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Elastomeric proteins: biological roles, structures and mechanisms
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Elastomeric proteins: biological roles, structures and mechanisms Arthur S. Tatham and Peter R. Shewry Elastomeric proteins are able to withstand significant deformations without rupture before returning to their original state when the stress is removed. Although elastomeric proteins differ considerably in their amino acid sequence, they all have a complex domain structure and share two common properties. Namely, they contain elastomeric domains, comprised of repeated sequences, and additional domains that form intermolecular crosslinks. Furthermore, several protein contain b-turns as a structural motif within the elastomeric domains. ELASTIC PROTEINS ARE present in a diverse range of animal species and tissues where they have evolved precise structures and properties to perform specific biological functions1. These proteins all possess rubber-like elasticity, undergoing high deformation without rupture, storing the energy involved in deformation, and then recovering to their original state when the stress is removed. The second stage of this process is, therefore, passive in that it does not require energy input. In contrast to animals, elasticity is a rare phenomenon in plants, the only welldocumented system being the gluten proteins of wheat2. Moreover, in these proteins the elastic properties have no known biological role, but are exploited by humankind to make bread and other foods. The ability of proteins to exhibit rubber-like elasticity relates to their polymer structure. Rubber-like materials must satisfy two criteria. First, the individual monomers must be flexible and conformationally free, so that they can respond quickly to an applied force. Second, the individual monomers must be crosslinked to form a network. The crosslinks can be covalent or noncovalent, and most elastic proteins have evolved to combine elastomeric domains with domains that form A.S. Tatham and P.R. Shewry are at the Institute of Arable Crops Research-Long Ashton Research Station, Dept of Agricultural Sciences, University of Bristol, Bristol, UK BS41 9AF. Email: [email protected]
crosslinks. Thus, the elastic properties of protein polymers can be influenced by the lengths and properties of the elastic domains and the degree of crosslinking.
Distribution and sequences of elastomeric proteins Elastomeric proteins are widely distributed in the animal kingdom, but only a small number have been characterized in detail. Their intrinsic insolubility and their nonglobular nature have limited direct analysis, whereas sequence information from molecular-genetic studies has only become available in recent years. We will, therefore, limit the present discussion to the small number of elastic proteins that have been characterized in some detail. Despite differing greatly in their structures, all these proteins have two features in common. They all contain distinct domains (Fig. 1), at least one of which comprises elastic repeated sequences (Table 1), and crosslinks can be formed between sites in the nonelastic domains (Table 1). Elastin is widely distributed in vertebrate tissues. It performs various functions, acting statically in dermis to resist long-term forces and dynamically in arteries to store and release energy rapidly. It is secreted as a soluble precursor, tropoelastin, before forming the amorphous component of elastic fibres3. Tropoelastin consists of alternating repetitive hydrophobic domains of variable length (the elastic repeats) and alanine-rich, lysine-containing domains that form crosslinks4.
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Abductin and byssus both occur in bivalve molluscs but have different properties and functions. Abductin forms the inner hinge ligament of the shell, acting as an antagonist to the action of the adductor muscle, opening the shell when the adductor muscle relaxes. This action has developed to form a swimming mechanism for scallops allowing them to swim a few metres at a time5. The processing of abductin is unclear; the N-terminal domain might be cleaved during secretion6. Byssal threads attach mussels to hard surfaces in the sea. They display a gradient of mechanical properties, from stiff to elastic, along the length of the fibre that provides sufficient flexibility to prevent brittle fracture in tidal zones. Threads exhibiting different properties comprise different arrangements of domains7,8. Figure 1 shows a precursor of a byssal thread protein (PreCol-P), the elastic domains of which contain highly conserved pentapeptide motifs that can be extended by glycine residues (Table 1)9. Both proteins appear to be crosslinked, but the precise mechanisms and sites are not known (Table 1). Spiders produce several silks with different mechanical properties. The dragline silks form the dropping line and web framework and are stiffer than the flagelliform silks that form the spiral, capturing part of the web. Whereas the dragline silks will extend by approximately 30%, the flagelliform silks extend by ~200% of their length without breaking. The flagelliform silks have similar properties to a slightly crosslinked rubber; that is, a combination of low stiffness and high extensibility10,11. The combination of different silk proteins allows the spider to produce fibres with different mechanical properties and allows the web to absorb the energy of the impacting insect without catapulting the insect out of the web. The two types of silk differ in that the dragline silks contain polyalanine domains which divide the elastic polypentapeptide repeats and can form noncovalent links between proteins12,13. Titin (or connectin) is a sarcomeric protein responsible for the elasticity of vertebrate striated muscle myofibrils and is involved in muscle assembly14. It is present in isoforms that have different properties in different tissues (for example, in skeletal and heart muscle)14. The elastic properties of titin are responsible for the force that is generated when passive muscle is stretched, but titin also maintains the integrity of the sarcomere and therefore ensures efficient muscle contraction in actively
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contracting muscle. Titin is a giant protein, with the human cardiac form comprising approximately 26 900 amino acid residues14. The sequence is divided into the Z-disc (~2000 residues), I-band (~5900 residues), A-band (~17 200 residues) and M-line (~1100 residues) regions, corresponding to the light and dark transverse bands observed by microscopy of myofibrils14. The I-band is thought to be primarily responsible for the elastic properties of titin15,16 and contains elastic repeated sequences rich in P, E, V and K (Refs 17,18). Wheat gluten corresponds to the seed storage proteins that form an elastic network in doughs19. Although the sole function of gluten proteins is to store carbon and nitrogen, they have elastic properties, which are probably a fortuitous consequence of structures that have evolved to facilitate their efficient storage and packaging into protein bodies in the seed. One group of gluten proteins, the highmolecular-weight (HMW) subunits of glutenin, appears to be largely responsible for the formation of the elastic gluten network20. They are characterized by an extensive central domain of repeated sequences and form disulphide crosslinks. Comparison of the structures and repeat motifs shows striking similarities as well as differences. In particular, all contain elastic domains comprising repeated sequences that can be flanked by or interspersed with other nonelastic domains such as the polyalanine repeats in tropoelastin and dragline silks and the collagen-like domain in byssus. The elastic repeat motifs are all rich in glycine residues that are accompanied by other hydrophobic residues. However, differences are also present allowing them to be divided into four groups (Table 1). In particular, the re-
1
136
Abductin 1
882
Byssus HMW subunit Flagelliform silk Dragline silk
1
827
1
1030
613
1
759
1 Tropoelastin
−5900 Titin I-band Nonrepetitive Acidic domain
’Spacer’ domain Histidine-rich domain
Collagen-like domain Elastic repeat
Fn3 Polyalanine repeat
Ig Ti BS
Figure 1 Domain structures of the elastomeric proteins. Abductin, high-molecular-weight (HMW) subunits and flagelliform silks show similar domain structures, with the addition of ‘spacer’ domains in the latter. The dragline silks and tropoelastin elastic repeats interspersed with polyalanine regions. Byssus shows a more complicated structure with elastomeric domains flanking a collagen-like domain with N- and C-terminal histidine-rich domains. The titin I-band consists predominantly of immunoglobulin (Ig)-like and fibronectin type III (Fn3)-like domains, the elastomeric domain forming a minor part of the sequence. Sequences taken from abductin6, byssus9, HMW subunit20, flagelliform silk13, dragline silk12, tropoelastin4 and titin I-band14.
