Synthesis and structural studies of a pentapeptide sequence of elastin. Poly (Val-Gly-Gly-Leu-Gly).

Poly (Val-Gly-Gly-Leu-Gly), a polypeptide mimicking the physico-chemical properties of the glycine-rich regions of elastin, has been synthesized and studied both in solution and in the aggregated state. By comparison, also the conformation of different "monomeric" units has been investigated. The polymer showed increased disorder with respect to the "monomers", the molecular conformation being accounted for by a more or less random collection of isolated beta-turns. Nevertheless, in the solid state the polymer is able to adopt supramolecular structures reminiscent of those found for elastin.     

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.     

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.     

Poly(Pro-Nle-Gly): Can an amorphus polypeptide take up a supramolecular elastinlike structure?

Poly(Pro-Nle-Gly) was found to have a CD spectrum characteristic of a random polypeptide and an x-ray powder pattern that does not reveal any regular secondary structure. In its electron micrograph, however, filaments and fibers were seen. This is a random polymer with supramolecular structure. It suggests that the supramolecular structure seen in electron micrographs of elastin preparations and related polypeptides does not necessarily imply an ordered structure at the molecular level.     

The infrastructure of aortic elastic fibers

Elastic fibers are composed of a central core of elastin that is amorphous and electron-lucent in conventional transmission electron micrographs and peripheral microfibrils. A complex infrastructure within the amorphous elastin of mature rat aorta is made visible by fixation and staining with a glutaraldehyde-ruthenium red mixture in phosphate buffer or osmium-ruthenium red in cacodylate buffer. The infrastructure is composed of at least two interlacing but distinct elastic structural components; a framework of circumferentially orientated microfibrils and a three-dimensional meshwork of filaments that permeate the fiber. The latter resembles a reticulum that has previously been observed in freeze-fractured and negatively stained elastin and attributed to the supramolecular organization of elastin. Microfibrils also extend from the core of the elastic fiber into the surrounding matrix where they appear to function as anchoring fibers. These observations indicate that the elastic properties of the arterial wall are an integrated function of both elastin and microfibrils.     

13C CPMAS NMR studies of the elastin-like polypeptide (LGGVG)n

The elucidation of structure-function relationships in insoluble elastin is often approached using elastin-like polypeptides. In this manner, the characterization of the different regions in this extensive biopolymer may be facilitated in a "piece-wise" manner. Our solid-state NMR experiments indicate that (LGGVG)n has structural similarities to elastin and some elastin peptides, providing support for the utility of the mimetic peptides. Furthermore, previous NMR and CD studies indicated that the structure of the elastin-like polypeptide (LGGVG)n in solution is best described as a "conformational ensemble" with a mixture of type I and II beta-turns, in addition to unfolded regions. Our data indicate that the peptide does not adopt a single conformation in the solid state, lending further support to models for elastin that involve significant conformational heterogeneity.     

A turning point in the knowledge of the structure-function-activity relations of elastin]

