Transformation of amyloid-like fibers, formed from an elastin-based biopolymer, into a hydrogel: an X-ray photoelectron spectroscopy and atomic force microscopy study

Previous studies have revealed the propensity of elastin-based biopolymers to form amyloid-like fibers when dissolved in water. These are of interest when considered as "ancestral units" of elastin in which they represent the simplest sequences in the hydrophobic regions of the general type XxxGlyGlyZzzGly (Xxx, Zzz = Val, Leu). We normally refer to these biopolymers based on elastin or related to elastin units as "elastin-like polypeptides". The requirement of water for the formation of amyloids seems quite interesting and deserves investigation, the water representing the natural transport medium in human cells. As a matter of fact, the "natural" supramolecular organization of elastin is in the form of beaded-string-like filaments and not in the form of amyloids whose "in vivo" deposition is associated with some important human diseases. Our work is directed, therefore, to understanding the mechanism by which such hydrophobic sequences form amyloids and any conditions by which they might regress to a non-amyloid filament. The elastin-like sequence here under investigation is the ValGlyGlyValGly pentapeptide that has been previously analyzed both in its monomer and polymer form. In particular, we have focused our investigation on the apparent stability of amyloids formed from poly(ValGlyGlyValGly), and we have observed these fibers evolving to a hydrogel after prolonged aging in water. We will show how atomic force microscopy can be combined with X-ray photoelectron spectroscopy to gain an insight into the spontaneous organization of an elastin-like polypeptide driven by interfacial interactions. The results are discussed also in light of fractal-like assembly and their implications from a biomedical point of view.     

Sequence and structure determinants for the self-aggregation of recombinant polypeptides modeled after human elastin

Elastin is a polymeric structural protein that imparts the physical properties of extensibility and elastic recoil to tissues. The mechanism of assembly of the tropoelastin monomer into the elastin polymer probably involves extrinsic protein factors but is also related to an intrinsic capacity of elastin for ordered assembly through a process of hydrophobic self-aggregation or coacervation. Using a series of simple recombinant polypeptides based on elastin sequences and mimicking the unusual alternating domain structure of native elastin, we have investigated the influence of sequence motifs and domain structures on the propensity of these polypeptides for coacervation. The number of hydrophobic domains, their context in the alternating domain structure of elastin, and the specific nature of the hydrophobic domains included in the polypeptides all had major effects on self-aggregation. Surprisingly, in polypeptides with the same number of domains, propensity for coacervation was inversely related to the mean Kyte-Doolittle hydropathy of the polypeptide. Point mutations designed to increase the conformational flexibility of hydrophobic domains had the unexpected effect of suppressing coacervation and promoting formation of amyloid-like fibers. Such simple polypeptides provide a useful model system for understanding the relationship between sequence, structure, and mechanism of assembly of polymeric elastin.     

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.     

Combined effects of solvation and aggregation propensity on the final supramolecular structures adopted by hydrophobic,glycine‐rich,elastin‐like polypeptides

Previous work on elastin‐like polypeptides (ELPs) made of hydrophobic amino acids of the type XxxGlyGlyZzzGly (Xxx, Zzz = Val, Leu) has consistently shown that differing dominant supramolecular structures were formed when the suspending media were varied: helical, amyloid‐like fibers when suspended in water and globules evolving into “string of bead” structures, poly(ValGlyGlyValGly), or cigar‐like bundles, poly(ValGlyGlyLeuGly), when suspended in methyl alcohol. Comparative experiments with poly(LeuGlyGlyValGly) have further indicated that the interface energy plays a significant role and that solvation effects act in concomitance with the intrinsic aggregation propensity of the repeat sequence. Continuing our investigation on ELPs using surface (X‐ray photoelectron spectroscopy, atomic force microscopy) and bulk (circular dichroism, Fourier transform infrared spectroscopy) techniques for their characterization, here we have compared the effect of suspending solvents (H₂O, dimethylsulfoxide, ethylene glycol, and MeOH) on poly(ValGlyGlyValGly), the polypeptide most inclined to form long and well‐refined helical fibers in water, searching for the signature of intermolecular interactions occurring between the polypeptide chains in the given suspension. The influence of sequence specificities has been studied by comparing poly(ValGlyGlyValGly) and poly(LeuGlyGlyValGly) with a similar degree of polymerization. Deposits on substrates of the polypeptides were characterized taking into account the differing evaporation rate of solvents, and tests on their stability in ultra high vacuum were performed. Finally, combining experimental and computational studies, we have revaluated the three‐dimensional modeling previously proposed for the supramolecular assembly in water of poly(ValGlyGlyValGly). The results were discussed and rationalized also in the light of published data. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 292–292, 2013.     

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.     