peats in the dragline silks and HMW subunits also contain glutamine, a bulky hydrophobic residue.
b-Turns as a structural feature of elastomeric proteins One requirement for polymer elasticity is that crosslinked molecules must be flexible and conformationally free. It might be expected, therefore, that this would preclude the formation of regular
secondary structures in the elastic domains, as these would not be as responsive to stress as unordered structures. However, the presence of regularly repeated sequences also implies a propensity to adopt a regular structure. Although direct determination of the structures of the elastic proteins is difficult, and limited information is available, the available data indicate that the repetitive domains do indeed form regular
Table 1. Repeat motifs and crosslinking mechanisms for elastomeric proteins Group
Protein
Elastic repeat motifsa
Nature of crosslinks
Ref.
1 2
Abductin Elastin
GGFGGMGGGx VPGG VPGVG APGVGV GPGGG
Unknown, disulphide (?) and/or via tyrosine residues (?) Via lysine residues in the alanine-rich regions
6 4
Unknown, metal complexation with histidine-rich regions (?) and/or via tyrosine residues (?) Unknown, noncovalent interactions in spacer regions (?) Noncovalent interactions between alanine-rich domains
8 13 28
Disulphide between N- and C-terminal domains
20
In Z-disc and M-line, mechanism unknown
18
Byssus
3
Flagelliform silk Dragline
HMW subunit
4
Titin (I-band)
GPGGx GPGQQ GPGGY GGYGPGS PGQGQQ GYYPTSPQQ GQQ PPAKVPEVPKKPVPEEKVPVPVPKKPEA
aGroup 1: glycine-rich; group 2: glycine- and proline-rich; group 3: glycine-, proline- and glutamine-rich; and group 4: proline-, glutamic acid-, valine- and lysine-rich. x is any amino acid.
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Figure 2 Computer-generated molecular model of a b-spiral for the backbone of the elastomeric domain of the high-molecular-weight (HMW) subunits of glutenin, showing the helical nature of the structure. The model was constructed from the 21-residue repeating motif (PGQGQQ GYYPTSPQQ PGQGQQ), with b-turns over tetrapeptide sequences QPGQ, YPTS, SPQQ and QGQQ generating a b-spiral with a diameter of 1.7 nm and a pitch of 1.3 nm. Red, oxygen atoms; blue, nitrogen atoms; green, carbon atoms. (A.S. Tatham, P.R. Shewry, D.J. Osguthorpe and O. Parchment, unpublished).
structures and that these might play important roles in the elastic mechanisms. Spectroscopic studies of elastin indicate that the backbone is highly mobile21 and that the alanine-rich regions, which contain the crosslinking sites, are a helical22. Urry et al. carried out spectroscopic studies of a-elastin. indicating the presence of b-turns as the dominant structural feature. However, the complex structure of elastin means that further information has been derived largely from studies of synthetic polymers based on the consensus tetra(VPGG), penta- (VPGVG) and hexapeptide (APGVGV) repeat motifs. In the synthetic polypentapeptide23 and polyhexapeptide24, the b-turns form a b-spiral, a loose water-containing helical structure25. However, only the polypentapeptide is elastic, with an elastic modulus (i.e. the force needed to elongate the material) similar to that of elastin. The dynamic b-spiral conformation of the polypentapeptide has no significant hydrogen bonding between the repeats, whereas the rigidity of the polyhexapeptide is thought to be due to an intramolecular hydrogen bonding between repeats and interlocking of the bspirals by hydrophobic interactions. By contrast, polymers based on the
tetrapeptide motif (VPGG) appeared to form a cross-b structure, where the bturns are separated by short b-strands, which exhibits little elasticity except at high (.40°C) temperatures26. The nonelastomeric structures formed by the tetra- and hexapeptides presumably play structural roles in elastin, but these are unclear at present. The alanine-rich regions of the spider dragline silks have been demonstrated by X-ray diffraction and NMR studies to form linked anti-parallel b-sheet regions27, compared with a-helical structures in the alanine-rich domains of elastin. These are thought to correspond to the crystalline regions that crosslink the protein molecules and provide the silks with their tensile strength. However, nothing is known about the structures formed by the nonrepetitive domains of the flagelliform silks, or by the ‘spacer regions’ within the repetitive domain13. In both types of silk, the repetitive domains have been proposed to form b-spiral structures. In the flagelliform silks, this structure might be based on b-turns formed by the pentapeptide repeat xPGGG (Table 1), which occurs up to 63 times in succession13,28. In dragline silks, the GGPGQ and YPGQQ pentapeptides are repeated up to nine
times and are also predicted to form bturns. The greater elasticity of the flagelliform silks could be related to their longer repetitive regions and the lower levels of crosslinking compared with the dragline silks. The repetitive domains of the HMW subunits of wheat glutenin comprise three repeats that are thought to be responsible for their elastic properties. Spectroscopic studies of linear and cyclic peptides corresponding to the repeat motifs indicate the presence of b-turns in the hexapeptide repeat (PGQGQQ) and nonapeptide repeat (GYYPTS[P/L]QQ) motifs29,30. Structure prediction indicates overlapping bturns, within and between repeat motifs29. Hydrodynamic studies show a rod-like molecule in solution31, and scanning tunnelling microscope images of HMW subunits deposited onto graphite reveal that the central repetitive domain adopts a helical structure with a diameter of ~1.9 nm and a pitch of ~1.5 nm32 (Fig. 2). Comparison of the I-band sequences of titin from heart, psoas (vertebral) and soleus (skeletal) muscle, each of which has different passive tensions, shows differences both in the tandem Ig-like and PEVK domains14. It was proposed initially that titin elasticity arose from the unfolding of the Ig-like b-barrel structure, but it is now thought to result from the properties of the PEVK domains15,33, which differ in length in the I-bands of different titin isoforms (from ~170 residues in cardiac muscle to ~2200 residues in human skeletal titin14). The PEVK region is more easily stretched than the Ig-like domains and is predicted to have an unstable structure due to the high charge density and high content of proline, preventing the formation of a stable tertiary structure14. Therefore, the elastomeric PEVK domain of titin has been described as a permanently unfolded nonglobular domain that functions as a stiff spring. However, this model is still controversial, and other studies have suggested a folded domain structure34,35. Prediction of the secondary structure of the PEVK domains suggests a polyproline-II-like structure (an extended left-handed helix with three residues per turn), because of the high content of proline residues and the formation of b-turns, so that the conformation could be an equilibrium between a b-spiral and an unfolded state. The secondary structures of the elastomeric domains of byssus and
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REVIEWS abductin have not yet been studied in detail. By analogy with elastin, the elastinlike domains of byssus are predicted to form b-turns, and a b-spiral structure could account for the elasticity. The predicted secondary structure of abductin indicates that the N-terminal domain adopts an a-helical structure, whereas the repetitive domain is a random coil. It can be concluded, therefore, that bturns are a common structural feature in elastomeric proteins or domains but are not universal.
