Structural determinants of cross-linking and hydrophobic domains for self-assembly of elastin-like polypeptides

Elastin is a major structural protein found in large blood vessels, lung, ligaments, and skin, imparting the physical properties of extensibility and elastic recoil to these tissues. To achieve the required structural durability of the elastic matrix, the elastin monomer, tropoelastin, undergoes ordered assembly into a covalently cross-linked, fibrillar polymeric structure. Human tropoelastin consists of 34 exons coding for alternating hydrophobic and cross-linking domains. Using a series of well-defined recombinant polypeptides based on human elastin sequences mimicking native elastin, we have previously investigated the role of sequence and context of hydrophobic domains in elastin self-assembly. Here, we demonstrate that the structure of both cross-linking and hydrophobic domains have significant effects on the assembly of these polypeptides. Removing a putative flexible hinge region in the center of a cross-linking domain substantially increased the alpha-helical content and strongly promoted their self-aggregation. However, while trifluoroethanol (TFE) promoted and urea inhibited self-assembly of these polypeptides, these effects were not predominantly due to altered alpha-helicity of the polypeptides. Our results suggest that, while increased alpha helicity also favors this process, the major effect of TFE to promote organized self-assembly of elastin-like polypeptides is likely related to direct effects of this cosolvent on hydrophobic domains. Such simple elastin polypeptide models can provide an important tool for understanding the relationships between sequence, structure, and polymeric assembly of elastin.     

Self-aggregation characteristics of recombinantly expressed human elastin polypeptides.

Elastin is an extracellular matrix protein found in tissues requiring extensibility and elastic recoil. Monomeric elastin has the ability to aggregate into fibrillar structures in vitro, and has been suggested to participate in the organization of its own assembly into a polymeric matrix in vivo. Although hydrophobic sequences in elastin have been suggested to be involved in this process of self-organization, the contributions of specific hydrophobic and crosslinking domains to the propensity of elastin to self-assemble have received less attention. We have used a series of defined, recombinant human elastin polypeptides to investigate the factors contributing to elastin self-assembly. In general, coacervation temperature of these polypeptides, used as a measure of their propensity to self-assemble, was influenced both by salt concentration and polypeptide concentration. In addition, hydrophobic domains appeared to be essential for the ability of these polypeptides to self-assemble. However, neither overall molecular mass, number of hydrophobic domains nor general hydropathy of the polypeptides provided a complete explanation for differences in coacervation temperature, suggesting that the specific nature of the sequences of these hydrophobic domains are an important determinant of the ability of elastin polypeptides to self-assemble.     

Conformational Transitions of the Cross-linking Domains of Elastin during Self-assembly

Elastin is the intrinsically disordered polymeric protein imparting the exceptional properties of extension and elastic recoil to the extracellular matrix of most vertebrates. The monomeric precursor of elastin, tropoelastin, as well as polypeptides containing smaller subsets of the tropoelastin sequence, can self-assemble through a colloidal phase separation process called coacervation. Present understanding suggests that self-assembly is promoted by association of hydrophobic domains contained within the tropoelastin sequence, whereas polymerization is achieved by covalent joining of lysine side chains within distinct alanine-rich, α-helical cross-linking domains. In this study, model elastin polypeptides were used to determine the structure of cross-linking domains during the assembly process and the effect of sequence alterations in these domains on assembly and structure. CD temperature melts indicated that partial α-helical structure in cross-linking domains at lower temperatures was absent at physiological temperature. Solid-state NMR demonstrated that β-strand structure of the cross-linking domains dominated in the coacervate state, although α-helix was predominant after subsequent cross-linking of lysine side chains with genipin. Mutation of lysine residues to hydrophobic amino acids, tyrosine or alanine, leads to increased propensity for β-structure and the formation of amyloid-like fibrils, characterized by thioflavin-T binding and transmission electron microscopy. These findings indicate that cross-linking domains are structurally labile during assembly, adapting to changes in their environment and aggregated state. Furthermore, the sequence of cross-linking domains has a dramatic effect on self-assembly properties of elastin-like polypeptides, and the presence of lysine residues in these domains may serve to prevent inappropriate ordered aggregation.     

