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Properties of Titin Immunoglobulin and Fibronectin-3 Domains

2004; Elsevier BV; Volume: 279; Issue: 45 Linguagem: Inglês

10.1074/jbc.r400023200

1083-351X

Larissa Tskhovrebova, John Trinick,

Cellular Mechanics and Interactions

Immunoglobulin (Ig) 1The abbreviations used are: Ig, immunoglobulin; Fn3, fibronectin-3; pN, piconewtons.and fibronectin-3 (Fn3) domains are common building blocks of many extracellular proteins involved in ligand recognition and cell adhesion. Ig and Fn3 domains are also the main components of a group of intracellular proteins associated with the contractile apparatus of muscles. The largest of the intracellular group is titin (∼3 MDa), which has key roles in the assembly and elasticity of vertebrate striated muscles. The titin molecule spans one-half of the sarcomere, the repeating contractile unit that gives striated muscle its characteristic striped appearance. Titin interacts with a majority of sarcomere proteins and is also likely to bind a variety of cytoplasmic signaling molecules. The Ig and Fn3 domains are important in titin interactions, whereas the levels of interdomain mobility and structural stability relate directly to mechanical functions. Titin and the other muscle Ig/Fn3 proteins are likely to have a common ancestry. It is plausible that the emergence and evolution of the muscle Ig/Fn3 proteins were closely related to the origin and evolution of multicellularity and cell differentiation. This review summarizes current knowledge regarding the properties of the Ig and Fn3 domains of titin. General properties of titins are presented only briefly and are described in detail in recent reviews (1Tskhovrebova L. Trinick J. Nat. Rev. Mol. Cell. Biol. 2003; 4: 679-689Crossref PubMed Scopus (284) Google Scholar, 2Granzier H.L. Labeit S. Circ. Res. 2004; 94: 284-295Crossref PubMed Scopus (469) Google Scholar, 3Miller M.K. Granzier H. Ehler E. Gregorio C.C. Trends Cell Biol. 2004; 14: 119-126Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Titins are formed from the largest polypeptides so far found in nature. The 363 exons estimated to comprise the human titin gene contain 114,114 bp, corresponding to a polypeptide of up to 38,138 amino acid residues long (4Labeit S. Kolmerer B. Science. 1995; 270: 293-296Crossref PubMed Scopus (1004) Google Scholar, 5Bang M.L. Centner T. Fornoff F. Geach A.J. Gotthardt M. McNabb M. Witt C.C. Labeit D. Gregorio C.C. Granzier H. Labeit S. Circ. Res. 2001; 89: 1065-1072Crossref PubMed Scopus (518) Google Scholar) (Swiss-Prot/TrEMBL accesion numbers Q10465, Q10466, and Q8WZ42). Isoforms sequenced from cardiac and skeletal muscle cDNAs contain between 240 and 300 of the Ig- and Fn-like domains. Assuming a linear arrangement of domains each ∼4 nm long, this indicates a molecule more than 1 μm long, which compares well with electron microscope measurements of the purified protein. The domain arrangement is also visible directly in a "string-of-beads" appearance in micrographs (6Trinick J. Knight P. Whiting A. J. Mol. Biol. 1984; 180: 331-356Crossref PubMed Scopus (142) Google Scholar). In situ the titin molecule spans one-half of the sarcomere, which is the repeating contractile unit of muscle, composed mainly of ordered arrays of thin (actin) and thick (myosin) filaments (Fig. 1). The N terminus is at the boundary of sarcomere, the Z-line, and the C terminus is in the M-line at the center of the sarcomere. More than one-half of the molecule (the A-band section) is an integral part of the myosin filament. This region is formed mainly by Ig and Fn3 domains. The I-band part of titin forms an elastic connection between the end of the thick filament and the Z-line, which changes its end-to-end distance when muscle contracts or extends. This part of titin does not contain Fn3 domains and has only Ig and unique sequences. Most of the Ig domains are arranged in tandem in two segments, "proximal" and "distal" to the Z-line, which flank the N2-PEVK region containing mainly unique sequences. On average, the titin Ig and Fn3 sequence motifs are less than 50% conserved within each family. However, each family has distinct subfamilies with sequence identity up to 90%. In the I-band part, the Ig domains constitutively expressed and those differentially expressed in isoforms fall into different subfamilies (7Witt C.C. Olivieri N. Centner T. Kolmerer B. Millevoi S. Morell J. Labeit D. Labeit S. Jockusch H. Pastore A. J. Struct. Biol. 1998; 122: 206-215Crossref PubMed Scopus (45) Google Scholar). The differentially expressed segment contains Ig domains from several different subfamilies that are arranged in long range patterns or "super-repeats" consisting of 6 and 10 domains (8Gautel M. Adv. Biophys. 