Mechanisms of elasticity Elastomeric force (f; the force developed on stretching an elastomeric material) has two components, an entropic component, fs, and an internal energy component, fe, with f 5 fs 1 fs. On deformation, fe is attributed to strain in the bonds and fs to a decrease in the number of conformations in the lowest energy state. As bond strain can lead to bond breakage, an ideal elastomer is one in which fe 5 0 and f 5 fs. For a predominantly entropic elastomer the value of fe/f is less than 0.5, for elastin the value is 0.26 and for natural rubber 0.18. A number of different mechanisms have been proposed to explain the elastic force of elastin. In the first, which is based on classical rubber theory (where rubber-like properties are attributed to a decrease in conformational entropy on deforming a network of kinetically free, random polymer molecules), elastin is a network of random chains of high entropy. Stress orders the chains and decreases entropy by limiting their conformational freedom, thus providing the restoring force to the relaxed state36. The second model, proposed by Urry and co-workers, is that the elasticity arises from the b-spiral structure37. In the polypentapeptide, the b-turns are repeated regularly and act as spacers between the turns of the spiral, effectively suspending chain segments in a relatively kinetically free state. The peptide segments can undergo large-amplitude, low-frequency rocking motions, called librations. On stretching, these librations decrease in amplitude and result in a decrease in entropy, thereby providing the restoring force37. In both models the restoring force is entropic26,37. Another mechanism is based on hydrophobic interactions, where stretching is proposed to increase hydrophobic side-chain exposure to an aqueous environment, decreasing the entropy of the surrounding water molecules, the restoring force arises from
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TIBS 25 – NOVEMBER 2000 the hydrophobic forces that stabilize folded protein structures38. Resilin and abductin also behave as entropic elastomers, returning almost all the energy stored in deformation5,39. The sequence of abductin consists of hydrophobic repeats, with a low proline content, but no b-turns are predicted, and the formation of a b-spiral is unlikely. The mechanism of elasticity can, therefore, be similar to that of a classical rubber. The amino acid composition of resilin is similar to that of elastin, with high contents of glycine, alanine, proline and aspartate, but lack of sequence data prevents further comparisons. Studies of contracted dragline silks indicate that the predominant force for retraction is entropic, accounting for ~85% of the total force40. However, in this case some of the energy of the impacting insect is absorbed and dissipated as heat and not recovered in elastic recoil10. This has two consequences: the insect is unlikely to be catapulted back out of the web by elastic recoil, and the mechanical energy converted to heat is not available to fracture the silk thread. The flagelliform silk that forms the capture spiral is highly extensible and covered with small droplets of glue41. This extensibility means that the struggling insect has no solid structure to push against. Titin elasticity has been explored using a number of different techniques, including mechanical studies on single molecules using laser tweezers42, forceextension atomic force microscopy43 and
fluorescently labelled antibodies in single myofibrils15. These studies indicate that there is a two-stage extension mechanism, the first stage involving straightening of the titin molecule and the second, extension of the PEVK domain. Under normal physiological conditions of stretch, the Ig-like domains of the I-band remain folded, but unfolding does occur when extreme stress is applied in vitro. The elastic properties of titin have been modelled as entropic polymers; at low force titin behaves as a purely entropic polymer, whereas at higher extensions there were deviations from the experimental data that indicated an enthalpic component (such as hydrophobic and electrostatic interactions)16,33. Enthalpic elasticity at high stretch can provide a ‘buffer’ before the potentially destructive effect of unfolding the Ig-like domains occurs. Within the PEVK domain the enthalpic component could arise both from electrostatic forces (lowering the ionic strength results in increased titin stiffness) and hydrophobic interactions (exposure of the valine residues to water)33. Crosslinked HMW subunits of glutenin show a similar modulus of elasticity to the crosslinked polymers of elastin. However, the mechanism of elastic recoil is not predominantly entropic but is apparently associated with strain in the chemical bonds within the repetitive domain of the protein. There has been no evolutionary pressure to develop elastic mechanisms in seed proteins, and their elasticity might have arisen as
Axel Innis is a PhD student in the Dept of Biochemistry, University of Cambridge, UK.
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TIBS 25 – NOVEMBER 2000 a consequence of the evolution of structures that facilitate efficient packaging of the proteins to act as reserves for seed germination.
What is the biological significance of different structures and mechanisms of elastomeric proteins? The entropic mechanism exhibited by elastomers such as elastin is ideal for elastomeric fibres, which are required to last the lifetime of an individual, undergoing many ‘stress–strain’ cycles. It is not surprising, therefore, that several unrelated animal proteins have evolved a similar entropic mechanism for different uses, such as flight and swimming. By contrast, stretching bonds in predominantly nonentropic systems, such as gluten, can lead to rupture. This is hardly ideal in constantly working systems but is not detrimental in gluten, as the elasticity plays no biological role. Similarly, spider silks do not exhibit an entirely entropic mechanism, as this would not serve their purpose in prey capture. It is clear, therefore, that the structures and mechanisms of elastic proteins have evolved to fulfil precise biological roles. However, the presence of both similarities and differences between the structural organization, sequences and secondary structures of different elastomeric proteins shows that the requirements can be satisfied in different ways. This is perhaps not surprising in view of the diverse range of organisms in which elastomeric proteins occur.
