Nanoscale structure of poly(ethylene glycol) hybrid block copolymers containing amphiphilic beta-strand peptide sequences

This paper discusses the solid state and melt nanoscale structure of a series of novel poly(ethylene glycol) (PEG) hybrid di- and triblock copolymers, which contain amphiphilic beta-strand peptide sequences. The block copolymers have been prepared via solid-phase synthesis, affording perfectly monodisperse peptide segments with a precisely defined alpha-amino acid sequence. Attenuated total reflection Fourier transform infrared spectroscopy and X-ray scattering experiments indicate that the self-assembly properties of the peptide sequences are retained upon conjugation to PEG and mediate the formation of an ordered superstructure consisting of alternating PEG layers and peptide domains with an highly organized antiparallel beta-sheet structure. The results suggest that combination of biological structural motifs with synthetic polymers may be a versatile strategy for the development of novel self-assembled materials with complex internal structures and the potential to interface with biology.     

Review: model peptides and the physicochemical approach to beta-amyloids

beta-Amyloid peptides are the main protein components of neuritic plaques and may be important in the pathogenesis of Alzheimer's Disease. The determination of the structure of beta-amyloid fibrils poses a challenge because of the limited solubility of beta-amyloid peptides and the noncrystalline nature of fibrils formed from these peptides. In this paper, we describe several physicochemical approaches which have been used to examine fibrils and the fibrillogenesis of peptide models of beta-amyloid. Recent advances in solid state NMR, such as the DRAWS pulse sequence, have made this approach a particularly attractive one for peptides such as beta-amyloid, which are not yet amenable to high-resolution solution phase NMR and crystallography. The application of solid state NMR techniques has yielded information on a model peptide comprising residues 10-35 of human beta-amyloid and indicates that in fibrils, this peptide assumes a parallel beta-strand conformation, with all residues in exact register. In addition, we discuss the use of block copolymers of Abeta peptides and polyethylene glycol as probes for the pathways of fibrillogenesis. These methods can be combined with other new methods, such as high-resolution synchrotron X-ray diffraction and small angle neutron and X-ray scattering, to yield structural data of relevance not only to disease, but to the broader question of protein folding and self-assembly.     

Self-assembly in aqueous solution of a modified amyloid beta peptide fragment

The self-assembly in films dried from aqueous solutions of a modified amyloid beta peptide fragment is studied. We focus on sequence Abeta(16-20), KLVFF, extended by two alanines at the N-terminus to give AAKLVFF. Self-assembly into twisted ribbon fibrils is observed, as confirmed by transmission electron microscopy (TEM). Dynamic light scattering reveals the semi-flexible nature of the AAKLVFF fibrils, while polarized optical microscopy shows that the peptide fibrils crystallize after an aqueous solution of AAKLVFF is matured over 5 days. The secondary structure of the fibrils is studied by FT-IR, circular dichroism and X-ray diffraction (XRD), which provide evidence for beta-sheet structure in the fibril. From high resolution TEM it is concluded that the average width of an AAKLVFF fibril is (63+/-18) nm, indicating that these fibrils comprise beta-sheets with multiple repeats of the unit cell, determined by XRD to have b and c dimensions 1.9 and 4.4 nm with an a axis 0.96 nm, corresponding to twice the peptide backbone spacing in the antiparallel beta-sheet.     

Amyloid fibril formation by A beta 16-22, a seven-residue fragment of the Alzheimer's beta-amyloid peptide, and structural characterization by solid state NMR

