Elasticity Of Elastin Research Articles

Structural and physical basis for the elasticity of elastin.

Elastin function is to endow vertebrate tissues with elasticity so that they can adapt to local mechanical constraints. The hydrophobicity and insolubility of the mature elastin polymer have hampered studies of its molecular organisation and structure-elasticity relationships. Nevertheless, a growing number of studies from a broad range of disciplines have provided invaluable insights, and several structural models of elastin have been proposed. However, many questions remain regarding how the primary sequence of elastin (and the soluble precursor tropoelastin) governs the molecular structure, its organisation into a polymeric network, and the mechanical properties of the resulting material. The elasticity of elastin is known to be largely entropic in origin, a property that is understood to arise from both its disordered molecular structure and its hydrophobic character. Despite a high degree of hydrophobicity, elastin does not form compact, water-excluding domains and remains highly disordered. However, elastin contains both stable and labile secondary structure elements. Current models of elastin structure and function are drawn from data collected on tropoelastin and on elastin-like peptides (ELPs) but at the tissue level, elasticity is only achieved after polymerisation of the mature elastin. In tissues, the reticulation of tropoelastin chains in water defines the polymer elastin that bears elasticity. Similarly, ELPs require polymerisation to become elastic. There is considerable interest in elastin especially in the biomaterials and cosmetic fields where ELPs are widely used. This review aims to provide an up-to-date survey of/perspective on current knowledge about the interplay between elastin structure, solvation, and entropic elasticity.

Detection of Labile Conformations of Elastin’s Prolines by Solid-State Nuclear Magnetic Resonance and Fourier Transform Infrared Techniques

Samples of native elastin are prepared with high levels of enrichment at its prolines, which are believed to play a major role in the elasticity of elastin. Major and minor populations of trans and cis isomers at the Xaa-Pro imide bonds are detected in two-dimensional 13C nuclear magnetic resonance (NMR) experiments. One- and two-dimensional 13C NMR and isotope-edited Fourier transform infrared experiments are also used to identify the prolines' folded and unfolded states, type II β-turn and random coil, respectively, at physiological temperatures. This study provides new details about elastin's conformational ensemble. In addition, the cis-trans isomerization of its abundant prolines provides an additional mechanism of fiber elongation in tissue.

Fabrication of triple-layered vascular grafts composed of silk fibers, polyacrylamide hydrogel, and polyurethane nanofibers with biomimetic mechanical properties

Mimicking the mechanical properties of native tissue is an important requirement for tissue engineering scaffolds. Blood vessels are subject to repetitive dilation and contraction and possess a special nonlinear mechanical property due to their triple-layered structure. Fabrication of vascular grafts consisting of bioresorbable materials with biomimetic mechanical properties is an urgent demand, as well as a critical challenge. Inspired by the configuration and function of collagen and elastin in native blood vessels, a new type of triple-layered vascular graft (TLVG) was developed in this study. The TLVGs were composed of braided silk as the inner layer, polyacrylamide (PAM) hydrogel as the middle layer, and electrospun thermoplastic polyurethane (TPU) as the outer layer. The woven-structured silk fibers were able to mimic the properties of the loosely distributed collagen fibers, while the highly elastic PAM hydrogel and TPU nanofibers mimicked the elasticity of elastin in the blood vessel. With this specially designed microstructure and combination of rigid and elastic materials, the TLVGs successfully mimicked the nonlinear mechanical property of native blood vessels. Moreover, TLVGs possess sufficient suture retention strength for surgical implantation. The introduction of a PAM hydrogel layer effectively solved the leaking issue for conventional porous vascular grafts and greatly enhanced the burst pressure. In addition, all materials used have high biocompatibility to human endothelial cells, which indicates that the developed TLVGs have high potential to be used as readily available vascular grafts.

