Brigida Bochicchio

Investigating by CD the molecular mechanism of elasticity of elastomeric proteins

Elastomeric proteins are widespread in the animal kingdom, and their main function is to confer elasticity and resilience to organs and tissues. Besides common functional properties, elastomeric proteins share a common sequence design. They are usually constituted by repetitive sequences with a high content of glycine residues. From a conformational point of view, all the elastomeric proteins since now analyzed show a dynamic equilibria between folded (mainly beta-turns) and extended (polyproline II and beta-strands) conformations that could be at the origin of the high entropy of the relaxed state. As a matter of fact, elastin, lamprin, abductin, as well as the PEVK domain of titin share the same conformational ensemble, thus pointing to a common molecular mechanism as the origin of elasticity. CD spectroscopy represents the proper spectroscopic technique to be used overall because of its particular sensitivity to the presence of PPII structure. Its use in the molecular studies of elastin, abductin, and lamprin as well as the recently analyzed protein resilin will be presented.

Design and production of a chimeric resilin-, elastin-, and collagen-like engineered polypeptide.

Protein-inspired biomaterials have gained great interest as an alternative to synthetic polymers, in particular, for their potential use as biomedical devices. The potential inspiring models are mainly proteins able to confer mechanical properties to tissues and organs, such as elasticity (elastin, resilin, spider silk) and strength (collagen, silk). The proper combination of repetitive sequences, each of them derived from different proteins, represents a useful tool for obtaining biomaterials with tailored mechanical properties and biological functions. In this report we describe the design, the production, and the preliminary characterization of a chimeric polypeptide, based on sequences derived from the highly resilient proteins resilin and elastin and from collagen-like sequences. The results show that the obtained chimeric recombinant material exhibits promising self-assembling properties. Youngs modulus of the fibers was determined by AFM image analysis and lies in the range of 0.1-3 MPa in agreement with the expectations for elastin-like and resilin-like materials.

Amyloid-like Fibrils in Elastin-Related Polypeptides : Structural Characterization and Elastic Properties

We report on the structural characterization of amyloid-like fibrils, self-assembled from synthetic polypentapeptides poly(ValGlyGlyLeuGly), whose monomeric sequence is a recurring, simple building block of elastin. This polymer adopts a beta-sheet structure as revealed by circular dichroism and Fourier transform infrared spectroscopy. Furthermore, Thioflavin-T and Congo red birefringence assays confirm the presence of amyloid-like structures. To analyze the supramolecular assembly and elastic properties of the fibrils, we employed atomic force microsocopy and spectroscopy, measuring also the elasticity of mature elastin for a comparative analysis. In the case of fibrils we estimated a Youngs modulus ranging from 3.5 to 7 MPa, whereas for elastin it is around 1 MPa. The possibility to section individual fibrils with nanometric control by the AFM tip, realizing biomolecular gaps in the 100 nm range, is also demonstrated. These results are expected to open interesting perspectives for the fabrication of protein-inspired nanostructures with specific physical and chemical properties for applications in biotechnology and tissue engineering.

Molecular and Supramolecular Structural Studies on Significant Repetitive Sequences of Resilin

Resilin is a member of the family of elastomeric proteins and is found in specialised regions of the cuticle of most insects, and provides low stiffness, high strain and efficient energy storage. It is best known for its role in insect flight and the remarkable jumping ability of fleas and spittle bugs. In common with other elastomeric proteins, the recently identified Drosophila melanogaster proresilin shows glycine‐rich repetitive sequences; in particular the N‐ and C‐terminal regions of the protein are dominated by 18 repeats of a 15‐residue sequence (SDTYGAPGGGNGGRP) and eleven repeats of a 13‐residue sequence (GYSGGRPGGQDLG), respectively. We synthesised and analysed the molecular and supramolecular structure of some polypeptides with sequences belonging to the glycine‐rich repeated domain of D. melanogaster resilin. The conformational studies performed by CD, FTIR and NMR spectroscopies pointed to the coexistence of two main conformational features, such as folded β‐turns and (quasi)extended structures (e.g., poly‐L‐proline II conformation) in common with other elastomeric proteins; this suggests an elasticity mechanism for resilin common to other elastomeric proteins. Our data show that also in the case of resilin, repetitive sequences are characterised by autonomous structures almost independent of the remaining parts of the molecule as already extensively found for elastin. From a supramolecular point of view, a great tendency to aggregate in fibrous structures is observed, particularly for the resilin‐ inspired polypeptide (PGGGN)10. This is encouraging for the development of resilin‐based biomaterials for the production of biocompatible medical devices, as well as high performing elastic materials.