AFM study of the elastin-like biopolymer poly(ValGlyGlyValGly)

In this paper, we report an AFM study on the supramolecular structures adopted by the synthetic polypentapeptide poly(ValGlyGlyValGly), whose monomeric sequence is an abundant, simple building block of elastin. The polypeptide was analyzed by deposition from both methanolic and aqueous suspensions, showing different behaviors. In methanol, the polypeptide is able to evolve, in a time-dependent way, from layers to ribbons to beaded filaments. When the equilibrium is reached, the formation of well-defined dendritic structures is also observed. This restructuring of the polypentapeptide seems to be reminiscent of a sort of Rayleigh instability. When deposited from aqueous suspensions, the polypeptide self-assembles either in fibrillar networks or in amyloid-like patterns, both of them being found in elastin or elastin-related polypeptides. As a general finding, poly(ValGlyGlyValGly) seems to constitute an excellent mimetic of the supramolecular properties of native elastin.     

The effects of the side chains of hydrophobic aliphatic amino acid residues in an amphipathic polypeptide on the formation of alpha helix and its association

The polypeptide alpha3, which was synthesized by us to produce an amphipathic helix structure, contains the regular three times repeated sequence (LETLAKA)(3), and alpha3 forms a fibrous assembly. To clarify how the side chains of amino acid residues affect the formation of alpha helix, Leu residues, which are located in the hydrophobic surface of an amphipathic helix, were replaced by other hydrophobic aliphatic amino acid residues systematically, and the characters of the resulting polypeptides were studied. According to the circular dichroism (CD) spectra, the Ile-substituted polypeptides formed alpha helix like alpha3. However, their helix formation ability was weaker than that of alpha3 under some conditions. The Val-substituted polypeptides formed alpha helix only under restricted condition. The Ala-substituted polypeptides did not form alpha helix under any condition. Thus, it is clear that the order of the alpha helix formation ability is as follows: Leu >or= Ile > Val > Ala. The formation of alpha helix was confirmed by Fourier Transform Infrared (FTIR) spectra. Through electron microscopic observation, it was clarified that the formation of the alpha helix structure correlates with the formation of a fibrous assembly. The amphipathic alpha helix structure would be stabilized by the formation of the fibrous assembly.     

Synthetic fragments and analogues of elastin. I. The synthesis

The synthesis of some repetitive sequences of elastin and their simplified analogues, all comprising the structural unit Gly-X-Gly (X = Val, Leu, Ala), is described. In particular, the following peptides and polypeptides were synthesized and characterized: Boc-Gly-Val-Gly-Gly-Leu-OMe, Boc-Gly-Leu-Gly-Gly-Val-OMe, Boc-(Gly-Val-Gly-Gly-Leu)2-OMe, Boc-(Gly-Val-Gly-Gly-Leu)3-OMe, Boc-Gly-Val-Gly-Gly-OEt, Boc-Leu-Gly-Gly-Leu-OMe, Boc-Val-Gly-Gly-Val-OMe, poly(Ala-Gly-Gly), poly(Val-Gly-Gly), and poly(Leu-Gly-Gly). In every case, the synthesis was accomplished by classical procedures in solution, by using the p-nitrophenyl ester method for the polycondensation step, and the mixed anhydride or the azide methods for the coupling steps.     

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.     

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.     

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.     

Synthetic fragments and analogues of elastin. II. Conformational studies

Conformational studies on synthetic repetitive sequences and analogues of elastin are described. CD and nmr measurements gave evidence of flexible beta-turns as the dominant structural feature whose stability was found to decrease by increasing the number of repetitive units. The sequences comprised the structural unit Gly-X-Gly (X = Val, Leu, Ala), with X-Gly or Gly-Gly located at the corners of the bend. Based on that, it is proposed that these regions of elastin, unlike the proline-containing sequences, contribute to the elasticity of the protein through a classical mechanism in terms of the rotational isomeric state theory.     

Design and synthesis of cationic Aib-containing antimicrobial peptides: conformational and biological studies.

Development of antimicrobial peptides has attracted considerable attention in recent years due to the excessive use of antibiotics, which has led to multiresistant bacteria. Cationic amphiphilic Aib-containing peptide models Ac-(Aib-Arg-Aib-Leu)(n)-NH2, n = 1-4, and sequential cationic polypeptides (Arg-X-Gly)(n), X = Ala, Val, Leu, were prepared and studied for their antimicrobial and hemolytic activity, as well as for their proteolytic stability. Ac-(Aib-Arg-Aib-Leu)(n)-NH2, n = 2, 3 and the polypeptide (Arg-Leu-Gly)(n) exhibited significant antimicrobial activity, and they were nontoxic at their MIC values and resistant, in particular the Aib-peptide models, to enzymatic degradation. The conformational characteristics of the peptide models were studied by circular dichroism (CD). Structure-activity relationship studies revealed the importance of the amphipathic alpha-helical conformation of the reported peptides in inducing antimicrobial effects. It is concluded that peptide models comprising cationic amino acids (Arg), helicogenic and noncoding residues (Aib) and/or hydrophobic and helix-promoting components (Leu) may lead to the development of antimicrobial therapeutics.     