1996; 33: 27-37Crossref PubMed Scopus (21) Google Scholar). In the A-band part, the Ig and Fn3 domains are arranged in 7- and 11-domain super-repeats, which are repeated 6 and 11 times, respectively. The domains at similar positions within different super-repeats have higher sequence homology than the domains from the same super-repeat, indicating that they belong to different subfamilies (9Muhle-Goll C. Pastore A. Nilges M. Structure. 1998; 6: 1291-1302Abstract Full Text Full Text PDF PubMed Google Scholar, 10Amodeo P. Fraternali F. Lesk A.M. Pastore A. J. Mol. Biol. 2001; 311: 283-296Crossref PubMed Scopus (28) Google Scholar). Structural modeling predicted that the subfamily-specific consensus sequences and residues would define the general surface and interface differences between domain subfamilies. Thus, in the I-band the constitutively and differentially expressed Ig domains are likely to differ systematically in exposed polar and charged residues and in the stiffness of domain interfaces (11Fraternali F. Pastore A. J. Mol. Biol. 1999; 290: 581-593Crossref PubMed Scopus (28) Google Scholar). Likewise, in the A-band the Fn3 domains are predicted to have subfamily-specific clusters of conserved residues, mostly charged, on their surfaces and interfaces (9Muhle-Goll C. Pastore A. Nilges M. Structure. 1998; 6: 1291-1302Abstract Full Text Full Text PDF PubMed Google Scholar, 10Amodeo P. Fraternali F. Lesk A.M. Pastore A. J. Mol. Biol. 2001; 311: 283-296Crossref PubMed Scopus (28) Google Scholar, 12Muhle-Goll C. Habeck M. Cazorla O. Nilges M. Labeit S. Granzier H. J. Mol. Biol. 2001; 313: 431-447Crossref PubMed Scopus (85) Google Scholar). The structures of titin domains inferred from sequence were confirmed by NMR and x-ray crystallography. The expressed domains that have been solved are representative of the M-line region, Ig-M5 (13Pfuhl M. Pastore A. Structure. 1995; 3: 391-401Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), the constitutive distal segment, Ig-I27 (14Improta S. Politou A.S. Pastore A. Structure. 1996; 4: 323-337Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), the A-band, Fn3-A71 (9Muhle-Goll C. Pastore A. Nilges M. Structure. 1998; 6: 1291-1302Abstract Full Text Full Text PDF PubMed Google Scholar), and the constitutive proximal Ig segment, Ig-I1 (16Mayans O. Wuerges J. Canela S. Gautel M. Wilmanns M. Structure. 2001; 9: 331-340Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In addition, atomic structures have been obtained of polydomain constructs (17Improta S. Krueger J.K. Gautel M. Atkinson R.A. Lefevre J.F. Moulton S. Trewhella J. Pastore A. J. Mol. Biol. 1998; 284: 761-777Crossref PubMed Scopus (74) Google Scholar), the kinase domain (18Mayans O. van der Ven P.F.M. Wilm M. Mues A. Young P. Furst D.O. Wilmanns M. Gautel M. Nature. 1998; 395: 863-869Crossref PubMed Scopus (315) Google Scholar), and a motif repeat of the unique PEVK region (19Ma K. Kan L.S. Wang K. Biochemistry. 2001; 40: 3427-3438Crossref PubMed Scopus (120) Google Scholar). The titin Ig structures have the β-sandwich fold characteristic of the immunoglobulin superfamily (Fig. 2). The fold contains 8–9 β-strands arranged in two sheets, ABED and A′GFC(C′), packed face-to-face. A disulfide bridge connecting the two sheets was observed only in one case, the I1 domain. This bridge, however, differs from the classical Ig disulfide, which links strands B and F; instead it connected strands C and E. Close inspection of the structures and sequences placed the domains in the intermediate I-set of the Ig superfamily (13Pfuhl M. Pastore A. Structure. 