In this review are presented the last new results of our research group dealing with the molecular structures (atomic level) of tropoelastin, elastin and elastin derived peptides studied by using essentially methods of bioinformatics (theoretical predictions and molecular modelling) linked to experimental circular dichroism spectroscopic studies. We already had characterized both the local secondary structure and some parts of the tertiary structure of the tropoelastin and elastin molecules (human, bovine...), by using either theoretical predictions (local secondary structure, linear epitopes...) and/or experimental data (optical spectroscopic methods: Raman scattering, infrared absorption, circular dichroism). Except the cross-linking regions which are in helical conformations, the whole tropoelastin structure displays a lot of beta-reverse turns which usually belong to irregular structures in proteins. These turns play a key role in other regularly structures orientation (alpha-helix, beta-strand), thus they are very important in the native protein 3D architecture. It is particularly true for human tropoelastin, because its sequence is rich in glycines and prolines, and these residues are frequently met in beta-turns (a beta-turn is made of four consecutive residues which are stabilized by an hydrogen bond). Several types of beta-turns can be defined with the dihedral angles values phi and psi of the two central residues. Thus, by using a very recent updated set of propensities for the amino acid residues to belong to given types of reverse beta-turns (extracted from a reference set of known 3-D structures of globular proteins), we have determined, (by using our home made software COUDES), for all possible tetrapeptides of the human tropoelastin sequence, the distribution and the characterization of the possible type of turns. Thus, it is shown that the locations and/or the types of these reverse beta-turns reveal a regularity and are not all random. This confirms our hypothesis that intra-molecular elasticity of tropoelastin could be explained by the possibility of transitions between conformations involving short beta-strands and beta-turns. This result is of great interest in the construction (by using molecular biology) of elastic biomaterials derived from the elastin sequence (particularly, the elastin derived peptides corresponding to the sequence exon 21--(exon 24--exon 24...). Our study permit also to predict the conformations of specific elastin derived peptides which could have interesting biological activity. Peptides resulting from the degradation of elastin, the insoluble polymer of tropoelastin and responsible for the elasticity of vertebrate tissues, can induce biological effects and notably the regulation of matrix metalloproteinases (MMP-s) activity. Recently, it was proposed that some elastin derived hexapeptides resulting from circular permutations of VGVAPG (a three fold repetition sequence in exon 24 of human tropoelastin) possess MMP-1 production and activation regulation properties. This effect depends on the presence of the tropoelastin specific membraneous receptor 67 KDa EBP (Elastin Binding Protein). Our results obtained by using both circular dichroism spectroscopy and linear predictions confirmed the hypothesis of a structure dependent mechanism with a possibly occurring type VIII beta-turn on the first four residues of the GXXPG sequence consensus which is only present among all active peptides. Thus, we have performed extensive molecular dynamics studies, in both implicit and explicit solvent, on these active and inactive elastin derived hexapeptides. Using our own analysis method of pattern recognition of the types of the beta-reverse-turns followed during the molecular dynamics trajectory, we found that active and inactive peptides effectively form two well distinct conformational groups in which active peptides preferentially adopt conformation close to type VIII GXXP (beta-reverse-turn. The structural role of the C terminal G residue could also be explained. Additional molecular simulations on (VGVAPG)2 and (VGVAPG)3 show the formation of two or three GXXP tetrapeptides adopting a structure close to type VIII beta-reverse-turn, suggesting a local conformational preference for this motif. This observation of a specific structural single and/or repeated motif is in agreement with the circular dichroism spectra of the involved (VGVAPG)1, (VGVAPG)2 and (VGVAPG)3 peptides and then it can be proposed that their biological activities have to be linear. The final aim of this type of work is to understand more about the sequence/structure/function/activity relationships of those structured peptides in order to propose specific sequences (corresponding to specific structures) for best biological activity results.     

Synthesis and Structural Studies of a Pentapeptide Sequence of Elastin. Poly (Val-Gly-Gly-Leu-Gly)

Abstract

Poly (Val-Gly-Gly-Leu-Gly), a polypeptide mimicking the physico-chemical properties of the glycine-rich regions of elastin, has been synthesized and studied both in solution and in the aggregated state. By comparison, also the conformation of different “monomeric” units has been investigated. The polymer showed increased disorder with respect to the “monomers”, the molecular conformation being accounted for by a more or less random collection of isolated β-turns. Nevertheless, in the solid state the polymer is able to adopt supramolecular structures reminiscent of those found for elastin.     

Analysis and prediction of the different types of beta-turn in proteins

beta-Turns have been extracted from 59 non-identical proteins (resolution 2 A) using the standard criterion that the distance between C alpha (i) and C alpha (i + 3) is less than 7 A (1 A = 0.1 nm). The beta-turns have been classified, using phi, psi angles, into seven conventional turn types (I, I', II, II', IV, VIa, VIb) and a new class of beta-turn, designated type VIII, in which the central residues (i + 1, i + 2) adopt an alpha R beta conformation. Most beta-turn types are found in various topological environments, with the exception of I' and II' beta-turns, where 83% and 50%, respectively, are found in beta-hairpins. Sufficient data have been gathered to enable, for the first time, the separate statistical analysis of type I and II beta-turns. The two turn types have been shown to be strikingly different in their sequence preferences. Type I turns favour Asp, Asn, Ser and Cys at i; Asp, Ser, Thr and Pro at i + 1; Asp, Ser, Asn and Arg at i + 2; Gly, Trp and Met at i + 3, whilst type II turns prefer Pro at i + 1; Gly and Asn at i + 2; Gln and Arg at i + 3. These preferences have been explained by the specific side-chain interactions observed within the X-ray structures. The positional trends for type I and II beta-turns have been incorporated into the simple empirical predictive algorithm originally developed by P.N. Lewis et al. The program has improved the positional prediction of beta-turns, and has enhanced and extended the method by predicting the type of beta-turn. Since the observed preferences reflect local interactions these predictions are applicable not only to proteins, but also to peptides, many of which are thought to contain beta-turns.     