Proline‐poor hydrophobic domains modulate the assembly and material properties of polymeric elastin

Elastin is a self‐assembling extracellular matrix protein that provides elasticity to tissues. For entropic elastomers such as elastin, conformational disorder of the monomer building block, even in the polymeric form, is essential for elastomeric recoil. The highly hydrophobic monomer employs a range of strategies for maintaining disorder and flexibility within hydrophobic domains, particularly involving a minimum compositional threshold of proline and glycine residues. However, the native sequence of hydrophobic elastin domain 30 is uncharacteristically proline‐poor and, as an isolated polypeptide, is susceptible to formation of amyloid‐like structures comprised of stacked β‐sheet. Here we investigated the biophysical and mechanical properties of multiple sets of elastin‐like polypeptides designed with different numbers of proline‐poor domain 30 from human or rat tropoelastins. We compared the contributions of these proline‐poor hydrophobic sequences to self‐assembly through characterization of phase separation, and to the tensile properties of cross‐linked, polymeric materials. We demonstrate that length of hydrophobic domains and propensity to form β‐structure, both affecting polypeptide chain flexibility and cross‐link density, play key roles in modulating elastin mechanical properties. This study advances the understanding of elastin sequence‐structure‐function relationships, and provides new insights that will directly support rational approaches to the design of biomaterials with defined suites of mechanical properties. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 563–573, 2015.     

Sequence and domain arrangements influence mechanical properties of elastin‐like polymeric elastomers

Elastin is the polymeric, extracellular matrix protein that provides properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Elastin assembles by crosslinking through lysine residues of its monomeric precursor, tropoelastin. Tropoelastin, as well as polypeptides based on tropoelastin sequences, undergo a process of self‐assembly that aligns lysine residues for crosslinking. As a result, both the full‐length monomer as well as elastin‐like polypeptides (ELPs) can be made into biomaterials whose properties resemble those of native polymeric elastin. Using both full‐length human tropoelastin (hTE) as well as ELPs, we and others have previously reported on the influence of sequence and domain arrangements on self‐assembly properties. Here we investigate the role of domain sequence and organization on the tensile mechanical properties of crosslinked biomaterials fabricated from ELP variants. In general, substitutions in ELPs involving similiar domain types (hydrophobic or crosslinking) had little effect on mechanical properties. However, modifications altering either the structure or the characteristic sequence style of these domains had significant effects on such properties. In addition, using a series of deletion and replacement constructs for full‐length hTE, we provide new insights into the role of conserved domains of tropoelastin in determining mechanical properties. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 392–407, 2013.     

Development of short and highly potent self‐assembling elastin‐derived pentapeptide repeats containing aromatic amino acid residues

Tropoelastin is the primary component of elastin, which forms the elastic fibers that make up connective tissues. The hydrophobic domains of tropoelastin are thought to mediate the self‐assembly of elastin into fibers, and the temperature‐mediated self‐assembly (coacervation) of one such repetitive peptide sequence (VPGVG) has been utilized in various bio‐applications. To elucidate a mechanism for coacervation activity enhancement and to develop more potent coacervatable elastin‐derived peptides, we synthesized two series of peptide analogs containing an aromatic amino acid, Trp or Tyr, in addition to Phe‐containing analogs and tested their functional characteristics. Thus, position 1 of the hydrophobic pentapeptide repeat of elastin (X¹P²G³V⁴G⁵) was substituted by Trp or Tyr. Eventually, we acquired a novel, short Trp‐containing elastin‐derived peptide analog (WPGVG)₃ with potent coacervation ability. From the results obtained during this process, we determined the importance of aromaticity and hydrophobicity for the coacervation potency of elastin‐derived peptide analogs. Generally, however, the production of long‐chain synthetic polypeptides in quantities sufficient for commercial use remain cost‐prohibitive. Therefore, the identification of (WPGVG)₃, which is a 15‐mer short peptide consisting simply of five natural amino acids and shows temperature‐dependent self‐assembly activity, might serve as a foundation for the development of various kinds of biomaterials. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Cooperativity between the hydrophobic and cross-linking domains of elastin