The seven-residue peptide N-acetyl-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH(2), called A beta(16-22) and representing residues 16-22 of the full-length beta-amyloid peptide associated with Alzheimer's disease, is shown by electron microscopy to form highly ordered fibrils upon incubation of aqueous solutions. X-ray powder diffraction and optical birefringence measurements confirm that these are amyloid fibrils. The peptide conformation and supramolecular organization in A beta(16-22) fibrils are investigated by solid state (13)C NMR measurements. Two-dimensional magic-angle spinning (2D MAS) exchange and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) measurements indicate a beta-strand conformation of the peptide backbone at the central phenylalanine. One-dimensional and two-dimensional spectra of selectively and uniformly labeled samples exhibit (13)C NMR line widths of <2 ppm, demonstrating that the peptide, including amino acid side chains, has a well-ordered conformation in the fibrils. Two-dimensional (13)C-(13)C chemical shift correlation spectroscopy permits a nearly complete assignment of backbone and side chain (13)C NMR signals and indicates that the beta-strand conformation extends across the entire hydrophobic segment from Leu17 through Ala21. (13)C multiple-quantum (MQ) NMR and (13)C/(15)N rotational echo double-resonance (REDOR) measurements indicate an antiparallel organization of beta-sheets in the A beta(16-22) fibrils. These results suggest that the degree of structural order at the molecular level in amyloid fibrils can approach that in peptide or protein crystals, suggest how the supramolecular organization of beta-sheets in amyloid fibrils can be dependent on the peptide sequence, and illustrate the utility of solid state NMR measurements as probes of the molecular structure of amyloid fibrils. A beta(16-22) is among the shortest fibril-forming fragments of full-length beta-amyloid reported to date, and hence serves as a useful model system for physical studies of amyloid fibril formation.     

Probing the importance of lateral hydrophobic association in self-assembling peptide hydrogelators

A class of peptides has been designed whose ability to self-assemble into hydrogel is dependent on their conformationally folded state. Under unfolding conditions aqueous peptide solutions are freely flowing having the viscosity of water. When folding is triggered by external stimuli, peptides adopt a β-hairpin conformation that self-assembles into a highly crosslinked network of fibrils affording mechanically rigid hydrogels. MAX 1, a 20 residue, amphiphilic hairpin self-assembles via a mechanism which entails both lateral and facial self-assembly events to form a network of fibrils whose local structure consists of a bilayer of hairpins hydrogen bonded in the direction of fibril growth. Lateral self-assembly along the long axis of the fibril is mainly facilitated by intermolecular hydrogen bonding between the strands of distinct hairpins and the formation of hydrophobic contacts between residue side chains of laterally associating hairpins. Facial assembly is driven by the hydrophobic collapse of the valine-rich faces of the amphiphilic hairpins affording a bilayer laminate. The importance of forming lateral hydrophobic contacts during hairpin self-assembly and the relative contribution these interactions have towards nano-scale morphology and material rigidity is probed via the study of: MAX1, a hairpin designed to exploit lateral hydrophobic interactions; MAX 4, a peptide with reduced ability to form these interactions; and MAX5, a control peptide. CD spectroscopy and rheological experiments suggest that the formation of lateral hydrophobic interactions aids the kinetics of assembly and contributes to the mechanical rigidity of the hydrogel. Transmission electron microscopy (TEM) shows that these interactions play an essential role in the self-assembly process leading to distinct nano-scale morphologies. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Self-assembling diblock copolymers of poly[N-(2-hydroxypropyl)methacrylamide] and a beta-sheet peptide

The self-assembly of hybrid diblock copolymers composed of poly(HPMA) and beta-sheet peptide P11 (CH(3)CO-QQRFQWQFEQQ-NH(2)) blocks was investigated. Copolymers were synthesized via thiol-maleimide coupling reaction, by conjugation of semitelechelic poly(HPMA)-SH with maleimide-modified beta-sheet peptide. As expected, CD and CR binding studies showed that the peptide block imposed its beta-sheet structural arrangement on the structure of diblock copolymers. TEM and AFM proved that peptide and these copolymers had the ability to self-assemble into fibrils.     

Review: Model Peptides and the Physicochemical Approach to β-Amyloids

β-Amyloid peptides are the main protein components of neuritic plaques and may be important in the pathogenesis of Alzheimer's Disease. The determination of the structure of β-amyloid fibrils poses a challenge because of the limited solubility of β-amyloid peptides and the noncrystalline nature of fibrils formed from these peptides. In this paper, we describe several physicochemical approaches which have been used to examine fibrils and the fibrillogenesis of peptide models of β-amyloid. Recent advances in solid state NMR, such as the DRAWS pulse sequence, have made this approach a particularly attractive one for peptides such as β-amyloid, which are not yet amenable to high-resolution solution phase NMR and crystallography. The application of solid state NMR techniques has yielded information on a model peptide comprising residues 10–35 of human β-amyloid and indicates that in fibrils, this peptide assumes a parallel β-strand conformation, with all residues in exact register. In addition, we discuss the use of block copolymers of Aβ peptides and polyethylene glycol as probes for the pathways of fibrillogenesis. These methods can be combined with other new methods, such as high-resolution synchrotron X-ray diffraction and small angle neutron and X-ray scattering, to yield structural data of relevance not only to disease, but to the broader question of protein folding and self-assembly.     