NMR Study of Elastin's Elasticity Mechanism

Elastin is the most abundant elastomer in nature. The recoil mechanism is believed to be entropically driven for which there re two molecular level theories. Recoil in one theory is a rubber-like mechanism in which the decrease in entropy with stretch is due to a decrease in conformational entropy from backbone ordering. The alternative is a decrease in solvent entropy with stretch from solvent ordering, i.e., the hydrophobic effect. Using a novel double quantum (2Q) pulse sequence in solid state NMR; water ordering at elastin fiber surface was studied quantitatively. We find that water is weakly oriented at the Elastin-water surface when the fiber is relaxed and stretching the fiber or increasing the temperature significantly increases the 2Q signal, i.e., increases the ordered water fraction. Furthermore, the 2Q signal decreases with the addition of high molecular weight polyethylene glycol (20kDa or 6kDa PEG) that is known to precipitate proteins and decrease their hydration in solution. We believe PEG causes less water to be available on the fibers surface and thus less ordered water. Also, this is likely the reason why the fibers become less flexible. Our preliminary results in both water and high molecular weight PEG experiments directly correlate the recoil mechanism with the hydrophobic effect.

The Liquid State of (Elastomeric) Proteins

Self-assembled elastomeric proteins make up an important and unusual class of structural proteins endowing biological tissues as diverse as spider silks and lung alveoli with extensibility and elastic recoil. In humans, elastin is the polymeric extracellular matrix protein responsible for the elasticity of lungs, skin, the bladder, the uterus, and large blood vessels. Elastin self-assembly involves a process of liquid-liquid phase separation called coacervation. Although the structure of elastin in the resulting matrix is unknown, the source of elastic recoil is known to be primarily entropic. Two different sources of entropy have been proposed in seemingly-contradictory models: rubber-like models attribute the elasticity of elastin to the entropy of the polypeptide chain in random-coil conformation, whereas “liquid drop” models ascribe the recoil of this highly-hydrophobic protein to the hydrophobic effect. Although its self-organization and mechanical properties have spurred interest in elastin as a model for useful biomimetic polymers, the molecular determinants of these properties are poorly understood.We use atomistic molecular dynamics simulations in explicit solvent to examine the structural basis for the self-assembly and mechanical properties of elastin-like polypeptides in water. Massive sampling over simulation times totalling 0.3 millisecond indicates that the peptides remain highly disordered (though not random) even in the aggregated state, and provides an ensemble description of phase-separated peptide aggregates in which the polypeptide chains exhibit liquid-like properties approaching those of a polymer melt. In this phase-separated state, the peptides are largely solvated by one another, although the polypeptide backbone remains significantly hydrated. Crucially, both conformational entropy and hydrophobic burial drive the formation of this protein-rich phase, which may be called the liquid state of proteins. These findings support a unified model of self-assembled elastomeric proteins in which these two entropic forces play essential roles in both self-assembly and elastic recoil.

Bioinspired Modular Synthesis of Elastin-Mimic Polymers To Probe the Mechanism of Elastin Elasticity

Bioinspired modular synthesis of elastin-mimic polymers (EMPs) is achieved via Cu-catalyzed alkyne-azide cyclization (CuAAC). By changing the module, EMPs with different secondary structures determined by circular dichroism (CD) spectra in trifluoroethanol (TFE) solution are obtained. The EMPs are characterized by measuring the lower critical solution temperatures (LSCTs) and the bulk mechanic properties under the conditions of both dry and hydrated forms. The unique molecular design enables us to probe mechanistic questions and assess the structure-property relationship of the EMPs. Our results indicate that, instead of a highly organized secondary structure, hydrophobic hydration is critical for the elasticity of EMPs.