The principal protein component of the elastic fiber found in elastic tissues is elastin, an amorphous, cross-linked biopolymer that is assembled from a high molecular weight monomer. The hydrophobic and cross-linking domains of elastin have been considered separate and independent, such that changes to one region are not thought to affect the other. However, results from these solid-state 13C NMR experiments demonstrate that cooperativity in protein folding exists between the two domain types. The sequence of the EP20-24-24 polypeptide has three hydrophobic sequences from exons 20 and 24 of the soluble monomer tropoelastin, interspersed with cross-linking domains constructed from exons 21 and 23. In the middle of each cross-linking domain is a "hinge" sequence. When this pentapeptide is replaced with alanines, as in EP20-24-24[23U], its properties are changed. In addition to the expected increase in alpha-helical content and the resulting increase in rigidity of the cross-linking domains, changes to the organization of the hydrophobic regions are also observed. Using one-dimensional CPMAS (cross-polarization with magic angle spinning) techniques, including spectral editing and relaxation measurements, evidence for a change in dynamics to both domain types is observed. Furthermore, it is likely that the methyl groups of the leucines of the hydrophobic domains are also affected by the substitution to the hinge region of the cross-linking sequences. This cooperativity between the two domain types brings new questions to the phenomenon of coacervation in elastin polypeptides and strongly suggests that functional models for the protein must include a role for the cross-linking regions.     

Recombinant human elastin polypeptides self-assemble into biomaterials with elastin-like properties

Processes involving self-assembly of monomeric units into organized polymeric arrays are currently the subject of much attention, particularly in the areas of nanotechnology and biomaterials. One biological example of a protein polymer with potential for self-organization is elastin. Elastin is the extracellular matrix protein that imparts the properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Tropoelastin, the approximately 70 kDa soluble monomeric form of elastin, is highly nonpolar in character, consisting essentially of 34 alternating hydrophobic and crosslinking domains. Crosslinking domains contain the lysine residues destined to form the covalent intermolecular crosslinks that stabilize the polymer. We and others have suggested that the hydrophobic domains are sites of interactions that contribute to juxtaposition of lysine residues in preparation for crosslink formation. Here, using recombinant polypeptides based on sequences in human elastin, we demonstrate that as few as three hydrophobic domains flanking two crosslinking domains are sufficient to support a self-assembly process that aligns lysines for zero-length crosslinking, resulting in formation of the crosslinks of native elastin. This process allows fabrication of a polymeric matrix with solubility and mechanical properties similar to those of native elastin.     

Proline periodicity modulates the self-assembly properties of elastin-like polypeptides

Elastin is a self-assembling protein of the extracellular matrix that provides tissues with elastic extensibility and recoil. The monomeric precursor, tropoelastin, is highly hydrophobic yet remains substantially disordered and flexible in solution, due in large part to a high combined threshold of proline and glycine residues within hydrophobic sequences. In fact, proline-poor elastin-like sequences are known to form amyloid-like fibrils, rich in β-structure, from solution. On this basis, it is clear that hydrophobic elastin sequences are in general optimized to avoid an amyloid fate. However, a small number of hydrophobic domains near the C terminus of tropoelastin are substantially depleted of proline residues. Here we investigated the specific contribution of proline number and spacing to the structure and self-assembly propensities of elastin-like polypeptides. Increasing the spacing between proline residues significantly decreased the ability of polypeptides to reversibly self-associate. Real-time imaging of the assembly process revealed the presence of smaller colloidal droplets that displayed enhanced propensity to cluster into dense networks. Structural characterization showed that these aggregates were enriched in β-structure but unable to bind thioflavin-T. These data strongly support a model where proline-poor regions of the elastin monomer provide a unique contribution to assembly and suggest a role for localized β-sheet in mediating self-assembly interactions.     