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.     

A neural network method for prediction of beta-turn types in proteins using evolutionary information

MOTIVATION: The prediction of beta-turns is an important element of protein secondary structure prediction. Recently, a highly accurate neural network based method Betatpred2 has been developed for predicting beta-turns in proteins using position-specific scoring matrices (PSSM) generated by PSI-BLAST and secondary structure information predicted by PSIPRED. However, the major limitation of Betatpred2 is that it predicts only beta-turn and non-beta-turn residues and does not provide any information of different beta-turn types. Thus, there is a need to predict beta-turn types using an approach based on multiple sequence alignment, which will be useful in overall tertiary structure prediction. RESULTS: In the present work, a method has been developed for the prediction of beta-turn types I, II, IV and VIII. For each turn type, two consecutive feed-forward back-propagation networks with a single hidden layer have been used where the first sequence-to-structure network has been trained on single sequences as well as on PSI-BLAST PSSM. The output from the first network along with PSIPRED predicted secondary structure has been used as input for the second-level structure-to-structure network. The networks have been trained and tested on a non-homologous dataset of 426 proteins chains by 7-fold cross-validation. It has been observed that the prediction performance for each turn type is improved significantly by using multiple sequence alignment. The performance has been further improved by using a second level structure-to-structure network and PSIPRED predicted secondary structure information. It has been observed that Type I and II beta-turns have better prediction performance than Type IV and VIII beta-turns. The final network yields an overall accuracy of 74.5, 93.5, 67.9 and 96.5% with MCC values of 0.29, 0.29, 0.23 and 0.02 for Type I, II, IV and VIII beta-turns, respectively, and is better than random prediction. AVAILABILITY: A web server for prediction of beta-turn types I, II, IV and VIII based on above approach is available at http://www.imtech.res.in/raghava/betaturns/ and http://bioinformatics.uams.edu/mirror/betaturns/ (mirror site).     

Role of elastin anisotropy in structural strain energy functions of arterial tissue

The vascular wall exhibits nonlinear anisotropic mechanical properties. The identification of a strain energy function (SEF) is the preferred method to describe its complex nonlinear elastic properties. Earlier constituent-based SEF models, where elastin is modeled as an isotropic material, failed in describing accurately the tissue response to inflation–extension loading. We hypothesized that these shortcomings are partly due to unaccounted anisotropic properties of elastin. We performed inflation–extension tests on common carotid of rabbits before and after enzymatic degradation of elastin and applied constituent-based SEFs, with both an isotropic and an anisotropic elastin part, on the experimental data. We used transmission electron microscopy (TEM) and serial block-face scanning electron microscopy (SBFSEM) to provide direct structural evidence of the assumed anisotropy. In intact arteries, the SEF including anisotropic elastin with one family of fibers in the circumferential direction fitted better the inflation–extension data than the isotropic SEF. This was supported by TEM and SBFSEM imaging, which showed interlamellar elastin fibers in the circumferential direction. In elastin-degraded arteries, both SEFs succeeded equally well in predicting anisotropic wall behavior. In elastase-treated arteries fitted with the anisotropic SEF for elastin, collagen engaged later than in intact arteries. We conclude that constituent-based models with an anisotropic elastin part characterize more accurately the mechanical properties of the arterial wall when compared to models with simply an isotropic elastin. Microstructural imaging based on electron microscopy techniques provided evidence for elastin anisotropy. Finally, the model suggests a later and less abrupt collagen engagement after elastase treatment.     

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. II. Characterization in solution and in the dry state.

