypertrophy. In this review, we ponder the evidence for signaling hotspots along the titin filament involved in mechanosensory control mechanisms. On the way, we distinguish between stress and strain as triggers of mechanical signaling events at the cardiac sarcomere. Whereas the Z-disk and M-band regions of titin may be prominently involved in sensing mechanical stress, signaling hotspots within the elastic I-band titin segment may respond primarily to mechanical strain. Common to both stress and strain sensor elements is their regulation by conformational changes in protein domains.

Designing Novel Proteins with Enhanced Mechanical Strengths

Designing novel proteins with specific properties remains a difficult goal to achieve, mainly because the understanding of the relationship between protein sequences and certain structures or properties is still incomprehensive. For the case of mechanical proteins, even though few successes are accomplished to enhance the mechanical strengths of protein domains, there are no rational and systematic methods to design proteins with specific mechanical properties. It has been shown that the Ig-like domains in the I-band of Titin need hierarchical external forces to unfold. Dr. Braun's group analyzed the sequences of the weak and strong Ig-like domains from different species, and identified unique motifs which may be important for the hierarchical strengths of Ig-like domains. In this study,we designed four novel proteins by swapping motifs or segments from strong domain I27 to weak domain I1. The hypothesis is that the unfolding forces of the mutants will be increased. We tested their unfolding forces by single molecular Atomic Force Microscope (AFM) experiments to verify the enhancements of mechanical strengths. Preliminary data indicate that the unfolding forces of some designed mutants are increased to the level of I27. Steered Molecular Dynamics (SMD) simulations for these mutants are in process to analyze the conformational changes during unfolding. The change of backbone hydrogen bonds of A-B, A’-G strands and their side-chain interactions with neighboring residues are monitored, since they were reported to be the key determinants of mechanical strengths for Ig-like domains in titin.

Titin Domains as Mechanical Reporters of UNC-45 Mediated Myosin Folding

Myosins are protein machines in which allosteric cross-talk between ATP-binding and hydrolysis and the movement of actin filaments requires precise changes in the polypeptide fold of the motor domain. UNC-45, a member of the UCS family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly for myosin into muscle thick filaments in vivo. However the molecular mechanisms by which UNC-45 interacts with myosin to promote proper folding of the myosin head domain are not known. We have devised a novel approach to analyze the mechanical refolding of the myosin motor domain at the single molecule level utilizing AFM techniques. By chemically coupling an I27 titin polyprotein to the motor domain of myosin, we introduced a “molecular reporter” into the motor domain, providing a specific attachment point and a well characterized mechanical fingerprint in the AFM measurements. This approach enabled us to study the folding of the motor domain and directly observe the effect of the chaperone UNC-45. The motor domain misfolded after mechanical unfolding, whereas the myosin rod refolded independently of the motor domain. The misfolded motor domain recruited the otherwise robustly folding I27 modules into a misfolded state. Thus, the I27 domains function as a folding sensor. UNC-45 prevents the detrimental interaction between the motor domain and the I27 modules, indicating that it bound to and stabilized the non-native myosin motor domain. Using truncated versions of UNC-45, we localized this protective effect to the C-terminal UCS domain of UNC-45. Our approach enables the study, for the first time, the chaperone effects of UNC-45 on myosin folding in mechanistic detail.This work was funded by NIH grants R01DK073394 (AFO), R01AR050051 (HFE), the Muscular Dystrophy Association and the Cecil and Ida Green Endowment (HFE).