1995; 3: 391-401Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). This implied that, unlike domains of the constant C2-set to which they were originally assigned, titin Ig domains are more similar to the V-set (variable) domains. The conserved pattern of residues specific to the I-set, the V-frame (20Harpaz Y. Chothia C. J. Mol. Biol. 1994; 238: 528-539Crossref PubMed Scopus (377) Google Scholar), had earlier been detected in other muscle Ig proteins and was also found in all the titin Ig domains. Thus I27, M5, and I1 all are similar to the typical I-set domain, telokin (20Harpaz Y. Chothia C. J. Mol. Biol. 1994; 238: 528-539Crossref PubMed Scopus (377) Google Scholar). The deviations from this pattern include inconsistency in structuring of the A, A′ and C′ strands and absence of some key interactions involving loops A′B and EF and strand D. There are also some variations between the domains in the length of the β-strands, in the size and conformation of loops, and in the overall shape. These differences seem to reflect general structural differences between the domain subfamilies (7Witt C.C. Olivieri N. Centner T. Kolmerer B. Millevoi S. Morell J. Labeit D. Labeit S. Jockusch H. Pastore A. J. Struct. Biol. 1998; 122: 206-215Crossref PubMed Scopus (45) Google Scholar, 11Fraternali F. Pastore A. J. Mol. Biol. 1999; 290: 581-593Crossref PubMed Scopus (28) Google Scholar). Titin domain A71, the only structure of an intracellular Fn3 domain so far solved, was confirmed to have the characteristic Fn3 family fold (9Muhle-Goll C. Pastore A. Nilges M. Structure. 1998; 6: 1291-1302Abstract Full Text Full Text PDF PubMed Google Scholar). The strongest resemblance is to the first Fn3 domain of Drosophila neuroglian and to Fn3 domains in fibronectin and tenascin. Sequence analysis indicated that all titin Fn3 domains contain key residues specific to the Fn3 family that form the hydrophobic core of the domain. In addition to these residues, titin domains were also found to contain titin-specific sets of conserved residues. Insights into the relative arrangement of domains and the overall conformation of the titin molecule have come from combined experimental and modeling studies. A model of the constitutively expressed I-band part, calculated from NMR data, supported the expected beads-on-a-string arrangement (17Improta S. Krueger J.K. Gautel M. Atkinson R.A. Lefevre J.F. Moulton S. Trewhella J. Pastore A. J. Mol. Biol. 1998; 284: 761-777Crossref PubMed Scopus (74) Google Scholar). This also suggested bending (∼155°) and twisting (∼90°) between successive domains, resulting in 10–17% shortening of the end-to-end distance from a fully extended conformation. The interdomain mobility appeared to be low, indicating a relatively well defined overall conformation of the constitutively expressed segment. The differentially expressed Ig segment was predicted to have an even more regular and stiff conformation, because of the presence of super-repeats and higher stiffness of interdomain interfaces suggested by sequence analysis. A-band titin is also likely to have an extended and relatively stiff conformation, based on modeling of the arrangement of the Fn3 domains, which are predominant in this part of the molecule (9Muhle-Goll C. Pastore A. Nilges M. Structure. 1998; 6: 1291-1302Abstract Full Text Full Text PDF PubMed Google Scholar, 10Amodeo P. Fraternali F. Lesk A.M. Pastore A. J. Mol. Biol. 2001; 311: 283-296Crossref PubMed Scopus (28) Google Scholar, 12Muhle-Goll C. Habeck M. Cazorla O. Nilges M. Labeit S. Granzier H. J. Mol. Biol. 2001; 313: 431-447Crossref PubMed Scopus (85) Google Scholar). The super-repeat pattern of A-band titin and the predicted high stiffness of the interdomain interfaces again suggest a regular conformation. Mobility between domains is likely to be the major source of the flexibility in titin. Flexibility is especially important in the I-band region that connects thick filaments to the Z-line. As described above, this region has important mechanical roles and is the origin of elasticity in relaxed sarcomeres. It also maintains the central position of the thick filament in the sarcomere, which ensures balance of forces between both its halves during the contractile cycle. As the sarcomere changes length during muscle contraction or passive extension, the I-band titin changes its conformation, coiling up or elongating. Although mobility between domains is predicted to be low throughout the molecule, its level appears to be enough to provide the considerable overall