Differences in stability and calcium sensitivity of the Ig domains in titin's N2A region

Titin is a large filamentous protein that spans half a sarcomere, from Z‐disk to M‐line. The N2A region within the titin molecule exists between the proximal immunoglobulin (Ig) region and the PEVK region and protein–protein interactions involving this region are required for normal muscle function. The N2A region consists of four Ig domains (I80–I83) with a 105 amino acid linker region between I80 and I81 that has a helical nature. Using chemical stability measurements, we show that predicted differences between the adjacent Ig domains (I81–I83) correlate with experimentally determined differences in chemical stability and refolding kinetics. Our work further shows that I83 has the lowest ΔG_unfolding, which is increased in the presence of calcium (pCa 4.3), indicating that Ca²⁺ plays a role in stabilizing this immunoglobulin domain. The characteristics of N2A's three Ig domains provide insight into the stability of the binding sites for proteins that interact with the N2A region. This work also provides insights into how Ca²⁺ might influence binding events involving N2A.     

Solution NMR Structure of Titin N2A Region Ig Domain I83 and Its Interaction with Metal Ions

Titin, the largest single chain protein known so far, has long been known to play a critical role in passive muscle function but recent studies have highlighted titin’s role in active muscle function. One of the key elements in this role is the Ca²⁺-dependent interaction between titin’s N2A region and the thin filament. An important element in this interaction is I83, the terminal immunoglobulin domain in the N2A region. There is limited structural information about this domain, but experimental evidence suggests that it plays a critical role in the N2A-actin binding interaction. We now report the solution NMR structure of I83 and characterize its dynamics and metal binding properties in detail. Its structure shows interesting relationships to other I-band Ig domains. Metal binding and dynamics data point towards the way the domain is evolutionarily optimized to interact with neighbouring domains. We also identify a calcium binding site on the N-terminal side of I83, which is expected to impact the interdomain interaction with the I82 domain. Together these results provide a first step towards a better understanding of the physiological effects associated with deletion of most of the I83 domain, as occurs in the mdm mouse model, as well as for future investigations of the N2A region.     

Molecular dissection of N2B cardiac titin's extensibility

Titin is a giant filamentous polypeptide of multidomain construction spanning between the Z- and M-lines of the cardiac muscle sarcomere. Extension of the I-band segment of titin gives rise to a force that underlies part of the diastolic force of cardiac muscle. Titin's force arises from its extensible I-band region, which consists of two main segment types: serially linked immunoglobulin-like domains (tandem Ig segments) interrupted with a proline (P)-, glutamate (E)-, valine (V)-, and lysine (K)-rich segment called PEVK segment. In addition to these segments, the extensible region of cardiac titin also contains a unique 572-residue sequence that is part of the cardiac-specific N2B element. In this work, immunoelectron microscopy was used to study the molecular origin of the in vivo extensibility of the I-band region of cardiac titin. The extensibility of the tandem Ig segments, the PEVK segment, and that of the unique N2B sequence were studied, using novel antibodies against Ig domains that flank these segments. Results show that only the tandem Igs extend at sarcomere lengths (SLs) below approximately 2.0 microm, and that, at longer SLs, the PEVK and the unique sequence extend as well. At the longest SLs that may be reached under physiological conditions ( approximately 2.3 microm), the PEVK segment length is approximately 50 nm whereas the unique N2B sequence is approximately 80 nm long. Thus, the unique sequence provides additional extensibility to cardiac titins and this may eliminate the necessity for unfolding of Ig domains under physiological conditions. In summary, this work provides direct evidence that the three main molecular subdomains of N2B titin are all extensible and that their contribution to extensibility decreases in the order of tandem Igs, unique N2B sequence, and PEVK segment.     

Single Molecule Force Spectroscopy of the Cardiac Titin N2B Element: EFFECTS OF THE MOLECULAR CHAPERONE ��B-CRYSTALLIN WITH DISEASE-CAUSING MUTATIONS*