Hydrophobic domains of human tropoelastin interact in a context-dependent manner

Tropoelastin is the soluble precursor of elastin, the major component of the extracellular elastic fiber. Tropoelastin undergoes self-association via an inverse temperature transition termed coacervation, which is a crucial step in elastogenesis. Coacervation of tropoelastin takes place through multiple intermolecular interactions of its hydrophobic domains. Previous work has implicated those hydrophobic domains located near the center of the polypeptide as playing a dominant role in coacervation. Short constructs of domains 18, 20, 24, and a mutated form of domain 26 were largely disordered at 20 degrees C but displayed increased order on heating that was consistent with the formation of beta-structures. However, their conformational transitions were not sensitive to physiological temperature in contrast to the observed behavior of the native domain 26. A polypeptide consisting of domains 17-27 of tropoelastin coacervated at temperatures above 60 degrees C, whereas individually expressed hydrophobic regions were not capable of coacervation. We conclude that coacervation depends on the hydrophobicity of the molecule and, by inference, the number of hydrophobic domains. Tropoelastin mutants were constructed to contain a Pro --> Ala mutation in domain 26, separate deletions of domains 18 and 26, and a displacement of domain 26. These constructs displayed unequal capacities for coacervation, even when they contained the same number of hydrophobic regions and comparable levels of secondary structure. Thus, the capability for coacervation is determined by contributions from individual hydrophobic domains for which function should be considered in the context of their positions in the intact tropoelastin molecule.     

Elastin as a self-organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self-assembly of elastin

Elastin is the major extracellular matrix protein of large arteries such as the aorta, imparting characteristics of extensibility and elastic recoil. Once laid down in tissues, polymeric elastin is not subject to turnover, but is able to sustain its mechanical resilience through thousands of millions of cycles of extension and recoil. Elastin consists of ca. 36 domains with alternating hydrophobic and cross-linking characteristics. It has been suggested that these hydrophobic domains, predominantly containing glycine, proline, leucine and valine, often occurring in tandemly repeated sequences, are responsible for the ability of elastin to align monomeric chains for covalent cross-linking. We have shown that small, recombinantly expressed polypeptides based on sequences of human elastin contain sufficient information to self-organize into fibrillar structures and promote the formation of lysine-derived cross-links. These cross-linked polypeptides can also be fabricated into membrane structures that have solubility and mechanical properties reminiscent of native insoluble elastin. Understanding the basis of the self-organizational ability of elastin-based polypeptides may provide important clues for the general design of self-assembling biomaterials.     

Domains in tropoelastin that mediate elastin deposition in vitro and in vivo

Elastic fiber assembly is a complicated process involving multiple different proteins and enzyme activities. However, the specific protein-protein interactions that facilitate elastin polymerization have not been defined. To identify domains in the tropoelastin molecule important for the assembly process, we utilized an in vitro assembly model to map sequences within tropoelastin that facilitate its association with fibrillin-containing microfibrils in the extracellular matrix. Our results show that an essential assembly domain is located in the C-terminal region of the molecule, encoded by exons 29-36. Fine mapping studies using an exon deletion strategy and synthetic peptides identified the hydrophobic sequence in exon 30 as a major functional element in this region and suggested that the assembly process is driven by the propensity of this sequence to form beta-sheet structure. Tropoelastin molecules lacking the C-terminal assembly domain expressed as transgenes in mice did not assemble nor did they interfere with assembly of full-length normal mouse elastin. In addition to providing important information about elastin assembly in general, the results of this study suggest how removal or alteration of the C terminus through stop or frameshift mutations might contribute to the elastin-related diseases supravalvular aortic stenosis and cutis laxa.     

Single nucleotide polymorphisms and domain/splice variants modulate assembly and elastomeric properties of human elastin. Implications for tissue specificity and durability of elastic tissue

Polymeric elastin provides the physiologically essential properties of extensibility and elastic recoil to large arteries, heart valves, lungs, skin and other tissues. Although the detailed relationship between sequence, structure and mechanical properties of elastin remains a matter of investigation, data from both the full‐length monomer, tropoelastin, and smaller elastin‐like polypeptides have demonstrated that variations in protein sequence can affect both polymeric assembly and tensile mechanical properties. Here we model known splice variants of human tropoelastin (hTE), assessing effects on shape, polymeric assembly and mechanical properties. Additionally we investigate effects of known single nucleotide polymorphisms in hTE, some of which have been associated with later‐onset loss of structural integrity of elastic tissues and others predicted to affect material properties of elastin matrices on the basis of their location in evolutionarily conserved sites in amniote tropoelastins. Results of these studies show that such sequence variations can significantly alter both the assembly of tropoelastin monomers into a polymeric network and the tensile mechanical properties of that network. Such variations could provide a temporal‐ or tissue‐specific means to customize material properties of elastic tissues to different functional requirements. Conversely, aberrant splicing inappropriate for a tissue or developmental stage or polymorphisms affecting polymeric assembly could compromise the functionality and durability of elastic tissues. To our knowledge, this is the first example of a study that assesses the consequences of known polymorphisms and domain/splice variants in tropoelastin on assembly and detailed elastomeric properties of polymeric elastin.     