flexibility. Studies of the purified protein indicate a value of persistence length in the range 13–19 nm, implying substantial bending over molecule segments containing 3–4 domains (21Higuchi H. Nakauchi Y. Maruyama K. Fujime S. Biophys. J. 1993; 65: 1906-1915Abstract Full Text PDF PubMed Scopus (67) Google Scholar, 22Tskhovrebova L. Trinick J. J. Mol. Biol. 2001; 310: 755-771Crossref PubMed Scopus (47) Google Scholar, 23Kellermayer M.S.Z. Bustamante C. Granzier H.L. Biochim. Biophys. Acta. 2003; 1604: 103-114Google Scholar). Thermal Stability—Thermal unfolding of individual expressed titin Ig domains has been shown to be reversible and to occur in a simple two-state reaction. The relatively wide range of the transition temperatures, 35–70 °C (24Politou A.S. Thomas D.J. Pastore A. Biophys. J. 1995; 69: 2601-2610Abstract Full Text PDF PubMed Scopus (107) Google Scholar), is similar to the range observed for extracellular Ig domains. Studies of domain pairs indicate independent unfolding of each domain, as well as mutual stabilization (25Politou A.S. Gautel M. Improta S. Vangelista L. Pastore A. J. Mol. Biol. 1996; 255: 604-616Crossref PubMed Scopus (70) Google Scholar). Confinement within narrow pores to mimic cellular environment also increases stability (26Bolis D. Politou A.S. Kelly G. Pastore A. Temussi P.A. J. Mol. Biol. 2004; 336: 203-212Crossref PubMed Scopus (67) Google Scholar). With the exception of M-line domains, the average domain stability appears to be higher in the I-band than in the A-band part of the molecule. Chemical Stability—Chemical denaturation of titin domains correlates with the thermal denaturation data. Unfolding again follows essentially a two-state process, although in some cases there is evidence of low stability intermediates (24Politou A.S. Thomas D.J. Pastore A. Biophys. J. 1995; 69: 2601-2610Abstract Full Text PDF PubMed Scopus (107) Google Scholar, 27Carrion-Vazquez M. Oberhauser A.F. Fowler S.B. Marszalek P.E. Broedel S.E. Clarke J. Fernandez J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3694-3699Crossref PubMed Scopus (893) Google Scholar, 28Scott K.A. Steward A. Fowler S.B. Clarke J. J. Mol. Biol. 2002; 315: 819-829Crossref PubMed Scopus (83) Google Scholar). The range of stabilities is relatively wide, with estimates of the free energy of unfolding between 2 and 10 kcal mol–1. The rates of folding and unfolding also vary widely. Unfolding rate constants are in the range 10–2 to 10–6 s–1, and folding rates are spread from 10–2 to 102 s–1 (28Scott K.A. Steward A. Fowler S.B. Clarke J. J. Mol. Biol. 2002; 315: 819-829Crossref PubMed Scopus (83) Google Scholar, 29Head J.G. Houmeida A. Knight P.J. Clarke A.R. Trinick J. Brady R.L. Biophys. J. 2001; 81: 1570-1579Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In multidomain constructs, stability increases slightly, and unfolding and refolding rates are slowed. I27 (from the distal Ig segment in the I-band) has been used extensively to study folding and unfolding mechanisms of Ig domains (30Fowler S.B. Clarke J. Structure. 2001; 9: 355-366Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 31Wright C.F. Lindorff-Larsen K. Randles L.G. Clarke J. Nat. Struct. Biol. 2003; 10: 658-662Crossref PubMed Scopus (151) Google Scholar, 32Wright C.F. Steward A. Clarke J. J. Mol. Biol. 2004; 338: 445-451Crossref PubMed Scopus (27) Google Scholar). Folding was shown to occur via a nucleation-condensation mechanism, in which the long range side-chain interactions of four key hydrophobic residues bring into close proximity the central regions of strands B, C, E, and F of the two β-sheets. These interactions define and stabilize the fold core, before the rest of the polypeptide is arranged around it. This folding pathway dominates in the physiological environment over alternative pathways controlled by local inter-residue interactions. Switching to the alternative pathways may occur in the presence of denaturants. Mechanical Stability—Titin and its expressed fragments were among the first macromolecules, the mechanical stabilities of which were explored at the single molecule level. Early availability of the atomic structure of I27 also led to it becoming popular for experimental and theoretical studies of mechanically induced unfolding. Studies of titin domains have provided