The structure of the central repetitive domain of high molecular weight HMW) wheat gluten proteins was characterized in solution and in the dry state using HMW proteins Bx6 and Bx7 and a subcloned, bacterially expressed part of the repetitive domain of HMW Dx5. Model studies of the HMW consensus peptides PGQGQQ and GYYPTSPQQ formed the basis for the data analysis (van Dijk AA et al., 1997, Protein Sci 6:637-648). In solution, the repetitive domain contained a continuous nonoverlapping series of both type I and type II II beta-turns at positions predicted from the model studies; type II beta-turns occurred at QPGQ and QQGY sequences and type I beta-turns at YPTS and SPQQ. The subcloned part of the HMW Dx5 repetitive domain sometimes migrated as two bands on SDS-PAGE; we present evidence that this may be caused by a single amino acid insertion that disturbs the regular structure of beta-turns. The type I beta-turns are lost when the protein is dried on a solid surface, probably by conversion to type II beta-turns. The homogeneous type II beta-turn distribution is compatible with the formation of a beta-spiral structure, which provides the protein with elastic properties. The beta-turns and thus the beta-spiral are stabilized by hydrogen bonds within and between turns. Reformation of this hydrogen bonding network after, e.g., mechanical disruption may be important for the elastic properties of gluten proteins.     

Towards an artificial model for Photosystem II: a manganese(II,II) dimer covalently linked to ruthenium(II) tris-bipyridine via a tyrosine derivative

In order to model the individual electron transfer steps from the manganese cluster to the photooxidized sensitizer P680+ in Photosystem II (PS II) in green plants, the supramolecular complex 4 has been synthesized. In this complex, a ruthenium(II) tris-bipyridine type photosensitizer has been linked to a manganese(II) dimer via a substituted L-tyrosine, which bridges the manganese ions. The trinuclear complex 4 was characterized by electron paramagnetic resonance (EPR) and electrospray ionization mass spectrometry (ESI-MS). The excited state lifetime of the ruthenium tris-bipyridine moiety in 4 was found to be about 110 ns in acetonitrile. Using flash photolysis in the presence of an electron acceptor (methylviologen), it was demonstrated that in the supramolecular complex 4 an electron was transferred from the excited state of the ruthenium tris-bipyridine moiety to methylviologen, forming a methylviologen radical and a ruthenium(III) tris-bipyridine moiety. Next, the Ru(III) species retrieved the electron from the manganese(II/II) dimer in an intramolecular electron transfer reaction with a rate constant kET > 1.0 x 10(7) s(-1), generating a manganese(II/III) oxidation state and regenerating the ruthenium(II) photosensitizer. This is the first example of intramolecular electron transfer in a supramolecular complex, in which a manganese dimer is covalently linked to a photosensitizer via a tyrosine unit, in a process which mimics the electron transfer on the donor side of PS II.     

Mimicry by asx- and ST-turns of the four main types of beta-turn in proteins

Hydrogen-bonded beta-turns in proteins occur in four categories: type I (the most common), type II, type II', and type I'. Asx-turns resemble beta-turns, in that both have an NH. . .OC hydrogen bond forming a ring of 10 atoms. Serine and threonine side chains also commonly form hydrogen-bonded turns, here called ST-turns. Asx-turns and ST-turns can be categorized into four classes, based on side chain rotamers and the conformation of the central turn residue, which are geometrically equivalent to the four types of beta-turns. We propose asx- and ST-turns be named using the type I, II, I', and II' beta-turn nomenclature. Using this, the frequency of occurrence of both asx- and ST-turns is: type II' > type I > type II > type I', whereas for beta-turns it is type I > type II > type I' > type II'. Almost all type II asx-turns occur as a recently described three residue feature named an asx-nest.     

Poly(ethylene oxide)-b-poly(L-lactide) diblock copolymer/carbon nanotube-based nanocomposites: LiCl as supramolecular structure-directing agent

This work relies on the CNT dispersion in either solution or a polymer matrix through the formation of a three-component supramolecular system composed of PEO-b-PLLA diblock copolymer, carbon nanotubes (CNTs), and lithium chloride. According to a one-pot procedure in solution, the "self-assembly" concept has demonstrated its efficiency using suspension tests of CNTs. Characterizations of the supramolecular system by photon correlation spectroscopy, Raman spectroscopy, and molecular dynamics simulations highlight the charge transfer interaction from the CNTs toward the PEO-b-PLLA/LiCl complex. Finally, this concept was successfully extended in bulk (absence of solvent) via melt-processing techniques by dispersing these complexes in a commercial polylactide (PLA) matrix. Electrical conductivity measurements and transmission electron microscopy attested for the remarkable dispersion of CNTs, confirming the design of high-performance PLA-based materials.     