Future directions for research Major advances in the study of elastic proteins have come from two areas. The first is molecular biology, in particular molecular cloning, which has provided amino acid sequences of proteins whose size, complexity, repetitive structure and insolubility allow restricted sequence analysis by conventional approaches. The second advance has been in the ability to determine directly the mechanical properties of single molecules using atomic force microscopy44 or laser tweezers45. These techniques were initially used to study the elastic properties of titin and have also been used to determine the mechanical properties of a number of non-elastic biological systems such as nacre (mollusc shell)46. There is no doubt that elastomeric proteins have great potential as novel industrial materials. Elastomeric ‘patches’ made from synthetic polymers
based on the polypentapeptide repeat motif of elastin can be used in surgery to prevent adhesion of wounds47, whereas spider silks are being studied to determine the basis for their tensile strength (allegedly greater than steel) and elasticity48. Similarly, marine adhesion proteins such as byssus are considered to have potential as adhesive8. The success of these and other applications will depend on our ability to understand and manipulate protein elasticity at the molecular level.
Acknowledgements IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC).
References 1 Gosline, J.M. (1987) Structure and mechanical properties of rubberlike proteins in animals. Rubber Chem. Technol. 60, 417–438 2 Tatham, A.S. et al. (1985) The beta-turn conformation in wheat gluten proteins – relationship to gluten elasticity. Cereal Chem. 62, 405–412 3 Rosenbloom, J. et al. (1993) Extracellular matrix 4: the elastic fibre. FASEB J. 7, 1209–1218 4 Indik, Z. et al. (1987) Alternative splicing of human elastin mRA indicated by sequence analysis of cloned genomic and complementary DNA. Proc. Natl. Acad. Sci. U. S. A. 84, 5680–5684 5 Bowie, M.A. et al. (1993) Damping in the hinge of the scallop Placopecten magellanicus. J. Exp. Bot. 175, 311–315 6 Cao, Q. et al. (1997) Sequence of abductin, the molluscan ‘rubber protein’. Curr. Biol. 7, R677–R678 7 Waite, J.H. et al. (1998) The peculiar collagens of mussel byssus. Matrix Biol. 17, 93–106 8 Deming, T.J. (1999) Mussel byssus and biomolecular materials. Curr. Opin. Chem. Biol. 3, 100–105 9 Coyne, K.J. (1997) Extensible collagen in mussel byssus: a natural block copolymer. Science 277, 1830–1832 10 Gosline, J.M. et al. (1986) The structure and properties of spider silk. Endeavour 10, 37–43 11 Lewis, R. (1992) Spider silk: the unravelling of a mystery. Acc. Chem. Res. 25, 392–398 12 Guerrette, P.A. et al. (1996) Silk properties determined by gland-specific expression of a spider fibroin gene family. Science 272, 112–115 13 Hayashi, C.Y. and Lewis, R.V. (1998) Evidence from flagelliform silk cDNA for the structure basis of elasticity and modular nature of spider silks. J. Mol. Biol. 275, 773–784 14 Labeit, S. and Kolmerer, B. (1995) Titins: giant proteins in charge of muscle ultrastructure and elasticity. Science 270, 293–296 15 Linke, W.A. et al. (1999) I-Band titin in cardiac muscle is a three-element molecular spring and is critical for maintaining thin filament structure. J. Cell Biol. 146, 631–644 16 Trombitás, K.M. et al. (1998) Titin extensibility in situ: entropic elasticity of permanently folded and permanently unfolded molecular segments. J. Cell Biol. 140, 853–859 17 Trinick, J. and Tskhovrebova, L. (1999) Titin: a molecular control freak. Trends Cell Biol. 9, 377–380 18 Greaser, M. et al. (2000) Sequence and mechanical implications of titin’s PEVK region. In Proceedings: Elastic Filaments of the Cell (Pollack, G.H. and Granzier, H., eds), Kluwer Academic Publishers/Plenum Press, The Netherlands 19 Shewry, P.R. and Tatham, A.S. (1990) The prolamin storage proteins of cereal seeds – structure and evolution. Biochem. J. 267, 1–12 20 Shewry, P.R. et al. (1992) High molecular weight subunits of wheat glutenin. Cereal Chem. 15, 105–115 21 Ellis, G.E. and Packer, K.J. (1976) Nuclear spinrelaxation studies of hydrated elastin. Biopolymers 15, 813–832 22 Foster, J.A. et al. (1976) Circular dichroism studies of an elastin crosslinked peptide. Biopolymers 15, 833–841
23 Urry, D.W. et al. (1984) Polypentapeptide of elastin: temperature dependence correlations of elastomeric force and dielectric permittivity. Biochim. Biophys. Res. Commun. 125, 1082–1088 24 Rapaka, R.S. et al. (1978) Non-elastomeric polypeptide models of elastin. Int. J. Pept. Protein Res. 11, 109–127 25 Urry, D.W. (1984) Protein elasticity based on conformations of sequential polypeptides – the biological elastic fibre. J. Prot. Chem. 3, 403–436 26 Urry, D.W. et al. (1986) Polytetrapeptide of elastin: temperature correlated elastomeric force and structure development. Int. J. Pept. Protein Res. 28, 649–660 27 Simmons, A. et al. (1996) Molecular orientation and two-component nature of the crystalline fraction of spider dragline silk. Science 271, 84–87 28 Hayashi, C.Y. et al. (1999) Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins. Int. J. Biol. Macromol. 24, 271–275 29 Tatham, A.S. et al. (1990) Conformational studies of peptides corresponding to the repetitive regions of the high molecular weight (HMW) glutenin subunits of wheat. J. Cereal Sci. 11, 189–200 30 Van Dijk, A. et al. (1997) Structure characterization of the central repetitive domain of high molecular weight gluten proteins. II. Characterization in solution and the dry state. Protein Sci. 6, 649–656 31 Field, J.M. et al. (1987) The structure of a high Mr subunit of durum wheat (Triticum durum) gluten. Biochem. J. 247, 215–221 32 Miles, M.J. et al. (1991) Scanning tunneling microscopy of a wheat seed storage protein reveals details of an unusual supersecondary structure. Proc. Natl. Acad. Sci. U. S. A. 88, 68–71 33 Linke, W.A. et al. (1998) Nature of PEVK-titin elasticity in skeletal muscle. Proc. Natl. Acad. Sci. U. S. A. 95, 8052–8057 34 Linke, W.A. et al. (1996) Towards a molecular understanding of the elasticity of titin. J. Mol. Biol. 261, 62–71 35 Zhang, B. et al. (1999) A kinetic molecular model of the reversible unfolding and refolding of titin under force extension. Biophys. J. 77, 1306–1315 36 Hoeve, C.A.J. and Flory, P.J. (1974) The elastic properties of elastin. Biopolymers 13, 677–686 37 Urry, D.W. (1988) Entropic elastic processes in protein mechanisms. 