Protein Unfolding and Chemical Reactions Under Force: Complexity Versus Simplicity

Elucidating the dynamics of reactant molecules along the particular pathway that connects them with the resulting products is a cornerstone of modern chemistry. In general, for small systems composed of only a few molecules, the valley connecting reactants with products is narrow and the position of the transition state is highly localized. By contrast, in the case of more complex reactions involving bigger molecular systems such as proteins, the potential energy surfaces become rougher, which typically results in heterogeneous reaction pathways with multiple transition state structures. In both cases, detailed information on the energy landscapes has been mainly restricted to the results obtained using computational methods. Single molecule force-clamp spectroscopy can be now used to directly monitor the individual unfolding pathways of single proteins and the reactive trajectories corresponding to the chemical reduction of a single disulfide bond embedded within the core of a protein. Herein, we compare the effect of a mechanical constant force on the energy landscape of two different reactions; the unfolding of a single protein and the reduction of a single disulfide bond, both occurring within the same single protein. Our results demonstrate that the native state of the 27th immunoglobulin-like domain of titin is rough and probably composed of a set of similar structures with slightly different energy. Interestingly, shortening of the I27 protein by the presence of disulfide bonds has a strong effect on the measured heterogeneity in the unfolding pathways. Our results are quantitatively discussed within the framework of the static disorder theory. In sharp contrast with these results, the energy landscape of an SN2 chemical reaction, occurring within a much shorter length-scale, is smooth, implying a high degree of homogeneity within the individual reactive pathways connecting the reactants with the reaction products.

Full Reconstruction of a Vectorial Protein Folding Pathway by Atomic Force Microscopy and Molecular Dynamics Simulations

During co-translational folding, the nascent polypeptide chain is extruded sequentially from the ribosome exit tunnel and is [corrected] under severe conformational constraints [corrected] dictated by the one-dimensional geometry of the tunnel. [corrected] How do such vectorial constraints impact the folding pathway? Here, we combine single-molecule atomic force spectroscopy and steered molecular dynamics simulations to examine protein folding in the presence of one-dimensional constraints that are similar to those imposed on the nascent polypeptide chain. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. We show that fully stretched and then relaxed NI6C starts folding by the formation of local secondary structures, followed by the nucleation of three N-terminal repeats. This rate-limiting step is then followed by the vectorial and sequential folding of the remaining repeats. However, after partial unfolding, when allowed to refold, the C-terminal repeats successively regain structures without any nucleation step by using the intact N-terminal repeats as a template. These results suggest a pathway for the co-translational folding of repeat proteins and have implications for mechanotransduction.

P2.13 Proteolytic processing of C-terminal titin

Mutations in the extreme C-terminal part of titin, residing in the sarcomeric M-band, lead to tibial muscular dystrophy (TMD), a dominant late-onset distal myopathy. In homozygotes, the same mutations cause the more severe recessive limb-girdle muscular dystrophy 2J (LGMD2J). The FINmaj mutation, underlying the titinopathies TMD/LGMD2J in Finnish patients, causes an exchange of four consecutive amino acids in the Ig-like domain M10, the most C-terminal domain of titin. Seven other missense or truncating mutations, all affecting M10 or the preceding is 7 region, are known to cause TMD/LGMD2J in other European populations.

Interactions with M-band Titin and Calpain 3 Link Myospryn (CMYA5) to Tibial and Limb-girdle Muscular Dystrophies

Mutations in the C terminus of titin, situated at the M-band of the striated muscle sarcomere, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy (LGMD) type 2J. Mutations in the protease calpain 3 (CAPN3), in turn, lead to LGMD2A, and secondary CAPN3 deficiency in LGMD2J suggests that the pathomechanisms of the diseases are linked. Yeast two-hybrid screens carried out to elucidate the molecular pathways of TMD/LGMD2J and LGMD2A resulted in the identification of myospryn (CMYA5, cardiomyopathy-associated 5) as a binding partner for both M-band titin and CAPN3. Additional yeast two-hybrid and coimmunoprecipitation studies confirmed both interactions. The interaction of myospryn and M-band titin was supported by localization of endogenous and transfected myospryn at the M-band level. Coexpression studies showed that myospryn is a proteolytic substrate for CAPN3 and suggested that myospryn may protect CAPN3 from autolysis. Myospryn is a muscle-specific protein of the tripartite motif superfamily, reported to function in vesicular trafficking and protein kinase A signaling and implicated in the pathogenesis of Duchenne muscular dystrophy. The novel interactions indicate a role for myospryn in the sarcomeric M-band and may be relevant for the molecular pathomechanisms of TMD/LGMD2J and LGMD2A.