Beaded nanofibers assembled from double‐hydrophobic elastin‐like block polypeptides: Effects of trifluoroethanol

A “double‐hydrophobic” elastin‐like triblock polypeptide GPG has been constructed by mimicking the localization of proline‐ and glycine‐rich hydrophobic domains of native elastin, a protein that provides elasticity and resilience to connective tissues. In this study, the effects of trifluoroethanol (TFE), an organic solvent that strongly affects secondary structures of polypeptides on self‐assembly of GPG in aqueous solutions were systematically studied. Beaded nanofiber formation of GPG , where nanoparticles are initially formed by coacervation of the polypeptides followed by their connection into one‐dimensional nanostructures, is accelerated by the addition of TFE at the concentrations up to 30% (v/v), whereas aggregates of nanoparticles are formed at 60% TFE. The concentration‐dependent assembly pattern discussed is based on the influence of TFE on the secondary structures of GPG . Well‐defined nanofibers whose diameter and secondary structures are controlled by TFE concentration may be ideal building blocks for constructing bioelastic materials in tissue engineering. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 175–185, 2015.     

Sequences and domain structures of mammalian, avian, amphibian and teleost tropoelastins: Clues to the evolutionary history of elastins

Tropoelastin is the monomeric form of elastin, a polymeric extracellular matrix protein responsible for properties of extensibility and elastic recoil in connective tissues of most vertebrates. As an approach to investigate how sequence and structural characteristics of tropoelastin assist in polymeric assembly and account for the elastomeric properties of this polymer, and to better understand the evolutionary history of elastin, we have identified and characterized tropoelastins from frog (Xenopus tropicalis) and zebrafish (Danio rerio), comparing these to their mammalian and avian counterparts. Unlike other species, two tropoelastin genes were expressed in zebrafish. All tropoelastins shared a predominant and characteristic alternating domain arrangement, as well as the fundamental crosslinking sequence motifs. However, zebrafish and frog tropoelastins had several unusual characteristics, including increased exon numbers and protein molecular weights, and decreased hydropathies. For all tropoelastins there was evidence of evolutionary expansion of the proteins by extensive replication of a hydrophobic-crosslinking exon pair. This was particularly apparent for zebrafish and frog tropoelastin genes, where remnants of sequence similarity were also seen in introns flanking the replicated exon pair. While overall alignment of mammalian, avian, frog and zebrafish tropoelastin sequences was not possible because of sequence variability, the C-terminal exon was well-conserved in all species. In addition, good sequence alignment was possible for several exons just upstream of the putative region of replication, suggesting that these conserved domains may represent 'primordial' core sequences present in the ancestral sequence common to all tropoelastins and in some way essential to the structure/function of elastin.     

Characterization of an unusual tropoelastin with truncated C-terminus in the frog

Tropoelastin is the monomeric form of elastin, a major polymeric protein of the extracellular elastic matrix of vertebrate tissues with properties of extensibility and elastic recoil. Mammalian and avian species contain a single gene for tropoelastin. A tropoelastin gene has also previously been identified in amphibians. In contrast, two tropoelastin genes with different tissue expression patterns have been described in teleosts. While general characteristics of tropoelastins, such as alternating arrangements of hydrophobic and crosslinking domains, are conserved across a wide phylogenetic range, sequences of these domains are highly variable, particularly when amphibian and teleost tropoelastins are included. For this reason exon-to-exon correspondence is not clear, and overall alignment of tropoelastin sequences across all species is not possible. An exception to this is the C-terminal exon, whose coding sequence has been very well-conserved across all species described to date. In mammalians this C-terminal domain has been shown to be important for interactions with cells and other matrix-associated proteins involved in matrix assembly. Here we identify and characterize a second tropoelastin gene in the frog with several unusual characteristics, the most striking of which is truncation of the C-terminal domain, deleting normally conserved sequence motifs. We demonstrate that, in spite of the absence of these motifs, both frog tropoelastin genes are expressed and incorporated into the elastic matrix, although with differential tissue localizations.     