Crystal structure of a tripeptide containing aminocyclododecane carboxylic acid: a supramolecular twisted parallel β‐sheet in crystals

The crystal structure of a tripeptide Boc‐Leu‐Val‐Ac₁₂c‐OMe ( 1 ) is determined, which incorporates a bulky 1‐aminocyclododecane‐1‐carboxylic acid (Ac₁₂c) side chain. The peptide adopts a semi‐extended backbone conformation for Leu and Val residues, while the backbone torsion angles of the C^α,α‐dialkylated residue Ac₁₂c are in the helical region of the Ramachandran map. The molecular packing of 1 revealed a unique supramolecular twisted parallel β‐sheet coiling into a helical architecture in crystals, with the bulky hydrophobic Ac₁₂c side chains projecting outward the helical column. This arrangement resembles the packing of peptide helices in crystal structures. Although short oligopeptides often assemble as parallel or anti‐parallel β‐sheet in crystals, twisted or helical β‐sheet formation has been observed in a few examples of dipeptide crystal structures. Peptide 1 presents the first example of a tripeptide showing twisted β‐sheet assembly in crystals. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Repeat polypeptide models of elastin as substrates for lysyl oxidase

Synthetic repeat polypeptides analogous in sequence to the valine-rich regions of elastin have been tested as substrates for purified bovine aorta lysyl oxidase. These polypeptides, HCO(phi-Pro-Gly-Gly)n-Val-OMe, HCO(Val-Pro-Gly-phi-Gly)n-Val-OMe, and HCO-Val-(Ala-Pro-Gly-phi-Gly-Val)n-OMe, where phi = Val or Lys at approximately a 4:1 ratio and where n greater than or equal to 40, are models of the tetra-, penta-, or hexapeptide repeat sequences found in elastin. alpha-Aminoadipic delta-semialdehyde is generated in each of these upon incubation with lysyl oxidase at 37 degrees C, whereas the aldol and anhydrolysinonorleucine bifunctional cross-linkages were formed only in the incubation of enzyme with polypentapeptide. Incubation of the polypentapeptide at 55 degrees C, which enhances coacervation of the peptide, increases aldehyde formation and generates a much higher ratio of cross-linkages to aldehyde than occurred at 37 degrees C. These results demonstrate that lysyl oxidase can oxidize lysine in synthetic polypeptides and suggest important conformational aspects of lysyl oxidase substrates which may control substrate potential as well as the ability of peptidyl aldehyde, once formed by the enzyme, to condense to cross-linkage products.     

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.     

Chemical synthesis of cross-linked poly(KGGVG), an elastin-like biopolymer

Previous studies afforded on peptides and polypeptides containing repetitive sequences of elastin have largely demonstrated that their molecular and supramolecular properties are fully representative of those of tropoelastin, the soluble, linear precursor of elastin itself. In the attempt to synthesize cross-linked elastin-mimetic polypeptides, the repeating sequence VGGVG (V: valine; G: glycine), typical of elastin, was modified to incorporate lysine residues, yielding the polymer poly(KGGVG) (K: lysine). This imparts primary amine functionality susceptible to cross-linking reaction with appropriate bifunctional cross-linking reagents. We report herein the chemical synthesis and cross-linking of poly(KGGVG) with glutaraldehyde (GTA) and with disuccinimidyl glutarate (DSG). In both cases, the characterization of the polymers, both linear and cross-linked, has been carried out by CD spectroscopy and transmission electron microscopy measurements. The obtained results, although not conclusive, demonstrate that poly(KGGVG), both linear and cross-linked, may be considered very similar to tropoelastin and mature elastin, as concerns its molecular and supramolecular properties.     

The action of neutrophil serine proteases on elastin and its precursor

This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P₁, which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P₁ in tropoelastin, whereas Lys was not identified at P₁ in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P₂ and P₄′. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG.     

Polypeptide models of elastin: CD and NMR studies on synthetic poly(X-Gly-Gly).

Poly(X-Gly-Gly), simple structural models for the hydrophobic, proline-devoid, regions of elastin, have been synthesized and studied by circular dichroism and NMR spectroscopies. The results gave evidence of type II beta-turns as the only ordered structure present in the polymers. The stability of the turns has been shown to decrease on hydration and to increase in the series Leu less than Ala less than Val less than Ile.