Cardiac Titin

Titin constitutes the third myofilament of cardiac muscle, with a single giant polypeptide spanning from Z-disk to the M-band region of the sarcomere1 (Fig. 1). The ∼1.0 MDa region in the I-band is extensible and consists of tandemly arranged immunoglobulin (Ig)-like domains that make up proximal (near Z-disk) and distal (near A-I junction) segments, interspersed by the PEVK sequence (rich in proline, glutamate, valine, and lysine residues) and the N2B element2. Each functions as a distinct spring element3. The C-terminal ∼2 MDa of titin is located in the A-band and is inextensible. It is composed of regular arrays of Ig and fibronectin type 3 (Fn3) modules forming so-called super-repeats2. A-band titin may function as a molecular ruler, regulating assembly of the thick filament2,4,5. Titin's ∼250-kDa COOH-terminal region is an integral part of the M-band and contains a kinase domain6,7. As in the Z-disk, where titin filaments from opposite sarcomeres overlap, titin filaments from opposite half-sarcomeres overlap within the M-band, where they are interconnected by M-band proteins8. Thus, titin filaments with opposite polarity overlap in both Z-disk and M-band, forming a contiguous filament along the myofibril. In this review, we discuss titin's functions in the heart, with an emphasis on its role in diastolic function and the various mechanisms whereby passive stiffness can be tuned. Due to space constraints, it has not been possible to provide inclusive references to all original articles in the field. Figure 1 Schematic of titin in sarcomere. Differential splicing Titin is encoded by a single gene containing 368 exons. Multiple splice pathways in the I-band encoding region (∼230 exons) give rise to isoforms with different spring composition9. The three cardiac isoform classes are shown in Fig. 1. The relatively small ∼3.0 MDa isoform is known as N2B titin (it contains the N2B element)9. A second class also contains the N2A element, and is termed N2BA titin. N2BA titins have a longer PEVK segment and a variable number of additional Ig domains resulting in a ∼3.3-3.5 MDa size9. The third class includes isoforms that predominate in fetal-neonatal life which contain additional spring elements in both tandem Ig and PEVK regions, resulting in a ∼3.6-3.8 MDa size protein10-12. These isoforms gradually disappear during postnatal development. Regulation of the spring composition of titin in fetal-neonatal myocardium allows adjustment of diastolic filling properties during development.

Theory of Biopolymer Stretching at High Forces.

We provide a unified theory for the high force entropic elasticity of biopolymers solely in terms of the persistence length, ξp , and the monomer spacing, a. When the force f>ℱ h ~ kBTξp /a2 the biopolymers behave as freely jointed chains (FJCs) while in the range ℱ l ~ kBT/ξp <f<ℱ h the worm-like chain (WLC) is a better model. We show that ξp can be estimated from the force extension curve (FEC) at the extension x ≈ 1/2 (normalized by the contour length of the biopolymer). After validating the theory using simulations, we provide a quantitative analysis of the FECs for a diverse set of biopolymers (dsDNA, ssRNA, ssDNA, polysaccharides, and unstructured PEVK domain of titin) for x ≥ 1/2. The success of a specific polymer model (FJC or WLC) to describe the FEC of a given biopolymer is naturally explained by the theory. Only by probing the response of biopolymers over a wide range of forces can the f-dependent elasticity be fully described.