Armadillo: domain boundary prediction by amino acid composition

The identification and annotation of protein domains provides a critical step in the accurate determination of molecular function. Both computational and experimental methods of protein structure determination may be deterred by large multi-domain proteins or flexible linker regions. Knowledge of domains and their boundaries may reduce the experimental cost of protein structure determination by allowing researchers to work on a set of smaller and possibly more successful alternatives. Current domain prediction methods often rely on sequence similarity to conserved domains and as such are poorly suited to detect domain structure in poorly conserved or orphan proteins. We present here a simple computational method to identify protein domain linkers and their boundaries from sequence information alone. Our domain predictor, Armadillo (http://armadillo.blueprint.org), uses any amino acid index to convert a protein sequence to a smoothed numeric profile from which domains and domain boundaries may be predicted. We derived an amino acid index called the domain linker propensity index (DLI) from the amino acid composition of domain linkers using a non-redundant structure dataset. The index indicates that Pro and Gly show a propensity for linker residues while small hydrophobic residues do not. Armadillo predicts domain linker boundaries from Z-score distributions and obtains 35% sensitivity with DLI in a two-domain, single-linker dataset (within +/-20 residues from linker). The combination of DLI and an entropy-based amino acid index increases the overall Armadillo sensitivity to 56% for two domain proteins. Moreover, Armadillo achieves 37% sensitivity for multi-domain proteins, surpassing most other prediction methods. Armadillo provides a simple, but effective method by which prediction of domain boundaries can be obtained with reasonable sensitivity. Armadillo should prove to be a valuable tool for rapidly delineating protein domains in poorly conserved proteins or those with no sequence neighbors. As a first-line predictor, domain meta-predictors could yield improved results with Armadillo predictions.     

Contribution of domain 30 of tropoelastin to elastic fiber formation and material elasticity

Elastin is a fibrous structural protein of the extracellular matrix that provides reversible elastic recoil to vertebrate tissues such as arterial vessels, lung, and skin. The elastin monomer, tropoelastin, contains a large proportion of intrinsically disordered and flexible hydrophobic sequences that collectively are responsible for the initial phase separation of monomers during assembly, and are essential for driving elastic recoil. While structural disorder of hydrophobic sequences is controlled by a high proline and glycine residue composition, hydrophobic domain 30 of human tropoelastin is atypically proline‐poor, and forms β‐sheet amyloid‐like fibrils as an individual peptide. We explored the contribution of confined regions of secondary structure at the location of domain 30 in human tropoelastin to fiber assembly and mechanical properties using a set of mutations designed to inhibit or enhance the propensity of β‐sheet formation at this location. Our data support a dual role for confined β‐sheet secondary structure in domain 30 of tropoelastin in guiding the formation of fibers, and as a determinant of stiffness and viscoelastic properties of cross‐linked materials. Together, these results suggest a mechanism for specificity in fiber assembly, and elucidate structure‐function relationships for the rational design of elastomeric biomaterials with defined mechanical properties. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 267–275, 2016.     

Domain 26 of tropoelastin plays a dominant role in association by coacervation

The temperature-dependent association of tropoelastin molecules through coacervation is an essential step in their assembly leading to elastogenesis. The relative contributions of C-terminal hydrophobic domains in coacervation were assessed. Truncated tropoelastins were constructed with N termini positioned variably downstream of domain 25. The purified proteins were assessed for their ability to coacervate. Disruption to domain 26 had a substantial effect and abolished coacervation. Circular dichroism spectroscopy of an isolated peptide comprising domain 26 showed that it undergoes a structural transition to a state of increased order with increasing temperature. Protease mapping demonstrated that domain 26 is flanked by surface sites and is likely to be in an exposed position on the surface of the tropoelastin molecule. These results suggest that the hydrophobic domain 26 is positioned to play a dominant role in the intermolecular interactions that occur during coacervation.