The small heat shock protein αB-crystallin interacts with N2B-Us, a large unique sequence found in the N2B element of cardiac titin. Using single molecule force spectroscopy, we studied the effect of αB-crystallin on the N2B-Us and its flanking Ig-like domains. Ig domains from the proximal tandem Ig segment of titin were also studied. The effect of wild type αB-crystallin on the single molecule force-extension curve was determined as well as that of mutant αB-crystallins harboring the dilated cardiomyopathy missense mutation, R157H, or the desmin-related myopathy mutation, R120G. Results revealed that wild type αB-crystallin decreased the persistence length of the N2B-Us (from ∼0.7 to ∼0.2 nm) but did not alter its contour length. αB-crystallin also increased the unfolding force of the Ig domains that flank the N2B-Us (by 51 ± 3 piconewtons); the rate constant of unfolding at zero force was estimated to be ∼17-fold lower in the presence of αB-crystallin (1.4 × 10^-4 s^-1 versus 2.4 × 10^-3 s^-1). We also found that αB-crystallin increased the unfolding force of Ig domains from the proximal tandem Ig segment by 28 ± 6 piconewtons. The effects of αB-crystallin were attenuated by the R157H mutation (but were still significant) and were absent when using the R120G mutant. We conclude that αB-crystallin protects titin from damage by lowering the persistence length of the N2B-Us and reducing the Ig domain unfolding probability. Our finding that this effect is either attenuated (R157H) or lost (R120G) in disease causing αB-crystallin mutations suggests that the interaction between αB-crystallin and titin is important for normal heart function.αB-crystallin is a member of the small heat shock protein family that by inhibiting denaturation and aggregation of proteins functions as a molecular chaperone (1). Although αB-crystallin has been most intensively studied in the vertebrate eye lens, it is also found in many other tissues (2) with cardiac muscle expressing αB-crystallin at 3-5% of the total soluble protein (3). Up-regulation of αB-crystallin occurs in a number of cardiac disorders, including familial cardiac hypertrophy, and overexpression appears to protect the cardiac cell from ischemia reperfusion injury (for a review see Ref. 4). An important binding partner of αB-crystallin in cardiac muscle is titin (5, 6). Titin is a large filamentous protein that forms a continuous filament along the myofibril, with single titin molecules spanning from the edge to the middle of the sarcomere, a distance of ∼1 μm (7). The I-band region of titin is extensible and functions as a molecular spring that, when extended, develops force (8, 9). This force is an important determinant of the passive stiffness of the heart that determines the filling characteristics during the diastolic part of the heart cycle (10). The interaction between αB-crystallin and titin could be important for maintaining heart function, especially when stressed, such as during ischemia (5), warranting studies of the effect of αB-crystallin on the biomechanical properties of titin.The molecular spring region of titin contains three distinct spring elements (7). The first element is the tandem Ig segment, consisting of serially linked Ig domains that form the so-called proximal tandem Ig segment (15 Ig domains) near the Z-disk of the sarcomere and a distal segment (22 Ig domains) near the A-band (11). The second spring element is the PEVK, a unique sequence that contains largely prolines, glutamates, valines, and lysines (11). The third element consists of a large unique sequence (in human 572 residues in size) named the N2B-Us; it is heart-specific and dominates the extension of titin near the upper limit of the physiological sarcomere length range (12). αB-crystallin appears to preferentially bind to the N2B-Us, although weak binding to Ig domains has also been detected (6). Previous studies have shown that αB-crystallin increases the unfolding force of Ig 91-98, a fragment that contains eight Ig domains from the distal tandem Ig segment of titin (6). However, the mechanical effect of αB-crystallin on the N2B-Us (its main binding partner in titin) has not been investigated.The association between αB-crystallin and titin has prompted a search for disease causing mutations in αB-crystallin. This revealed in patients with dilated cardiomyopathy (DCM),² a missense mutation, R157H, that affects an evolutionarily conserved amino acid residue; the mutation decreases the binding to the N2B domain without affecting distribution of the mutant crystallin protein in cardiomyocytes (13). In another disease, the desmin-related myopathy mutation R120G (14) decreases the binding of αB-crystallin to the N2B element and causes intracellular aggregates of the mutant protein (13).In the present study, we used single molecule force spectroscopy and determined the contour length (CL; end-to-end length when stretched with infinite force) and persistence length (PL; a measure of the bending rigidity) of the N2B-Us. We also studied the unfolding force of Ig domains, those that flank the N2B-Us and those that make up the proximal tandem Ig segment. In addition, we investigated the effect of wild type and R157H and R120G αB-crystallin on the molecular mechanics of the N2B-Us, its flanking Ig domains, and the Ig domains in the proximal tandem Ig segment. Findings support that αB-crystallin functions as a chaperone that lowers the probability of Ig domain unfolding and the persistence length of the titin N2B-Us spring region. Importantly, this chaperone function is significantly reduced by the R157H mutation and abolished by the R120G mutation.     

Molecular Characterisation of Titin N2A and Its Binding of CARP Reveals a Titin/Actin Cross-linking Mechanism

Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remains poorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress.     

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.     

Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.     

1H, 15N and 13C backbone chemical shift assignment of titin domains A59–A60 and A60 alone

The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is >1 μm long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or ‘super-repeats’. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619–628, 2012). Here we report backbone ¹⁵N, ¹³C and ¹H chemical shift and ¹³Cβ assignments for the A59–A60 domain tandem from the titin A59–A69 large super-repeat, completed using triple resonance NMR. Since, some regions of the backbone remained unassigned in A60 domain of the complete A59–A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59–A60.     