Synthesis and self-assembly of chitosan-based copolymer with a pair of hydrophobic/hydrophilic grafts of polycaprolactone and poly(ethylene glycol)

Chitosan-based copolymers with binary grafts of hydrophobic polycaprolactone and hydrophilic poly(ethylene glycol) (CS-g-PCL&PEG) were prepared by a homogeneous coupling reaction of phthaloyl-protected chitosan with functional PCL-COOH and PEG-COOH, following deprotection to regenerate free amino groups back to chitosan backbone. They were characterized by ¹H NMR, Fourier transform infrared and X-ray diffraction analysis. These CS-g-PCL&PEG copolymers could form nano-size self-aggregates in acidic aqueous solution without a specific processing technique, which were investigated using dynamic light scattering and transmission electron microscopy. The formed self-aggregates become smaller with weakened stability upon pH increasing. Moreover, the aggregates of copolymer with higher content of PEG and PCL grafts could remain stable for over 30 days in both acid and neutral condition. A possible mechanism was proposed for the formation of self-aggregates from CS-g-PCL&PEG and their structural changes as pH. It is warranted to find promising application of these self-aggregates based on chitosan as drug carriers.     

Charge attraction and beta propensity are necessary for amyloid fibril formation from tetrapeptides

Amyloid fibrils in which specific proteins have polymerized into a cross-beta-sheet structure are found in about 20 diseases. In contrast to the close structural similarity of fibrils formed in different amyloid diseases, the structures of the corresponding native proteins differ widely. We show here that peptides as short as 4 residues with the sequences KFFE or KVVE can form amyloid fibrils that are practically identical to fibrils formed in association with disease, as judged by electron microscopy and Congo red staining. In contrast, KLLE or KAAE do not form fibrils. The fibril-forming KFFE and KVVE show partial beta-strand conformation in solution, whereas the non-fibril-forming KLLE and KAAE show random structure only, suggesting that inherent propensity for beta-strand conformation promotes fibril formation. The peptides KFFK or EFFE do not form fibrils on their own but do so in an equimolar mixture. Thus, intermolecular electrostatic interactions, either between charged dipolar peptides or between complementary charges of co-fibrillating peptides favor fibril formation.     

Synthesis and characterization of hydroxypropyl methylcellulose and ethyl acrylate graft copolymers

The synthesis of hydroxypropyl methylcellulose-g-poly (ethyl acrylate) was carried out by potassium persulfate induced graft copolymerization in homogeneous aqueous medium. By varying the reaction conditions, graft copolymers with different percentage of grafting were prepared. These graft copolymers were characterized by fourier transform infrared spectra (FTIR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), thermogravimetric analyses (TGA), X-ray diffraction analysis (XRD), and dynamic light scattering (DLS) methods. The molecular weight of grafted and ungrafted polymer chains determined by gel permeation chromatography (GPC) increased with increasing monomer and matrix concentration but decreased with increasing initiator concentration and reaction temperature. The mechanical properties of graft copolymers were measured as function of the percentage of grafting. In addition, the equilibrium humidity adsorption behavior and the disintegration time of the grafted copolymer films were also studied.     