Supramolecular organization of elastin and elastin-related nanostructured biopolymers.

The ultrastructure of elastin has been extensively analyzed by different methodologies. Starting from the first descriptions, where elastin was depicted as an amorphous structure, more complex and, in some cases, varied morphologies were revealed. The supramolecular structures found for elastin have been compared with those found for other elastin-related polypeptides, such as alpha-elastin and tropoelastin, and very similar features emerged. This review will deal with the supramolecular organization exhibited by many elastin-related compounds, starting from elastin, going through polypeptides constituted by different domains of tropoelastin, up to polymers containing repetitive sequences of elastin. In particular, recent developments on biopolymers of general type poly(Val-Pro-Gly-Xaa-Gly) and poly(Xaa-Gly-Gly-Zaa-Gly) (Xaa, Zaa = Val, Leu, Lys, Glu, Orn) obtained either by chemical synthesis or recombinant DNA techniques will be discussed in detail. The general aim is to describe the supramolecular features useful for the identification of elastin-inspired nanostructured biopolymers for developing highly functional and biocompatible vascular grafts as well as scaffolds for tissue regeneration.

On (GGLGY) synthetic repeating sequences of lamprin and analogous sequences

The repetitive sequence GGLGY was found in lamprin, the most important matrix protein of lamprey annular cartilage by Keeley and co-workers. Similar sequences appear also in other proteins, i.e. elastin, spidroin, spider minor ampullate silk proteins, in matrix proteins of the chorion or egg shell membrane of insects and others. We synthesized (GGLGY)n, n=1, 2, 6, because the sequence is repeated six times in the aggregated protein. The peptides were studied both in solution and in the solid state. Because the CD spectra were dominated by aromatic contribution, we synthesized GGLGF and GGLGA in order to carefully interpret the CD spectra. The conformational analysis suggests that all synthetic peptides do adopt the same secondary structure. In solution the peptides present a flexible conformation with a significant amount of PPII structure. In the solid state PPII, beta-pleated-sheets and beta-turns possibly co-exist.

Structural characterization and biological properties of the amyloidogenic elastin-like peptide (VGGVG)3

The peculiar and unique properties of elastin are due to the abundance of hydrophobic residues and of repetitive sequences as XGGZG (X, Z=V, L or A). Unexpectedly, these sequences not only provide elasticity to the whole protein, but are also able to form amyloid-like fibrils. Even though amyloid fibrils have been associated for a long time to the development of serious disorders as Alzheimers disease, recent evidence suggests that toxicity may be related to oligomeric species or to pre-fibrillar intermediates, rather than to mature fibrils. In addition, a number of studies highlighted the potential of bio-inspired materials based on amyloid-like nanostructures. The present study has been undertaken with the aim to characterize a chemically synthesized elastin-like peptide (VGGVG)3. Structural and biological features were compared with those of peptides as poly(VGGVG) and VGGVG that, having the same amino acid sequence, but different length and supramolecular structure have been previously investigated for their amyloidogenic properties. Results demonstrate that a minimum sequence of 15 amino acids is sufficient to aggregate into short amyloid-like fibrils, whose formation is however strictly dependent on the specific VGGVG repeated sequence. Moreover, in the attempt to elucidate the relationship among aggregation properties, fibers morphology and biocompatibility, 3T3 fibroblasts were grown in the presence of VGGVG-containing elastin-like peptides (ELPs) and analyzed for their ability to proliferate, attach and spread on ELPs-coated surfaces. Data clearly show that amyloid-like fibrils made of (VGGVG)3 are not cytotoxic at least up to the concentration of 100 μg/ml, even after several days of culture, and are a good support for cell attachment and spreading.