1. Elastic structures due to an inverse temperature transition and elasticity due to internal chain dynamics. J. Prot. Chem. 7, 1–34 38 Gosline, J.M. (1978) Hydrophobic interaction and a model for the elasticity of elastin. Biopolymers 17, 677–695 39 Weis-Fogh, T. (1961) Thermodynamic properties of resilin, a rubber-like protein. J. Mol. Biol. 3, 520–531 40 Gosline, J.M. et al. (1984) Spider silk as rubber. Nature, 309, 551–552 41 Vollrath, F. and Edmonds, D.T. (1989) Modulation of the mechanical properties of spider silk by coating with water. Nature 340, 305–307 42 Kellermeyer, M.S.Z. et al. (1997) Folding-unfolding transitions in single titin molecules characterized with laser tweezers. Science 276, 1112–1116 43 Rief, M. et al. (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 44 Fisher, T.E. et al. (1999) The study of protein mechanics with the atomic force microscope. Trends Biochem. Sci. 24, 379–384 45 Sterba, R.E. and Sheetz, M.P. (1998) Basic laser tweezers. Meth. Cell Biol. 55, 29–41 46 Smith, B.L. et al. (1999) Molecular mechanisitic origin of the toughness of natural adhesives, fibres and composites. Nature 399, 761–763 47 Urry, D.W. (1995) Elastic biomolecular machines. Sci. Am. 272, 44–49 48 Termonia, Y. (1994) Molecular modeling of spider silk elasticity. Macromolecules 27, 7378–7381
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Elastomeric proteins: biological roles, structures and mechanisms Arthur S. Tatham and Peter R. Shewry Elastomeric proteins are able to withstand significant deformations without rupture before returning to their original state when the stress is removed. Although elastomeric proteins differ considerably in their amino acid sequence, they all have a complex domain structure and share two common properties. Namely, they contain elastomeric domains, comprised of repeated sequences, and additional domains that form intermolecular crosslinks. Furthermore, several protein contain b-turns as a structural motif within the elastomeric domains. ELASTIC PROTEINS ARE present in a diverse range of animal species and tissues where they have evolved precise structures and properties to perform specific biological functions1. These proteins all possess rubber-like elasticity, undergoing high deformation without rupture, storing the energy involved in deformation, and then recovering to their original state when the stress is removed. The second stage of this process is, therefore, passive in that it does not require energy input. In contrast to animals, elasticity is a rare phenomenon in plants, the only welldocumented system being the gluten proteins of wheat2. Moreover, in these proteins the elastic properties have no known biological role, but are exploited by humankind to make bread and other foods. The ability of proteins to exhibit rubber-like elasticity relates to their polymer structure. Rubber-like materials must satisfy two criteria. First, the individual monomers must be flexible and conformationally free, so that they can respond quickly to an applied force. Second, the individual monomers must be crosslinked to form a network. The crosslinks can be covalent or noncovalent, and most elastic proteins have evolved to combine elastomeric domains with domains that form A.S. Tatham and P.R. Shewry are at the Institute of Arable Crops Research-Long Ashton Research Station, Dept of Agricultural Sciences, University of Bristol, Bristol, UK BS41 9AF. Email: [email protected]
crosslinks. Thus, the elastic properties of protein polymers can be influenced by the lengths and properties of the elastic domains and the degree of crosslinking.
Distribution and sequences of elastomeric proteins Elastomeric proteins are widely distributed in the animal kingdom, but only a small number have been characterized in detail. Their intrinsic insolubility and their nonglobular nature have limited direct analysis, whereas sequence information from molecular-genetic studies has only become available in recent years. We will, therefore, limit the present discussion to the small number of elastic proteins that have been characterized in some detail. Despite differing greatly in their structures, all these proteins have two features in common. They all contain distinct domains (Fig. 1), at least one of which comprises elastic repeated sequences (Table 1), and crosslinks can be formed between sites in the nonelastic domains (Table 1). Elastin is widely distributed in vertebrate tissues. It performs various functions, acting statically in dermis to resist long-term forces and dynamically in arteries to store and release energy rapidly. It is secreted as a soluble precursor, tropoelastin, before forming the amorphous component of elastic fibres3. Tropoelastin consists of alternating repetitive hydrophobic domains of variable length (the elastic repeats) and alanine-rich, lysine-containing domains that form crosslinks4.
0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved.
Abductin and byssus both occur in bivalve molluscs but have different properties and functions. Abductin forms the inner hinge ligament of the shell, acting as an antagonist to the action of the adductor muscle, opening the shell when the adductor muscle relaxes. This action has developed to form a swimming mechanism for scallops allowing them to swim a few metres at a time5. The processing of abductin is unclear; the N-terminal domain might be cleaved during secretion6. Byssal threads attach mussels to hard surfaces in the sea. They display a gradient of mechanical properties, from stiff to elastic, along the length of the fibre that provides sufficient flexibility to prevent brittle fracture in tidal zones. Threads exhibiting different properties comprise different arrangements of domains7,8. Figure 1 shows a precursor of a byssal thread protein (PreCol-P), the elastic domains of which contain highly conserved pentapeptide motifs that can be extended by glycine residues (Table 1)9. Both proteins appear to be crosslinked, but the precise mechanisms and sites are not known (Table 1). Spiders produce several silks with different mechanical properties. The dragline silks form the dropping line and web framework and are stiffer than the flagelliform silks that form the spiral, capturing