powerful insights into the relationship between protein internal architecture and the levels of force and deformation it can withstand. Application of mechanical force deforms the domains and initiates unfolding by breaking the inter- and intrasheet bonds and stretching the polypeptide. Unfolding of multidomain constructs generates a series of steps or peaks in force-distance curves, reflecting sequential unfolding of the domains. Values of peak unfolding force and the pattern of unfolding depend on various factors, including not only the rate of extension, as was shown first, but also structural variations between domains, the pulling geometry (33Brockwell D.J. Paci E. Zinober R.C. Beddard G.S. Olmsted P.D. Smith D.A. Perham R.N. Radford S.E. Nat. Struct. Biol. 2003; 10: 731-737Crossref PubMed Scopus (328) Google Scholar) and overall compliance (34Zinober R.C. Brockwell D.J. Beddard G.S. Blake A.W. Olmsted P.D. Radford S.E. Smith D.A. Protein Sci. 2002; 11: 2759-2765Crossref PubMed Scopus (73) Google Scholar). One of the major differences observed between the unfolding patterns of different Ig domains relates to unfolding intermediates. Domains from the distal Ig segment of the I-band titin, such as I27, unfold in a three-state process (35Marszalek P.E. Lu H. Li H.B. Carrion-Vazquez M. Oberhauser A.F. Schulten K. Fernandez J.M. Nature. 1999; 402: 100-103Crossref PubMed Scopus (708) Google Scholar), whereas domains from the proximal Ig segment, such as I1, unfold by a two-state process (36Li H. Fernandez J.M. J. Mol. Biol. 2003; 334: 75-86Crossref PubMed Scopus (81) Google Scholar). Differences in unfolding behavior were predicted and explained by theoretical modeling as resulting from differences in the relative density of hydrogen bonding networks connecting pairs of the strands A and B and A′ and G in the N- and C-terminal parts of the domains (37Gao M. Lu H. Schulten K. J. Muscle Res. Cell Motil. 2002; 23: 513-521Crossref PubMed Scopus (59) Google Scholar). Stronger bonding network between strands A′ and G, compared with strands A and B in I27, acts as a "mechanical clamp" that stabilizes the intermediate state after early detachment of the A strand. Indeed, a mutant I27 lacking the A strand was found to be an adequate model of this intermediate, maintaining the architecture and integrity of the wild type protein with only a little loss of stability (38Fowler S.B. Best R.B. Herrera J.L.T. Rutherford T.J. Steward A. Paci E. Karplus M. Clarke J. J. Mol. Biol. 2002; 322: 841-849Crossref PubMed Scopus (171) Google Scholar). An important question raised by the stability studies concerns the similarity of unfolding mechanisms probed by different methods (27Carrion-Vazquez M. Oberhauser A.F. Fowler S.B. Marszalek P.E. Broedel S.E. Clarke J. Fernandez J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3694-3699Crossref PubMed Scopus (893) Google Scholar). It was realized that the same C-terminal region of the domain, stabilized by a network of interactions centered on the A′ and G strands, defines the level of its kinetic stability in resisting both mechanically and chemically induced unfolding (39Best R.B. Fowler S.B. Herrera J.L. Steward A. Paci E. Clarke J. J. Mol. Biol. 2003; 330: 867-877Crossref PubMed Scopus (154) Google Scholar, 40.Deleted in proofGoogle Scholar). However, the interactions in the core important for thermodynamic stability of the domain do not contribute to its mechanical properties (30Fowler S.B. Clarke J. Structure. 2001; 9: 355-366Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 41Brockwell D.J. Beddard G.S. Clarkson J. Zinober R.C. Blake A.W. Trinick J. Olmsted P.D. Smith D.A. Radford S.E. Biophys. J. 2002; 83: 458-472Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Also, the routes of unfolding induced by different methods are different. This was first suggested by the apparent absence of the mechanical unfolding intermediate of I27 in chemical denaturation data. Theoretical modeling also indicated that the earliest events are either different or differ in the proportions of conformational changes occurring in parallel (42Paci E. Karplus M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6521-6526Crossref PubMed Scopus (275) Google Scholar). Further experiments illustrated that I27 unfolds differently with