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.     

Titin Extensibility In Situ: Entropic Elasticity of Permanently Folded and Permanently Unfolded Molecular Segments

Abstract. Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293–296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres (~2.0 μm) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from ~2.0 to 2.7 μm, the “contracted” tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond ~2.7 μm, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112–1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force–SL relation that is not achievable with a single elastic segment.     

Method for isolation of intact titin (connectin) molecules from mammalian cardiac muscle

Cardiac titin was isolated from rabbit and ground squirrel ventricular muscles by a method that was used earlier to obtain myofibrils with intact minor proteins located in A-bands of sarcomeres (Podlubnaya, Z. A., et al. (1989) J. Mol. Biol., 210, 655–658). Small pieces of cardiac muscle were incubated for 2–3 weeks at 4°C in Ca²⁺-depleting solution before their homogenization to decrease activity of Ca²⁺-dependent proteases. Then the muscle was homogenized, and titin was isolated by the method of Soteriou, A., et al. (1993) J. Cell Sci., 14, 119–123. In control experiments, titin was isolated from cardiac muscle without its preincubation in Ca²⁺-depleting solution. Sometimes control titin preparations contained only T2-fragment, but generally they contained ～5–20% N2B-isoform of titin along with its T2-fragment. Preparations of titin obtained from rabbit cardiac muscle by our method contained ～30–50% of N2BA- and N2B-titin isoforms along with its T2-fragment. The content of α-structures in titin isolated by our method was increased. Actomyosin ATPase activity in vitro increased in the presence of titin preparations containing more intact molecules. This result confirms the significant role of titin in the regulation of actin-myosin interaction in muscles. The method used by us to preserve titin might be used for isolation of other proteins that are substrates of Ca²⁺-dependent proteases.     

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.     

Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca²⁺ sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb²⁺, a structural and functional surrogate of Ca²⁺, to generate the protein states with well-defined protein-metal ion stoichiometry. NMR experiments revealed that binding of one divalent metal ion per C2 domain results in loss of conformational plasticity of the loop regions, potentially pre-organizing them for additional metal-ion and membrane-binding events. In C2A, a divalent metal ion in site 1 is sufficient to drive its weak association with phosphatidylserine-containing membranes, whereas in C2B, it enhances the interactions with the signaling lipid phosphatidylinositol-4,5-bisphosphate. In full-length Syt1, both Pb²⁺-complexed C2 domains associate with phosphatidylserine-containing membranes. Electron paramagnetic resonance experiments show that the extent of membrane insertion correlates with the occupancy of the C2 metal ion sites. Together, our results indicate that upon partial metal ion saturation of the intra-loop region, Syt1 adopts a dynamic, partially membrane-bound state. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, “prime” Syt1 for cooperative binding of a full complement of metal ions and deeper membrane insertion.     

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.     

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.     

Structural evidence for a possible role of reversible disulphide bridge formation in the elasticity of the muscle protein titin

BACKGROUND: The giant muscle protein titin contributes to the filament system in skeletal and cardiac muscle cells by connecting the Z disk and the central M line of the sarcomere. One of the physiological functions of titin is to act as a passive spring in the sarcomere, which is achieved by the elastic properties of its central I band region. Titin contains about 300 domains of which more than half are folded as immunoglobulin-like (Ig) domains. Ig domain segments of the I band of titin have been extensively used as templates to investigate the molecular basis of protein elasticity. RESULTS: The structure of the Ig domain I1 from the I band of titin has been determined to 2.1 A resolution. It reveals a novel, reversible disulphide bridge, which is neither required for correct folding nor changes the chemical stability of I1, but it is predicted to contribute mechanically to the elastic properties of titin in active sarcomeres. From the 92 Ig domains in the longest isoform of titin, at least 40 domains have a potential for disulphide bridge formation. CONCLUSIONS: We propose a model where the formation of disulphide bridges under oxidative stress conditions could regulate the elasticity of the I band in titin by increasing sarcomeric resistance. In this model, the formation of the disulphide bridge could refrain a possible directed motion of the two beta sheets or other mechanically stable entities of the I1 Ig domain with respect to each other when exposed to mechanical forces.     