Self-assembly and hydrogelation of an amyloid peptide fragment

The self-assembly of a fragment of the amyloid beta peptide that has been shown to be critical in amyloid fibrillization has been studied in aqueous solution. There are conflicting reports in the literature on the fibrillization of Abeta (16-20), i.e., KLVFF, and our results shed light on this. In dilute solution, self-assembly of NH 2-KLVFF-COOH is strongly influenced by aromatic interactions between phenylalanine units, as revealed by UV spectroscopy and circular dichroism. Fourier transform infrared (FTIR) spectroscopy reveals beta-sheet features in spectra taken for more concentrated solutions and also dried films. X-ray diffraction and cryo-transmission electron microscopy (cryo-TEM) provide further support for beta-sheet amyloid fibril formation. A comparison of cryo-TEM images with those from conventional dried and negatively stained TEM specimens highlights the pronounced effects of sample preparation on the morphology. A comparison of FTIR data for samples in solution and dried samples also highlights the strong effect of drying on the self-assembled structure. In more concentrated phosphate-buffered saline (PBS) solution, gelation of NH 2-KLVFF-COOH is observed. This is believed to be caused by screening of the electrostatic charge on the peptide, which enables beta sheets to aggregate into a fibrillar gel network. The rheology of the hydrogel is probed, and the structure is investigated by light scattering and small-angle X-ray scattering.     

The folding transition state of the cold shock protein is strongly polarized

It has been shown recently that an 11-residue peptide fragment of transthyretin, TTR(105-115), can form amyloid fibrils in vitro by adopting an extended beta-strand conformation. We used molecular dynamics simulations on systems of TTR(105-115) peptides, for a total length of about 5 micros, to explore the process of self-assembly and the structures of the resulting aggregates. Our results suggest that an antiparallel association of the beta-strands is more probable than a parallel one and that the central residues (T106-L111) in a beta-strand have a high propensity to form inter-peptide hydrogen bonds. The study of the dynamics of self-association indicated that, for this peptide, trajectories leading to conformations with high alpha-helical content are off-pathway from those leading to aggregates with high beta-structure content. We also show that the diverse oligomeric structures that form spontaneously in the molecular dynamics simulations are, to a large extent, compatible with solid-state NMR experimental measurements, including chemical shifts, on fully formed fibrils. The strategy that we present may therefore be used in the design of new experiments to determine the structure of amyloid fibrils, such as those involving site-specific isotope labelling schemes to measure key inter-atomic distances.     

Amyloids protect the silkmoth oocyte and embryo

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of proteins that have remarkable mechanical and chemical properties protecting the oocyte and the developing embryo from a wide range of environmental hazards. We present data from electron microscopy (negative staining and shadowing), X-ray diffraction and modeling studies of synthetic peptide analogues of silkmoth chorion proteins indicating that chorion is a natural amyloid. The folding and self-assembly models of chorion peptides strongly support the beta-sheet helix model of amyloid fibrils proposed recently by Blake and Serpell [Structure 4 (1996) 989-998].     

Acceleration of protein aggregation by amphiphilic peptides: transformation of supramolecular structure of the aggregates

Protein self-assembly and aggregation represent a special tool in biomedicine and biotechnology to produce biological materials for a wide range of applications. The protein aggregates are very different morphologically, varying from soluble amorphous aggregates to highly ordered amyloid-like fibrils, the latter being associated with molecular structures able to perform specific functions in living systems. Fabrication of novel biomaterials resembling natural protein assemblies has awakened interest in identification of low-molecular-weight biogenic agents as regulators of transformation of aggregation-prone proteins into fibrillar structures. Short amphiphilic peptides can be considered for this role. Using dynamic light scattering, turbidimetry, fluorescence spectroscopy, and transmission electron microscopy (TEM), we have demonstrated that the Arg-Phe dipeptide dramatically accelerates the aggregation of a model protein, α-lactalbumin, to generate morphologically different structures. TEM revealed transformation of spherical particles observed in the control samples into branched chains of fibril-like nanostructures in the presence of the peptide, suggesting that amphiphilic peptides can induce changes in the physicochemical properties of a protein substrate (net charge, hydrophobicity, and tendency to β-structure formation) resulting in accumulation of peptide-protein complexes competent to self-assembly into supramolecular structures. A number of other short amphiphilic peptides have also been shown to accelerate the aggregation process, using alternative complementary protein substrates for identification of molecular recognition modules. Peptide-protein assemblies are suggested to play the role of building blocks for formation of supramolecular structures profoundly differing from those of the individual protein substrate in type, size, and shape.     