part of the web. Whereas the dragline silks will extend by approximately 30%, the flagelliform silks extend by ~200% of their length without breaking. The flagelliform silks have similar properties to a slightly crosslinked rubber; that is, a combination of low stiffness and high extensibility10,11. The combination of different silk proteins allows the spider to produce fibres with different mechanical properties and allows the web to absorb the energy of the impacting insect without catapulting the insect out of the web. The two types of silk differ in that the dragline silks contain polyalanine domains which divide the elastic polypentapeptide repeats and can form noncovalent links between proteins12,13. Titin (or connectin) is a sarcomeric protein responsible for the elasticity of vertebrate striated muscle myofibrils and is involved in muscle assembly14. It is present in isoforms that have different properties in different tissues (for example, in skeletal and heart muscle)14. The elastic properties of titin are responsible for the force that is generated when passive muscle is stretched, but titin also maintains the integrity of the sarcomere and therefore ensures efficient muscle contraction in actively
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contracting muscle. Titin is a giant protein, with the human cardiac form comprising approximately 26 900 amino acid residues14. The sequence is divided into the Z-disc (~2000 residues), I-band (~5900 residues), A-band (~17 200 residues) and M-line (~1100 residues) regions, corresponding to the light and dark transverse bands observed by microscopy of myofibrils14. The I-band is thought to be primarily responsible for the elastic properties of titin15,16 and contains elastic repeated sequences rich in P, E, V and K (Refs 17,18). Wheat gluten corresponds to the seed storage proteins that form an elastic network in doughs19. Although the sole function of gluten proteins is to store carbon and nitrogen, they have elastic properties, which are probably a fortuitous consequence of structures that have evolved to facilitate their efficient storage and packaging into protein bodies in the seed. One group of gluten proteins, the highmolecular-weight (HMW) subunits of glutenin, appears to be largely responsible for the formation of the elastic gluten network20. They are characterized by an extensive central domain of repeated sequences and form disulphide crosslinks. Comparison of the structures and repeat motifs shows striking similarities as well as differences. In particular, all contain elastic domains comprising repeated sequences that can be flanked by or interspersed with other nonelastic domains such as the polyalanine repeats in tropoelastin and dragline silks and the collagen-like domain in byssus. The elastic repeat motifs are all rich in glycine residues that are accompanied by other hydrophobic residues. However, differences are also present allowing them to be divided into four groups (Table 1). In particular, the re-
1
136
Abductin 1
882
Byssus HMW subunit Flagelliform silk Dragline silk
1
827
1
1030
613
1
759
1 Tropoelastin
−5900 Titin I-band Nonrepetitive Acidic domain
’Spacer’ domain Histidine-rich domain
Collagen-like domain Elastic repeat
Fn3 Polyalanine repeat
Ig Ti BS
Figure 1 Domain structures of the elastomeric proteins. Abductin, high-molecular-weight (HMW) subunits and flagelliform silks show similar domain structures, with the addition of ‘spacer’ domains in the latter. The dragline silks and tropoelastin elastic repeats interspersed with polyalanine regions. Byssus shows a more complicated structure with elastomeric domains flanking a collagen-like domain with N- and C-terminal histidine-rich domains. The titin I-band consists predominantly of immunoglobulin (Ig)-like and fibronectin type III (Fn3)-like domains, the elastomeric domain forming a minor part of the sequence. Sequences taken from abductin6, byssus9, HMW subunit20, flagelliform silk13, dragline silk12, tropoelastin4 and titin I-band14.
peats in the dragline silks and HMW subunits also contain glutamine, a bulky hydrophobic residue.
b-Turns as a structural feature of elastomeric proteins One requirement for polymer elasticity is that crosslinked molecules must be flexible and conformationally free. It might be expected, therefore, that this would preclude the formation of regular
secondary structures in the elastic domains, as these would not be as responsive to stress as unordered structures. However, the presence of regularly repeated sequences also implies a propensity to adopt a regular structure. Although direct determination of the structures of the elastic proteins is difficult, and limited information is available, the available data indicate that the repetitive domains do indeed form regular
Table 1. Repeat motifs and crosslinking mechanisms for elastomeric proteins Group
Protein
Elastic repeat motifsa
Nature of crosslinks
Ref.
1 2
Abductin Elastin
GGFGGMGGGx VPGG VPGVG APGVGV GPGGG
Unknown, disulphide (?) and/or via tyrosine residues (?) Via lysine residues in the alanine-rich regions
6 4
Unknown, metal complexation with histidine-rich regions (?) and/or via tyrosine residues (?) Unknown, noncovalent interactions in spacer regions (?) Noncovalent interactions between alanine-rich domains
8 13 28
Disulphide between N- and C-terminal domains
20
In Z-disc and M-line, mechanism unknown
18
Byssus
3
Flagelliform silk Dragline
HMW subunit
4
Titin (I-band)
GPGGx GPGQQ GPGGY GGYGPGS PGQGQQ GYYPTSPQQ GQQ PPAKVPEVPKKPVPEEKVPVPVPKKPEA
aGroup 1: glycine-rich; group 2: glycine- and proline-rich; group 3: glycine-, proline- and glutamine-rich; and group 4: proline-, glutamic acid-, valine- and lysine-rich. x is any amino acid.
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Figure 2 Computer-generated molecular model of a b-spiral for the backbone of the elastomeric domain of the high-molecular-weight (HMW) subunits of glutenin, showing the helical nature of the structure. The model was constructed from the 21-residue repeating motif (PGQGQQ GYYPTSPQQ PGQGQQ), with b-turns over tetrapeptide sequences QPGQ, YPTS, SPQQ and QGQQ generating a b-spiral with a diameter of 1.7 nm and a pitch of 1.3 nm. Red, oxygen atoms; blue, nitrogen atoms; green, carbon atoms. (A.S. Tatham, P.R. Shewry, D.J. Osguthorpe and O. Parchment, unpublished).