The dissection of human tropoelastin: from the molecular structure to the self-assembly to the elasticity mechanism

After a historical introduction the authors describe their most recent results on the structure, assembly and elasticity of elastin. Recent results obtained by analyzing the conformation of polypeptide sequences encoded by the single exons of human tropoelastin demonstrated the presence of labile conformations such as poly-proline II helix (PPII) and β-turns whose stability is strongly dependent on the microenvironment. Stable, periodic structures, such as α-helices, are only present in the poly-alanine cross-linking domains. These findings give a strong experimental basis to the understanding of the molecular mechanism of elasticity of elastin. In particular, they strongly support the description of the native relaxed state of the protein in terms of trans-conformational equilibria between extended and folded structures as previously proposed [Int. J. Biochem. Cell. Biol. 31 (1999) 261]. The same polypeptide sequences have been analyzed for their ability to coacervate and to self-assembly. Although the great majority of them were shown to be able to adopt more or less organized structures, only a few were indeed able to coacervate. Studies carried out by transmission electron microscopy showed the polypeptides to adopt a variety of supramolecular structures going from a filamentous organization (typical of elastin) to amyloid-like fibers. On the whole, the results obtained gave significant insight to the roles played by specific polypeptide sequences in self-assembly and possibly in elasticity.

Structure distribution in an elastin-mimetic peptide (VPGVG)3 investigated by solid-state NMR.

Elastin is an extracellular-matrix protein that imparts elasticity to tissues. We have used solid-state NMR to determine a number of distances and torsion angles in an elastin-mimetic peptide, (VPGVG)3, to understand the structural basis of elasticity. C-H and C-N distances between the V6 carbonyl and the V9 amide segment were measured using 13C-15N and 13C-1H rotational-echo double-resonance experiments. The results indicate the coexistence of two types of intramolecular distances: a third of the molecules have short C-H and C-N distances of 3.3 +/- 0.2 and 4.3 +/- 0.2 A, respectively, while the rest have longer distances of about 7 A. Complementing the distance constraints, we measured the (phi, psi ) torsion angles of the central pentameric unit using dipolar correlation NMR. The -angles of P7 and G8 are predominantly ~150, thus restricting the majority of the peptide to be extended. Combining all torsion angles measured for the five residues, the G8 C chemical shift, and the V6-V9 distances, we obtained a bimodal structure distribution for the PG residues in VPGVG. The minor form is a compact structure with a V6-V9 C=O-HN hydrogen bond and can be either a type II -turn or a previously unidentified turn with Pro (phi = -70, psi= 20 +/- 20) and Gly ( phi= -100 +/- 20, psi = -20 +/- 20). The major form is an extended and distorted beta-strand without a V6-V9 hydrogen bond and differs from the ideal parallel and antiparallel beta-strands. The other three residues in the VPGVG unit mainly adopt antiparallel beta-sheet torsion angles. Since (VPGVG)3 has the same 13C and 15N isotropic and anisotropic chemical shifts as the elastin-mimetic protein (VPGXG)n (X = V and K, n = 195), the observed conformational distribution around Pro and Gly sheds light on the molecular mechanism of elastin elasticity.

Spectroscopic evidence revealing polyproline II structure in hydrophobic, putatively elastomeric sequences encoded by specific exons of human tropoelastin.

Elastin, the protein responsible for tissue elasticity, is contained in arterial walls, lungs, and skin. Given the cassette like organization of the human tropoelastin gene, giving rise to alternating exons encoding for crosslink domains and elastomeric domains, it is tempting to suggest that polypeptides encoded by different exons could adopt structures independent of the other exons. The results obtained with the polypeptide sequences encoded by exons 3, 7, and 30 of human tropoelastin are described. It is shown that these hydrophobic exons may partly assume the polyproline II (PPII) structure, as found by circular dichroism studies in aqueous solution. Classical Raman spectroscopy evidences a specific sharp band at 1314 cm(-1), which is assigned to the PPII structure adopted by these exons in the solid state. As these sequences are among those putatively responsible for elastomeric properties, these findings are of particular interest in relation to the current models of the elasticity of elastin.

Investigation of the dynamics of an elastin-mimetic polypeptide using solid-state NMR.