Circular dichroism studies on repeating polypeptide sequences of abductin

The secondary structure of abductin was investigated by CD and NMR studies of several synthetic peptides. Results obtained with these peptides showed the dominant conformations to be the polyproline II (PPII) structure in aqueous solution and different types of beta-turns in the less polar solvent trifluoroethanol. Accordingly, a preliminary structure-elasticity relationship for abductin, not unlike that currently accepted for elastin, is proposed.     

Prediction of the location and type of beta-turns in proteins using neural networks.

A neural network has been used to predict both the location and the type of beta-turns in a set of 300 nonhomologous protein domains. A substantial improvement in prediction accuracy compared with previous methods has been achieved by incorporating secondary structure information in the input data. The total percentage of residues correctly classified as beta-turn or not-beta-turn is around 75% with predicted secondary structure information. More significantly, the method gives a Matthews correlation coefficient (MCC) of around 0.35, compared with a typical MCC of around 0.20 using other beta-turn prediction methods. Our method also distinguishes the two most numerous and well-defined types of beta-turn, types I and II, with a significant level of accuracy (MCCs 0.22 and 0.26, respectively).     

Evidence of PPII-like helical conformation and glass transition in a self-assembled solid-state polypeptide-surfactant complex: poly(L-histidine)/docylbenzenesulfonic acid

We present lamellar self-assembly of cationic poly(L-histidine) (PLH) stoichiometrically complexed with an anionic surfactant, dodecyl benzenesulfonic acid (DBSA), which allows a stabilized conformation reminiscent of polyproline type II (PPII) left-handed helices. Such a conformation has no intrapeptide hydrogen bonds, and it has previously been found to be one source of flexibility, e.g., in collagen and elastin, as well as an intermediate in silk processing. PLH(DBSA)1.0 complexes were characterized by Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC). The PPII-like conformation in PLH(DBSA)1.0 is revealed by characteristic CD and FTIR spectra, where the latter indicates absence of intrachain peptide hydrogen bonds. In addition, a glass transition was directly verified by DSC at ca. 135 degrees C for PLH(DBSA)1.0 and indirectly by SAXS and TEM in comparison to pure PLH at 165 degrees C, thus indicating plasticization. Glass transitions have not been observed before in polypeptide-surfactant complexes. The present results show that surfactant binding can be a simple scheme to provide steric crowding to stabilize PPII conformation to tune the polypeptide properties, plasticization and flexibility.     

Optical spectroscopic elucidation of beta-turns in disulfide bridged cyclic tetrapeptides

Vibrational circular dichroism (VCD) spectroscopic features of type II beta-turns were characterized previously, but, criteria for differentiation between beta-turn types had not been established yet. Model tetrapeptides, cyclized through a disulfide bridge, were designed on the basis of previous experimental results and the observed incidence of amino acid residues in the i + 1 and i + 2 positions in beta-turns, to determine the features of VCD spectra of type I and II beta-turns. The results were correlated with electronic circular dichroism (ECD) spectra and VCD spectra calculated from conformational data obtained by molecular dynamics (MD) simulations. All cyclic tetrapeptides yielded VCD signals with a higher frequency negative and a lower frequency positive couplet with negative lobes overlapping. MD simulations confirmed the conformational homogeneity of these peptides in solution. Comparison with ECD spectroscopy, MD, and quantum chemical calculation results suggested that the low frequency component of VCD spectra originating from the tertiary amide vibrations could be used to distinguish between types of beta-turn structures. On the basis of this observation, VCD spectroscopic features of type II and VIII beta-turns and ECD spectroscopic properties of a type VIII beta-turn were suggested. The need for independent experimental as well as theoretical investigations to obtain decisive conformational information was recognized.