structures and that these might play important roles in the elastic mechanisms. Spectroscopic studies of elastin indicate that the backbone is highly mobile21 and that the alanine-rich regions, which contain the crosslinking sites, are a helical22. Urry et al. carried out spectroscopic studies of a-elastin. indicating the presence of b-turns as the dominant structural feature. However, the complex structure of elastin means that further information has been derived largely from studies of synthetic polymers based on the consensus tetra(VPGG), penta- (VPGVG) and hexapeptide (APGVGV) repeat motifs. In the synthetic polypentapeptide23 and polyhexapeptide24, the b-turns form a b-spiral, a loose water-containing helical structure25. However, only the polypentapeptide is elastic, with an elastic modulus (i.e. the force needed to elongate the material) similar to that of elastin. The dynamic b-spiral conformation of the polypentapeptide has no significant hydrogen bonding between the repeats, whereas the rigidity of the polyhexapeptide is thought to be due to an intramolecular hydrogen bonding between repeats and interlocking of the bspirals by hydrophobic interactions. By contrast, polymers based on the
tetrapeptide motif (VPGG) appeared to form a cross-b structure, where the bturns are separated by short b-strands, which exhibits little elasticity except at high (.40°C) temperatures26. The nonelastomeric structures formed by the tetra- and hexapeptides presumably play structural roles in elastin, but these are unclear at present. The alanine-rich regions of the spider dragline silks have been demonstrated by X-ray diffraction and NMR studies to form linked anti-parallel b-sheet regions27, compared with a-helical structures in the alanine-rich domains of elastin. These are thought to correspond to the crystalline regions that crosslink the protein molecules and provide the silks with their tensile strength. However, nothing is known about the structures formed by the nonrepetitive domains of the flagelliform silks, or by the ‘spacer regions’ within the repetitive domain13. In both types of silk, the repetitive domains have been proposed to form b-spiral structures. In the flagelliform silks, this structure might be based on b-turns formed by the pentapeptide repeat xPGGG (Table 1), which occurs up to 63 times in succession13,28. In dragline silks, the GGPGQ and YPGQQ pentapeptides are repeated up to nine
times and are also predicted to form bturns. The greater elasticity of the flagelliform silks could be related to their longer repetitive regions and the lower levels of crosslinking compared with the dragline silks. The repetitive domains of the HMW subunits of wheat glutenin comprise three repeats that are thought to be responsible for their elastic properties. Spectroscopic studies of linear and cyclic peptides corresponding to the repeat motifs indicate the presence of b-turns in the hexapeptide repeat (PGQGQQ) and nonapeptide repeat (GYYPTS[P/L]QQ) motifs29,30. Structure prediction indicates overlapping bturns, within and between repeat motifs29. Hydrodynamic studies show a rod-like molecule in solution31, and scanning tunnelling microscope images of HMW subunits deposited onto graphite reveal that the central repetitive domain adopts a helical structure with a diameter of ~1.9 nm and a pitch of ~1.5 nm32 (Fig. 2). Comparison of the I-band sequences of titin from heart, psoas (vertebral) and soleus (skeletal) muscle, each of which has different passive tensions, shows differences both in the tandem Ig-like and PEVK domains14. It was proposed initially that titin elasticity arose from the unfolding of the Ig-like b-barrel structure, but it is now thought to result from the properties of the PEVK domains15,33, which differ in length in the I-bands of different titin isoforms (from ~170 residues in cardiac muscle to ~2200 residues in human skeletal titin14). The PEVK region is more easily stretched than the Ig-like domains and is predicted to have an unstable structure due to the high charge density and high content of proline, preventing the formation of a stable tertiary structure14. Therefore, the elastomeric PEVK domain of titin has been described as a permanently unfolded nonglobular domain that functions as a stiff spring. However, this model is still controversial, and other studies have suggested a folded domain structure34,35. Prediction of the secondary structure of the PEVK domains suggests a polyproline-II-like structure (an extended left-handed helix with three residues per turn), because of the high content of proline residues and the formation of b-turns, so that the conformation could be an equilibrium between a b-spiral and an unfolded state. The secondary structures of the elastomeric domains of byssus and
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REVIEWS abductin have not yet been studied in detail. By analogy with elastin, the elastinlike domains of byssus are predicted to form b-turns, and a b-spiral structure could account for the elasticity. The predicted secondary structure of abductin indicates that the N-terminal domain adopts an a-helical structure, whereas the repetitive domain is a random coil. It can be concluded, therefore, that bturns are a common structural feature in elastomeric proteins or domains but are not universal.
Mechanisms of elasticity Elastomeric force (f; the force developed on stretching an elastomeric material) has two components, an entropic component, fs, and an internal energy component, fe, with f 5 fs 1 fs. On deformation, fe is attributed to strain in the bonds and fs to a decrease in the number of conformations in the lowest energy state. As bond strain can lead to bond breakage, an ideal elastomer is one in which fe 5 0 and f 5 fs. For a predominantly entropic elastomer the value of fe/f is less than 0.5, for elastin the value is 0.26 and for natural rubber 0.18. A number of different mechanisms have been proposed to explain the elastic force of elastin. In the first, which is based on classical rubber theory (where rubber-like properties are attributed to a decrease in conformational entropy on deforming a network of kinetically free, random polymer molecules), elastin is a network of random chains of high entropy. Stress orders the chains and decreases entropy by limiting their conformational freedom, thus providing the restoring force to the relaxed state36. The second model, proposed by Urry and co-workers, is that the elasticity arises from the b-spiral structure37. In the polypentapeptide, the b-turns are repeated regularly and act as spacers between the turns of the spiral, effectively suspending chain segments in a relatively kinetically free state. The peptide segments can undergo large-amplitude, low-frequency rocking motions, called librations. On stretching, these librations decrease in amplitude and result in a decrease in entropy, thereby providing the restoring force37. In both models the restoring force is entropic26,37. Another mechanism is based on hydrophobic interactions, where stretching is proposed to increase hydrophobic side-chain exposure to an aqueous environment, decreasing the entropy of the surrounding water molecules, the restoring force arises from
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TIBS 25 – NOVEMBER 2000 the hydrophobic forces that stabilize folded protein structures38. Resilin and abductin also behave as entropic elastomers, returning almost all the energy stored in deformation5,39. The sequence of abductin consists of hydrophobic repeats, with a low proline content, but no b-turns are predicted, and the formation of a b-spiral is unlikely. The mechanism of elasticity can, therefore, be similar to that of a classical rubber. The amino acid composition of resilin is similar to that of elastin, with high contents of glycine, alanine, proline and aspartate, but lack of sequence data prevents further comparisons. Studies of contracted dragline silks indicate that the predominant force for retraction is entropic, accounting for ~85% of the total force40. However, in this case some of the energy of the impacting insect is absorbed and dissipated as heat and not recovered in elastic recoil10. This has two consequences: the insect is unlikely to be catapulted back out of the web by elastic recoil, and the mechanical energy converted to heat is not available to fracture the silk thread. The flagelliform silk that forms the capture spiral is highly extensible and covered with small droplets of glue41. This extensibility means that the struggling insect has no solid structure to push against. Titin elasticity has been explored using a number of different techniques, including mechanical studies on single molecules using laser tweezers42, forceextension atomic force microscopy43 and
fluorescently labelled antibodies in single myofibrils15. These studies indicate that there is a two-stage extension mechanism, the first stage involving straightening of the titin molecule and the second, extension of the PEVK domain. Under normal physiological conditions of stretch, the Ig-like domains of the I-band remain folded, but unfolding does occur when extreme stress is applied in vitro. The elastic properties of titin have been modelled as entropic polymers; at low force titin behaves as a purely entropic polymer, whereas at higher extensions there were deviations from the experimental data that indicated an enthalpic component (such as hydrophobic and electrostatic interactions)16,33. Enthalpic elasticity at high stretch can provide a ‘buffer’ before the potentially destructive effect of unfolding the Ig-like domains occurs. Within the PEVK domain the enthalpic component could arise both from electrostatic forces (lowering the ionic strength results in increased titin stiffness) and hydrophobic interactions (exposure of the valine residues to water)33. Crosslinked HMW subunits of glutenin show a similar modulus of elasticity to the crosslinked polymers of elastin. However, the mechanism of elastic recoil is not predominantly entropic but is apparently associated with strain in the chemical bonds within the repetitive domain of the protein. There has been no evolutionary pressure to develop elastic mechanisms in seed proteins, and their elasticity might have arisen as
Axel Innis is a PhD student in the Dept of Biochemistry, University of Cambridge, UK.