Elastin is the main structural protein that provides elasticity to various tissues and organs in vertebrates. Molecular motions are believed to play a significant role in its elasticity. We have used solid-state NMR spectroscopy to characterize the dynamics of an elastin-mimetic protein as a function of hydration to better understand the origin of elastin elasticity. Poly(Lys-25), [(VPGVG)(4)(VPGKG)](39), has a repeat sequence common to natural elastin. (13)C cross-polarization and direct polarization spectra at various hydration levels indicate that water enhances the protein motion in a non-uniform manner. Below 20% hydration, the backbone motion increases only slightly whereas above 30% hydration, both the backbone and the side-chains undergo large-amplitude motions. The motional amplitudes are extracted from (13)C-(1)H and (1)H-(1)H dipolar couplings using 2D isotropic-anisotropic correlation experiments. The root mean square fluctuation angles are found to be 11-18 degrees in the dry protein and 16-21 degrees in the 20% hydrated protein. Dramatically, the amplitudes increase to near isotropic at 30% hydration. Field-dependent (1)H rotating-frame spin-lattice relaxation times (T(1rho)) indicate that significant motions occur on the microsecond time-scale (1.2-2.3 micros). The large-amplitude and low-frequency motion of poly(Lys-25) at relatively mild hydration indicates that the conformational entropy of the protein in the relaxed state contributes significantly to the elasticity.

Editorial

The papers in this issue of Peptide Science reflect the scientific identity of the journal, as well as the truly international character of the field of peptide chemistry and biology. We are very pleased to present a review solicited from one of the recipients of the 2004 Vincent du Vigneaud Awards from the American Peptide Society. Dieter Seebach of the ETH, Zurich, was honored for “the discovery of peptides consisting of homologated proteinogenic amino acids and of their unexpected chemical, structural, and biological properties.” His review in this issue provides an extensive discussion of β-amino acids, specifically those with side chains in the carbonyl position, including their occurrence in nature, their synthesis, and their uses in design of novel peptide properties. We congratulate Dieter for this richly deserved recognition, and we thank him for providing an exciting review to Peptide Science. The original papers appearing in this issue epitomize the field of peptide science and illustrate the power of peptide synthesis and conformational study: Chakrabarty and coworkers present results of peptide design aimed at deducing the principles for helical bundle formation. Their work explores factors important in packing of α-helices and shows how peptide studies can shed light on fundamental ‘rules’ for the formation of protein architectures. The use of synthetic peptides to elucidate the origins of biological activity is the theme of the paper contributed by Epand and coworkers, who are exploring how the biological activity of calcitonin analogues correlates with their biophysical properties. Lastly, Alix and coworkers present synergistic experimental and computational studies of the conformational preferences of short peptides responsible for the elasticity of elastin, a major component of the extracellular matrix.

Dissection of human tropoelastin: exon-by-exon chemical synthesis and related conformational studies.

Polypeptide sequences encoding the single exons of human tropoelastin were synthesized and their conformations were studied in different solvents and at different temperatures by CD and (1)H NMR. The results demonstrated the presence of labile conformations such as poly-proline II helix (PPII) and beta-turns whose stability is strongly dependent on the microenvironment. Stable, periodic structures, such as alpha-helices, are only present in the poly-alanine cross-linking domains. These findings give a strong experimental basis to the understanding of the molecular mechanism of elasticity of elastin. In particular, they strongly support the description of the native relaxed state of the protein in terms of trans-conformational equilibria between extended and folded structures as previously proposed [Debelle, L., and Tamburro, A. M. (1999) Int. J. Biochem. Cell. Biol. 31, 261-272].

Hydrophobic hydration is an important source of elasticity in elastin-based biopolymers.

Molecular dynamics simulations with explicit waters have been employed to investigate the dominant source of elastin's elasticity. An elastin-like peptide, (VPGVG)(18), was pulled and released in molecular dynamics simulations, at 10 and 42 degrees C, lasting several nanoseconds, which is consistent with the experimentally determined dielectric and NMR relaxation time scales. At elastin's physiological temperature and degree of extension, the simulations indicate that the orientational entropy of waters hydrating hydrophobic groups decreases during pulling of the molecule, but it increases upon release. In contrast, the main-chain fluctuations and other measures of mobility suggest that elastin's backbone is more dynamic in the extended than released state. These results and the agreement between the simulations with various experimental observations suggest that hydrophobic hydration is an important source of the entropy-based elasticity of elastin. Moreover, elastin tends to reorder itself to form a hydrophobic globule when it was held in its extended state, indicating that the hydrophobic effect also contributes in the holding process. On the whole, our simulations support the hydrophobic mechanism of elasticity and provide a framework for description of the molecular basis of this phenomenon.