these two methods (38Fowler S.B. Best R.B. Herrera J.L.T. Rutherford T.J. Steward A. Paci E. Karplus M. Clarke J. J. Mol. Biol. 2002; 322: 841-849Crossref PubMed Scopus (171) Google Scholar, 41Brockwell D.J. Beddard G.S. Clarkson J. Zinober R.C. Blake A.W. Trinick J. Olmsted P.D. Smith D.A. Radford S.E. Biophys. J. 2002; 83: 458-472Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Forces that unfold titin Fn3 domains were in the region 100–200 pN, suggesting that these domains are weaker than the Ig domains, which needed 150–300 pN at similar force-loading rates (43Rief M. Gautel M. Schemmel A. Gaub H.E. Biophys. J. 1998; 75: 3008-3014Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Studies of homologous domains in tenascin and fibronectin also showed generally lower mechanical strength. Overall, the experiments agree with conclusions drawn from comparative modeling of Ig and Fn3 domains, which emphasize how the details of domain structure influence unfolding (42Paci E. Karplus M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6521-6526Crossref PubMed Scopus (275) Google Scholar). Domain Stability and Mechanical Functions of Titin—Domain stability, especially mechanical stability in the I-band region, directly relates to the mechanical function of titin in the sarcomere. Recent reports show how the molecule structure has adapted to functional demands. First, as already outlined, Ig domains are strongest. The weaker Fn3 domains are present only in the A-band part of the molecule, which is stabilized within the thick filament structure. Second, within the I-band part, Ig domains differing in stability and unfolding properties are arranged in distinct patterns. Adjacent to the Z-line, the constitutive proximal Ig segment contains most of the weak Ig domains, with average unfolding forces of ∼150 pN (36Li H. Fernandez J.M. J. Mol. Biol. 2003; 334: 75-86Crossref PubMed Scopus (81) Google Scholar, 44Li H.B. Linke W.A. Oberhauser A.F. Carrion-Vazquez M. Kerkviliet J.G. Lu H. Marszalek P.E. Fernandez J.M. Nature. 2002; 418: 998-1002Crossref PubMed Scopus (425) Google Scholar). The differentially expressed domains, which are centrally positioned in the I-band, have intermediate stability and unfolding forces of ∼200 pN (43Rief M. Gautel M. Schemmel A. Gaub H.E. Biophys. J. 1998; 75: 3008-3014Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 45Watanabe K. Muhle-Goll C. Kellermayer M.S.Z. Labeit S. Granzier H. J. Struct. Biol. 2002; 137: 248-258Crossref PubMed Scopus (74) Google Scholar). Near the I/A junction, the constitutive distal segment contains the strongest domains, with an unfolding force of ∼250 pN (43Rief M. Gautel M. Schemmel A. Gaub H.E. Biophys. J. 1998; 75: 3008-3014Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 44Li H.B. Linke W.A. Oberhauser A.F. Carrion-Vazquez M. Kerkviliet J.G. Lu H. Marszalek P.E. Fernandez J.M. Nature. 2002; 418: 998-1002Crossref PubMed Scopus (425) Google Scholar, 45Watanabe K. Muhle-Goll C. Kellermayer M.S.Z. Labeit S. Granzier H. J. Struct. Biol. 2002; 137: 248-258Crossref PubMed Scopus (74) Google Scholar). Finally, the average unfolding rate constant of the proximal Ig domains is higher (∼10–3 s–1) than that of the distal Ig domains (∼10–4 s–1) (44Li H.B. Linke W.A. Oberhauser A.F. Carrion-Vazquez M. Kerkviliet J.G. Lu H. Marszalek P.E. Fernandez J.M. Nature. 2002; 418: 998-1002Crossref PubMed Scopus (425) Google Scholar). This implies that, if muscle stretch initiates multiple unfolding of I-band Ig domains, the process is likely to start near the Z-line region and then spread toward the A-band part of the molecule. For refolding to begin, the polypeptide has to collapse to almost the size of the intact domain to form the early native contacts (27Carrion-Vazquez M. Oberhauser A.F. Fowler S.B. Marszalek P.E. Broedel S.E. Clarke J. Fernandez J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3694-3699Crossref PubMed Scopus (893) Google Scholar, 30Fowler S.B. Clarke J. Structure. 2001; 9: 355-366Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 46Tskhovrebova L. Trinick J. Sleep J.A. Simmons R.M. Nature. 1997; 387: 308-312Crossref PubMed Scopus (667) Google Scholar). Refolding times, estimated from mechanical experiments, are on the order of seconds, with faster rates for the distal than the proximal domains (44Li H.B. Linke W.A. Oberhauser A.F. Carrion-Vazquez M. Kerkviliet J.G. Lu H. Marszalek P.E. Fernandez J.M. Nature. 