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TIBS 25 – NOVEMBER 2000 a consequence of the evolution of structures that facilitate efficient packaging of the proteins to act as reserves for seed germination.
What is the biological significance of different structures and mechanisms of elastomeric proteins? The entropic mechanism exhibited by elastomers such as elastin is ideal for elastomeric fibres, which are required to last the lifetime of an individual, undergoing many ‘stress–strain’ cycles. It is not surprising, therefore, that several unrelated animal proteins have evolved a similar entropic mechanism for different uses, such as flight and swimming. By contrast, stretching bonds in predominantly nonentropic systems, such as gluten, can lead to rupture. This is hardly ideal in constantly working systems but is not detrimental in gluten, as the elasticity plays no biological role. Similarly, spider silks do not exhibit an entirely entropic mechanism, as this would not serve their purpose in prey capture. It is clear, therefore, that the structures and mechanisms of elastic proteins have evolved to fulfil precise biological roles. However, the presence of both similarities and differences between the structural organization, sequences and secondary structures of different elastomeric proteins shows that the requirements can be satisfied in different ways. This is perhaps not surprising in view of the diverse range of organisms in which elastomeric proteins occur.
Future directions for research Major advances in the study of elastic proteins have come from two areas. The first is molecular biology, in particular molecular cloning, which has provided amino acid sequences of proteins whose size, complexity, repetitive structure and insolubility allow restricted sequence analysis by conventional approaches. The second advance has been in the ability to determine directly the mechanical properties of single molecules using atomic force microscopy44 or laser tweezers45. These techniques were initially used to study the elastic properties of titin and have also been used to determine the mechanical properties of a number of non-elastic biological systems such as nacre (mollusc shell)46. There is no doubt that elastomeric proteins have great potential as novel industrial materials. Elastomeric ‘patches’ made from synthetic polymers
based on the polypentapeptide repeat motif of elastin can be used in surgery to prevent adhesion of wounds47, whereas spider silks are being studied to determine the basis for their tensile strength (allegedly greater than steel) and elasticity48. Similarly, marine adhesion proteins such as byssus are considered to have potential as adhesive8. The success of these and other applications will depend on our ability to understand and manipulate protein elasticity at the molecular level.
Acknowledgements IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC).
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23 Urry, D.W. et al. (1984) Polypentapeptide of elastin: temperature dependence correlations of elastomeric force and dielectric permittivity. Biochim. Biophys. Res. Commun. 125, 1082–1088 24 Rapaka, R.S. et al. (1978) Non-elastomeric polypeptide models of elastin. Int. J. Pept. Protein Res. 11, 109–127 25 Urry, D.W. (1984) Protein elasticity based on conformations of sequential polypeptides – the biological elastic fibre. J. Prot. Chem. 3, 403–436 26 Urry, D.W. et al. (1986) Polytetrapeptide of elastin: temperature correlated elastomeric force and structure development. Int. J. Pept. Protein Res. 28, 649–660 27 Simmons, A. et al. (1996) Molecular orientation and two-component nature of the crystalline fraction of spider dragline silk. Science 271, 84–87 28 Hayashi, C.Y. et al. (1999) Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins. Int. J. Biol. Macromol. 24, 271–275 29 Tatham, A.S. et al. (1990) Conformational studies of peptides corresponding to the repetitive regions of the high molecular weight (HMW) glutenin subunits of wheat. J. Cereal Sci. 11, 189–200 30 Van Dijk, A. et al. (1997) Structure characterization of the central repetitive domain of high molecular weight gluten proteins. II. Characterization in solution and the dry state. Protein Sci. 6, 649–656 31 Field, J.M. et al. (1987) The structure of a high Mr subunit of durum wheat (Triticum durum) gluten. Biochem. J. 247, 215–221 32 Miles, M.J. et al. (1991) Scanning tunneling microscopy of a wheat seed storage protein reveals details of an unusual supersecondary structure. Proc. Natl. Acad. Sci. U. S. A. 88, 68–71 33 Linke, W.A. et al. (1998) Nature of PEVK-titin elasticity in skeletal muscle. Proc. Natl. Acad. Sci. U. S. A. 95, 8052–8057 34 Linke, W.A. et al. (1996) Towards a molecular understanding of the elasticity of titin. J. Mol. Biol. 261, 62–71 35 Zhang, B. et al. (1999) A kinetic molecular model of the reversible unfolding and refolding of titin under force extension. Biophys. J. 77, 1306–1315 36 Hoeve, C.A.J. and Flory, P.J. (1974) The elastic properties of elastin. Biopolymers 13, 677–686 37 Urry, D.W. (1988) Entropic elastic processes in protein mechanisms. 1. Elastic structures due to an inverse temperature transition and elasticity due to internal chain dynamics. J. Prot. Chem. 7, 1–34 38 Gosline, J.M. (1978) Hydrophobic interaction and a model for the elasticity of elastin. Biopolymers 17, 677–695 39 Weis-Fogh, T. (1961) Thermodynamic properties of resilin, a rubber-like protein. J. Mol. Biol. 3, 520–531 40 Gosline, J.M. et al. (1984) Spider silk as rubber. Nature, 309, 551–552 41 Vollrath, F. and Edmonds, D.T. (1989) Modulation of the mechanical properties of spider silk by coating with water. Nature 340, 305–307 42 Kellermeyer, M.S.Z. et al. (1997) Folding-unfolding transitions in single titin molecules characterized with laser tweezers. Science 276, 1112–1116 43 Rief, M. et al. (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 44 Fisher, T.E. et al. (1999) The study of protein mechanics with the atomic force microscope. Trends Biochem. Sci. 24, 379–384 45 Sterba, R.E. and Sheetz, M.P. (1998) Basic laser tweezers. Meth. Cell Biol. 55, 29–41 46 Smith, B.L. et al. (1999) Molecular mechanisitic origin of the toughness of natural adhesives, fibres and composites. Nature 399, 761–763 47 Urry, D.W. (1995) Elastic biomolecular machines. Sci. Am. 272, 44–49 48 Termonia, Y. (1994) Molecular modeling of spider silk elasticity. Macromolecules 27, 7378–7381
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