Analysis of biomolecular chaos in aqueous solution

The tropoelastin peptide CH3CO-Gly-Leu-Gly-Gly-NHCH3 has been modeled in aqueous solution by means of force-field molecular dynamics simulations and its motion characterized using nonlinear dynamics theory. The trajectory R(t) of the representative system point in configurational space has been considered. Fractional Brownian motion with anomalous diffusion is observed resulting from chaotic dynamics of molecules on fractal media. The chaos of the peptide is a consequence of nonlinear effects such as hydrodynamic interactions of the chain due to the poor solvent role of water. The viscous drag is pointed out and should be due to the percolation network of hydrogen-bonded water molecules. The method of reconstruction of the phase space using the embedding theorem is applied to the trajectory Dee(t) of the peptide end-to-end distance. The existence of a low-dimensional chaotic attractor for dissipative systems has been demonstrated. The dynamical high-entropy state of the peptide in solution strengthens the transition-to-chaos mechanism for the elastin elasticity.

On the occurrence of polyproline II structure in elastin

To shed light on the occurrence of the polyproline II (PP II) structure in the elastomeric protein elastin, the octapeptide sequence ALGGGALG of the N-terminal region of human elastin was studied in its monomeric and polymeric form, both in solution and in the solid state. Furthermore, the polymer poly(PG), chosen by us as an a priori reference compound for investigating the stability of PP II structure in presence of alternating proline and glycine residues along the polypeptide chain, was studied by circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. Its “monomeric” form Boc-PG-OH, was also analyzed by X-ray diffraction. It was shown that, in the solid state the presence of PG or GGG sequences in polypeptide chains and even in a short peptide as Boc-PG-OH induces the acquisition of the PP II structural motif. However, in solution this conformation appears to be much more unstable even in the case of long polypeptide chains. The finding that at room temperature the PP II structure is always in equilibrium with other conformers suggests that its dynamics could also contribute to the molecular mechanism of elastin elasticity.

Conformational chaos and biomolecular instability in aqueous solution

The glycine-rich tropoelastin tetrapeptide Ac-Gly-Leu-Gly-Gly-NMe has been modeled in aqueous solution by means of molecular dynamics simulations and the conformational motions have been characterized using nonlinear dynamics theory. Large amplitude fluctuations of the peptide backbone and H-bond patterns are detected. The end-to-end vector Ree undergoes anomalous diffusion with antipersistent fractional Brownian motion according to chaotic motions of molecules on fractal media. The vibrational picture of the intramolecular vectors shows a spatiotemporal self-similar disorder along the peptide chain on large scale observation demonstrating a high entropy state. The conformational chaos of the peptide is a consequence of the nonlinear effects of the attractive interactions between residues developed in aqueous solution due to water being a poor solvent. The viscous drag is high-lighted and is thought to be due to the percolation network of disordered H-bonded water molecules. The method of the reconstruction of the phase-space using the embedding theorem is presented and the invariant properties of the peptide are calculated. The existence of a low dimensional chaotic attractor according to dissipative systems has been demonstrated. The dynamical high entropy state of the peptide in solution is in agreement with the proposed mechanism of the transition-to-chaos for the elastin elasticity.