Mechanoenzymatics and Protective Mechanisms of Titins’ Catalytic and IG Domains

The giant titin filament controls many structural and functional properties of the sarcomere. Titin filaments connect M-line and Z-disc of the sarcomere and consist of four regions: the M-line, A-band, I-band, and Z-line. The different domains of titin (mainly immunoglobulin Ig and fibronectin-3 domains, and the catalytic domain titin kinase (TK)) exhibit dramatically different mechanical properties. We used atomistic molecular dynamics simulations to explore the coupling of mechanical stability with the enzymatic activity of titin kinase and the protective properties of Ig-domains. We showed that a unique autoinhibitory mechanism allows TK to act as a molecular force sensor, as relatively low forces already remove the autoinhibitory tail and prime the molecule for ATP binding. At much higher forces, the mechanical stability of Ig27 becomes important: In our studies, extensive dynamic force spectroscopy (DFS), Brownian dynamics, and molecular dynamics simulations worked together to examine mechanical stability of Ig27 under different loading rates. Our results suggest that Ig27 is perfectly suited to act as a molecular force buffer over a wide range of loading rates.

Non-Native Interactions Are Critical for Mechanical Strength in PKD Domains

SummaryExperimental observation has led to the commonly held view that native state protein topology is the principle determinant of mechanical strength. However, the PKD domains of polycystin-1 challenge this assumption: they are stronger than predicted from their native structure. Molecular dynamics simulations suggest that force induces rearrangement to an intermediate structure, with nonnative hydrogen bonds, that resists unfolding. Here we test this hypothesis directly by introducing mutations designed to prevent formation of these nonnative interactions. We find that these mutations, which only moderately destabilize the native state, reduce the mechanical stability dramatically. The results demonstrate that nonnative interactions impart significant mechanical stability, necessary for the mechanosensor function of polycystin-1. Remarkably, such nonnative interactions result from force-induced conformational change: the PKD domain is strengthened by the application of force.

The first Italian family with tibial muscular dystrophy caused by a novel titin mutation

Tibial muscular dystrophy (TMD) or Udd myopathy is an autosomal dominant distal myopathy with late onset, at first described in the Finnish population. We report here the first Italian cases of TTN mutated titinopathy. The proband, a 60 year-old female, had the first muscular signs at the age of 59 years, with difficulty in walking and right foot drop. Muscle imaging showed selective fatty degenerative change in the anterior compartment of leg muscles. Her 67 year-old brother, started to show muscle weakness, pain at lower limbs and hypertrophy of calf muscles at the age of 66 years. Their mother began to show foot drop and impaired walking from the age of 60 years. Other relatives are reported to be affected in a similar way. Because the phenotype appeared compatible with TMD, we analyzed the TTN gene in the DNA of the proband and we identified a heterozygous mutation 293326A>C. This mutation is also present in the brother and in the other affected individuals of the same family. The mutation predicts a His33378Pro change located next to the previously known Belgian TMD mutation. The mutation was not found in 100 Italian control DNA samples. Then, since the introduction of a proline in the last domain of titin was previously known to cause TMD in French families, we can conclude that this missense mutation is the obvious pathogenetic mutation in the affected patients. No other disease causing mutations in the TTN gene have so far been reported in the Italian population.

Bin1 Src Homology 3 Domain Acts as a Scaffold for Myofiber Sarcomere Assembly

In skeletal muscle development, the genes and regulatory factors that govern the specification of myocytes are well described. Despite this knowledge, the mechanisms that regulate the coordinated assembly of myofiber proteins into the functional contractile unit or sarcomere remain undefined. Here we explored the hypothesis that modular domain proteins such as Bin1 coordinate protein interactions to promote sarcomere formation. We demonstrate that Bin1 facilitates sarcomere organization through protein-protein interactions as mediated by the Src homology 3 (SH3) domain. We observed a profound disorder in myofiber size and structural organization in a murine model expressing the Bin1 SH3 region. In addition, satellite cell-derived myogenesis was limited despite the accumulation of skeletal muscle-specific proteins. Our experiments revealed that the Bin1 SH3 domain formed transient protein complexes with both actin and myosin filaments and the pro-myogenic kinase Cdk5. Bin1 also associated with a Cdk5 phosphorylation domain of titin. Collectively, these observations suggest that Bin1 displays protein scaffold-like properties and binds with sarcomeric factors important in directing sarcomere protein assembly and myofiber maturation.