Association of the chaperone alphaB-crystallin with titin in heart muscle

alphaB-crystallin, a major component of the vertebrate lens, is a chaperone belonging to the family of small heat shock proteins. These proteins form oligomers that bind to partially unfolded substrates and prevent denaturation. alphaB-crystallin in cardiac muscle binds to myofibrils under conditions of ischemia, and previous work has shown that the protein binds to titin in the I-band of cardiac fibers (Golenhofen, N., Arbeiter, A., Koob, R., and Drenckhahn, D. (2002) J. Mol. Cell. Cardiol. 34, 309-319). This part of titin extends as muscles are stretched and is made up of immunoglobulin-like modules and two extensible regions (N2B and PEVK) that have no well defined secondary structure. We have followed the position of alphaB-crystallin in stretched cardiac fibers relative to a known part of the titin sequence. alphaB-crystallin bound to a discrete region of the I-band that moved away from the Z-disc as sarcomeres were extended. In the physiological range of sarcomere lengths, alphaB-crystallin bound in the position of the N2B region of titin, but not to PEVK. In overstretched myofibrils, it was also in the Ig region between N2B and the Z-disc. Binding between alphaB-crystallin and N2B was confirmed using recombinant titin fragments. The Ig domains in an eight-domain fragment were stabilized by alphaB-crystallin; atomic force microscopy showed that higher stretching forces were needed to unfold the domains in the presence of the chaperone. Reversible association with alphaB-crystallin would protect I-band titin from stress liable to cause domain unfolding until conditions are favorable for refolding to the native state.     

Membrane-Binding Cooperativity and Coinsertion by C2AB Tandem Domains of Synaptotagmins 1 and 7

Synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7) contain analogous tandem C2 domains, C2A and C2B, which together sense Ca²⁺ to bind membranes and promote the stabilization of exocytotic fusion pores. Syt-1 triggers fast release of neurotransmitters, whereas Syt-7 functions in processes that involve lower Ca²⁺ concentrations such as hormone secretion. Syt-1 C2 domains are reported to bind membranes cooperatively, based on the observation that they penetrate farther into membranes as the C2AB tandem than as individual C2 domains. In contrast, we previously suggested that the two C2 domains of Syt-7 bind membranes independently, based in part on measurements of their liposome dissociation kinetics. Here, we investigated C2A-C2B interdomain cooperativity with Syt-1 and Syt-7 using directly comparable measurements. Equilibrium Ca²⁺ titrations demonstrate that the Syt-7 C2AB tandem binds liposomes lacking phosphatidylinositol-4,5-bisphosphate (PIP₂) with greater Ca²⁺ sensitivity than either of its individual domains and binds to membranes containing PIP₂ even in the absence of Ca²⁺. Stopped-flow kinetic measurements show differences in cooperativity between Syt-1 and Syt-7: Syt-1 C2AB dissociates from PIP₂-free liposomes much more slowly than either of its individual C2 domains, indicating cooperativity, whereas the major population of Syt-7 C2AB has a dissociation rate comparable to its C2A domain, suggesting a lack of cooperativity. A minor subpopulation of Syt-7 C2AB dissociates at a slower rate, which could be due to a small cooperative component and/or liposome clustering. Measurements using an environment-sensitive fluorescent probe indicate that the Syt-7 C2B domain inserts deeply into membranes as part of the C2AB tandem, similar to the coinsertion previously reported for Syt-1. Overall, coinsertion of C2A and C2B domains is coupled to cooperative energetic effects in Syt-1 to a much greater extent than in Syt-7. The difference can be understood in terms of the relative contributions of C2A and C2B domains toward membrane binding in the two proteins.     

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.     

Electron Microscopy and 3D Reconstruction Reveals Filamin Ig Domain Binding to F-Actin

Filamin A (FLNa) is an actin-binding protein that cross-links F-actin into networks of orthogonally branched filaments. FLNa also directs the networks to integrins while responding to mechanochemical signaling pathways. Flexible, 160-nm-long FLNa molecules are tail-to-tail dimers, each subunit of which contains an N-terminal calponin homology (CH)/actin-binding domain connected by a series of 24 immunoglobulin (Ig) repeats to a dimerization site at their C-terminal end. Whereas the contribution of the CH domains to F-actin affinity is weak (apparent K_a ~ 10⁵), the binding of the intact protein to F-actin is strong (apparent K_a ~ 10⁸), suggesting involvement of additional parts of the molecule in this association. Indeed, previous results indicate that Ig repeats along FLNa contribute significantly to the strength of the actin filament interaction. In the current study, we used electron microscopy and three-dimensional reconstruction to elucidate the structural basis of the Ig repeat–F-actin binding. We find that FLNa density is clearly delineated in reconstructions of F-actin complexed either with a four-Ig-repeat segment of FLNa containing Ig repeat 10 or with immunoglobulin-like filamin A repeat (IgFLNa)10 alone. The mass attributable to IgFLNa10 lies peripherally along the actin helix over the N-terminus of actin subdomain 1. The IgFLNa10 interaction appears to be specific, since no other individual Ig repeat or fragment of the FLNa molecule examined, besides ones with IgFLNa10 or CH domains, decorated F-actin filaments or were detected in reconstructions. We conclude that the combined interactions of CH domains and the IgFLNa10 repeat provide the binding strength of the whole FLNa molecule and propose a model for the association of IgFLNa10 on actin filaments.