The structures of elastins and their function

Elastin structures and their significance towards elastic recoil properties have been reviewed. Starting from the initial hypothesis that elastin conformation is conditioned by that of its monomer, the structure of tropoelastin was first described using theoretical and experimental methods and a β class folding type was evidenced for the isolated unbound tropoelastin molecules. The structure of elastin in the solid state was consistent with that of its monomer and consequently, fibrous elastin appeared constituted of globular tropoelastin molecules. Finally, theoretical and experimental considerations have led us to the conclusion that the functional form of the elastomer, water swollen elastin, could be a triphasic system comprising the protein chains, hydration water and solvent water. Following this description, the dynamic structural equilibria occurring within elastin hydrophobic domains and the plastisizing effect of water could explain elastin elasticity, in keeping with a classical entropic mechanism.

Biochemistry of tropoelastin.

Elastic fibres are an important component of the extracellular matrix and are made of two major components : the more abundant cross‐linked elastic protein elastin and the multi‐component microfibrils. The biosynthesis of elastic fibres is a complex process involving the interplay of many diverse proteins and genes with elastin as the major component. Tropoelastin is the soluble precursor of elastin and as such it plays a dominant role in elastogenesis. The expression of tropoelastin is under a complex control mechanism, with many isoforms existing. Numerous other components, including the microfibrillar proteins, the elastin‐binding protein and lysyl oxidase, the enzyme which initiates elastin cross‐linking, are involved in elastogenesis. Tropoelastin undergoes self‐association under physiological conditions in a process referred to as coacervation, and this is thought to be a vital process during elastic fibre formation and in providing elasticity. Although various models explaining the elasticity of elastin have been put forward, only the fibrillar model is based on the coacervation ability of tropoelastin. With the molecular cloning of a number of components of the elastic fibre, the availability of these components is increasing and paves the way for in vitro modelling of complex interactions of the elastic fibre. This review emphasises the biochemistry of tropoelastin and its role in elastic fibre structure and assembly.

Simulated annealing and molecular dynamics of an elastin-related tetrapeptide in aqueous solution

For the first time a fully unrestrained simulated annealing of an aqueous solution is reported: a dilute solution of the elastinrelated tetrapeptide AcGlyLeuGlyGlyNMe has been heated to high temperature and cooled slowly to room temperature and pressure by using a heuristic molecular dynamics approach in the absence of fictitious potential. The peptide conformational patterns during the final equilibrium stages of the annealed states thus obtained have been characterized. The time constants τ T and τ P , which control the temperature and pressure relaxation, respectively, and thus the energy dissipation rate, have been varied and successful criteria fixed. The procedure is amenable, although attention must be paid to prevent vaporization of the system for particularly slow coolings at high temperature. The tetrapeptide in solution at ambient conditions is characterized by conformational disorder with large-amplitude motions giving rise to endless formation and breakage of γ- and β-turns, according to known experimental evidence. This dynamic, high-entropy behavior is in agreement with the mechanism proposed for the elasticity of elastin, of which the peptide represents a relevant block, and with the dynamic nonlinear behavior recently found by us for the isolated molecule.

Conformational non-linear dynamical behavior of the peptide Boc-Gly-Leu-Gly-Gly-NMe

The conformational motion of a peptide has been investigated by means of molecular dynamics simulations and has been characterized in the time and the frequency domains. The tetrapeptide Boc-Gly-Leu-Gly-Gly-NMe has been considered as a model for some repetitive amino acid sequences of glycine-rich regions of elastin, which could play a key role in the entropic elasticity mechanism. The classical tools of dynamical analysis (time series, spectral density, time correlation functions, delay maps) have been used and new methods, introducing the idea of travelling trajectory packets and trajectories in the frequency space, have been developed. In vacuo, non-ergodic, essentially quasiperiodic motion has been revealed and solitons have been observed according to the non-linear dynamical behavior of systems with small anharmonic perturbation. This particularly concerns the dynamics of the end-to-end distance, a global variable very sensitive to the whole conformational flexibility of the model molecule. The dynamical picture has been joined with the experimentally observed conformational disorder and with the amplitude instability of KAM theory for non-linear systems: a transition is hypothesized from quasiperiodic (solitons) to ergodic motion (Hamiltonian chaos) for the peptide in solution and the entropic mechanism of elastin elasticity is interpreted from a new and unitary dynamical point of view.