Investigating by circular dichroism some amyloidogenic elastin-derived polypeptides.

Tamburro and coworkers have demonstrated that some elastin-derived polypeptide sequences are able to give rise, in vitro, to amyloid-like fibers. The biological relevance of this finding could be explained by the recent detection of some amyloidogenic material found in arteries of old patients affected by atherosclerosis and demonstrated to be elastin derived. In this context, the comprehension of the mechanism responsible for the amyloid-like fibrillogenesis of elastin-derived sequences is of crucial importance for the design of drugs that could inhibit the amyloidogenic process. To gain further insights into the elastin amyloidogenic process, we studied the polypeptide sequences encoded by Exon 7 and Exon 32 of the human tropoelastin gene, and we demonstrated that only Exon 32 is able to aggregate in amyloid-like fibers. Vis-UV Thioflavin T circular dichroism (CD) spectroscopy rapidly and unambiguously detected the amyloidogenic propensity of the polypeptides. To gain additional insights into the aggregation mechanism of elastin-derived amyloidogenic peptides, we carried out the kinetics of EX32 amyloid-like aggregates by using ThT dye. CD spectroscopy was also used for investigating the secondary structure of the polypeptides, thus giving useful insights into the conformations involved in amyloid-like fiber formation. Furthermore, complementary techniques such as fluorescence spectroscopy, spectral shift, and binding Congo red UV assays as well as atomic force microscopy were also used to confirm the amyloidogenic behavior of the studied polypeptides.

Conformational and thermal characterization of a synthetic peptidic fragment inspired from human tropoelastin: Signature of the amyloid fibers.

OBJECTIVESnThis work deals with the conformational and thermal characterization of a synthetic peptide (S4) released during the proteolysis of human tropoelastin by the matrix metalloproteinase-12 that was shown to form amyloid-like fibres under certain conditions.nnnMATERIALS AND METHODSnS4 peptides were synthesized by solid-phase methodology and aggregated in solution at 80°C. Fourier transform-infrared spectroscopy (FT-IR) was used to access the secondary structure. Thermal characterization was performed by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC).nnnRESULTSnThe DSC study of the soluble linear peptide S4 in solution in TBS reveals the irreversible aggregation into amyloid fibres. FT-IR, DSC and TGA analyses performed on freeze-dried samples evidence differences between the linear peptide and its associated amyloid-like fibres, both on the conformation and the physical structure. When S4 peptides are aggregated, the prominent conformation scanned by FT-IR is the cross β-structure, corresponding to TGA to an increase of the thermal stability. Moreover, the DSC thermograms of S4 fibres are characteristic of a highly ordered structure, in contrast to the DSC thermograms of S4 linear peptides, characteristic of an amorphous structure. Finally, the DSC analysis of differently hydrated S4 fibres brings to the fore the specific thermal answer of the wet interfaces of the cross β-fibres.nnnCONCLUSIONnFT-IR and thermal techniques are well suited to evidence conformational and structural differences between the soluble peptide and its amyloid form.

Formation of nanostructures by self-assembly of an elastin peptide

Elastin and elastin-related peptides have great potential in the biomaterial field, because of their peculiar mechanical properties and spontaneous self-assembling behavior. Depending on their sequences and under appropriate experimental conditions, they are able to self-assemble in different fiber morphologies, including amyloid-like fibers. Temperature-triggered self-assembly of a small elastin peptide shows a novel complex aggregation mechanism as revealed by different microscopy techniques. The conformations of the peptide have been investigated in solution and in the aggregated state by different spectroscopic techniques (CD, NMR, FT-IR) and revealed that the conformations adopted by the peptides in water in the prefibrillar state correspond to those populated by other elastin peptides, mainly polyproline II helix (PPII) and random coil. Conversely, the aggregated state shows evidence for antiparallel cross-β structures. Our molecular studies highlight the important role of PPII conformation on the prefibrillar state, putting forward the hypothesis that aggregation takes place through addition of the monomer in the PPII conformation with preformed β-sheet aggregates and/or through direct interaction of PPII helices.