2002; 418: 998-1002Crossref PubMed Scopus (425) Google Scholar). This indicates refolding may occur in situ, provided that after stretch the muscle is allowed to relax to its resting length and the time interval before the next extension is not too short. The relatively low probability of simultaneous unfolding of contiguous domains will minimize misfolding (28Scott K.A. Steward A. Fowler S.B. Clarke J. J. Mol. Biol. 2002; 315: 819-829Crossref PubMed Scopus (83) Google Scholar). The probability of domain unfolding during normal sarcomere function is relatively low. Domains are protected by the limits of normal muscle extension and by the high compliance N2-PEVK region of titin. However, if muscle is overextended, reversible unfolding of domains may protect titin and the sarcomere structure from permanent damage. Titin, with its giant size and hundreds of Ig and Fn3 domains, illustrates a natural "molecular platform," capable of binding diverse ligands according to a strict spatial pattern (Fig. 3). Among the first titin ligands identified were the thick filament proteins, myosin and C-protein. Since then, many other potential binding partners have been found, mostly by yeast two-hybrid assay. Overall, the data suggest that titin provides scaffolding not only for most structural proteins in the sarcomere, but also for many cytoplasmic proteins. Interactions of titin in the Z- and M-line regions have two roles: they contribute to the structural integrity of these parts of the sarcomere and they provide site-specific links with regulatory systems. The regulatory links are likely to differ in cardiac and skeletal muscles, in accordance with the structural and biochemical differences of these muscle types. Within the elastic I-band part of titin, which spans between the Z-line and the tip of thick filament, most of the interactions appear to be concentrated in the N2-PEVK region. Interactions here are mainly with cytosolic proteins of various signaling systems. The A-band part of titin is integral with the thick filament. Two major partners here, myosin and C-protein, are bound in a regular manner correlating with the periodic structure of the thick filament. Attempts have been made to trace evolutionary relationships between titin domains and other members of the muscle Ig family (47Higgins D.G. Labeit S. Gautel M. Gibson T.J. J. Mol. Evol. 1997; 38: 395-404Crossref Scopus (29) Google Scholar, 48Kenny P.A. Liston E.M. Higgins D.G. Gene (Amst.). 1999; 232: 11-23Crossref PubMed Scopus (55) Google Scholar). Major conclusions of this analysis are as follows. Firstly, the different regions of titin, the A-band part and the constitutively and differentially expressed I-band parts, evolved independently from common ancestral domains. Evolution of these regions involved a series of gene duplication events, including duplications at the level of groups of domains, giving rise to the domain super-repeats. Secondly, titin and the other vertebrate and invertebrate members of the muscle Ig family have a common ancestry, and a primordial protein must have existed before the divergence of vertebrates and nematodes. The evolutionary origin and expansion of multimodular proteins are thought to be closely related to the emergence and expansion of multicellularity and the animal world (15Patthy L. Genetica. 2003; 118: 217-231Crossref PubMed Scopus (132) Google Scholar). The Ig-like domains apparently considerably predated multicellular organisms and were some of the first to be recruited to produce adhesion molecules, which were needed for cell-cell and cell-substrate communications. It seems plausible that, later in evolution, increased complexity of multicellular organisms and cell differentiation necessitated new generations of intracellular "adhesive" proteins. Evolution of striated muscle cells, in particular, would have needed such proteins to order and stabilize the growing network of the contractile apparatus. The ancient adhesive Ig fold, with its low requirements for sequence conservation, and thus high ability to maintain structural framework while changing ligand-binding properties, was well suited to these tasks in muscle.