Amyloid fibril formation from sequences of a natural beta-structured fibrous protein, the adenovirus fiber

Amyloid fibrils are fibrous beta-structures that derive from abnormal folding and assembly of peptides and proteins. Despite a wealth of structural studies on amyloids, the nature of the amyloid structure remains elusive; possible connections to natural, beta-structured fibrous motifs have been suggested. In this work we focus on understanding amyloid structure and formation from sequences of a natural, beta-structured fibrous protein. We show that short peptides (25 to 6 amino acids) corresponding to repetitive sequences from the adenovirus fiber shaft have an intrinsic capacity to form amyloid fibrils as judged by electron microscopy, Congo Red binding, infrared spectroscopy, and x-ray fiber diffraction. In the presence of the globular C-terminal domain of the protein that acts as a trimerization motif, the shaft sequences adopt a triple-stranded, beta-fibrous motif. We discuss the possible structure and arrangement of these sequences within the amyloid fibril, as compared with the one adopted within the native structure. A 6-amino acid peptide, corresponding to the last beta-strand of the shaft, was found to be sufficient to form amyloid fibrils. Structural analysis of these amyloid fibrils suggests that perpendicular stacking of beta-strand repeat units is an underlying common feature of amyloid formation.     

Effect of environmental conditions on aggregation and fibril formation of barstar

The dependence on environmental conditions of the assembly of barstar into amyloid fibrils was investigated starting from the nonnative, partially folded state at low pH (A-state). The kinetics of this process was monitored by CD spectroscopy and static and dynamic light scattering. The morphology of the fibrils was visualized by electron microscopy, while the existence of the typical cross- structure substantiated by solution X-ray scattering. At room temperature, barstar in the A-state is unable to form amyloid fibrils, instead amorphous aggregation is observed at high ionic strength. Further destabilization of the structure is required to transform the polypeptide chain into an ensemble of conformations capable of forming amyloid fibrils. At moderate ionic strength (75 mM NaCl), the onset and the rate of fibril formation can be sensitively tuned by increasing the temperature. Two types of fibrils can be detected differing in their morphology, length distribution and characteristic far UV CD spectrum. The formation of the different types depends on the particular environmental conditions. The sequence of conversion: A-statefibril type Ifibril type II appears to be irreversible. The transition into fibrils is most effective when the protein chain fulfills particular requirements concerning secondary structure, structural flexibility and tendency to cluster.Abbreviations CD circular dichroism - DLS dynamic light scattering - EM electron microscopy - SLS static light scattering - SAXS small-angle X-ray scattering - SOXS solution X-ray scattering     

Structural transformation of collagen fibrils in corneal stroma during drying. An x-ray scattering study.

X-ray scattering experiments were performed on human corneas during drying. In a first stage the collagen interfibrillar distance decreased considerably. Then, at a critical point of dehydration, a structural transformation of the collagen fibrils was observed. This finding leads to a two-stage drying model, which explains the discrepancy between the collagen fibril diameters determined by x-ray scattering and by electron microscopy. Our results strongly suggest that the collagen fibrils in the corneal stroma are surrounded by a cylindrical coating made mainly of proteoglycans. The coating appears as a three-dimensional fractal network with fractal dimension of 2.7 +/- 0.1.     

Solution structural characterization of coiled-coil peptide-polymer side-conjugates

Detailed structural characterization of protein-polymer conjugates and understanding of the interactions between covalently attached polymers and biomolecules will build a foundation to design and synthesize hybrid biomaterials. Conjugates based on simple protein structures are ideal model system to achieve these ends. Here we present a systematic structural study of coiled-coil peptide-poly(ethylene glycol) (PEG) side-conjugates in solution, using circular dichroism, dynamic light scattering, and small-angle X-ray scattering, to determine the conformation of conjugated PEG chains. The overall size and shape of side-conjugates were determined using a cylindrical form factor model. Detailed structural information of the covalently attached PEG chains was extracted using a newly developed model where each peptide-PEG conjugate was modeled as a Gaussian chain attached to a cylinder, which was further arranged in a bundle-like configuration of three or four cylinders. The peptide-polymer side-conjugates were found to retain helix bundle structure, with the polymers slightly compressed in comparison with the conformation of free polymers in solution. Such detailed structural characterization of the peptide-polymer conjugates, which elucidates the conformation of conjugated PEG around the peptide and assesses the effect of PEG on peptide structure, will contribute to the rational design of this new family of soft materials.     

Organic solvents mediate self-assembly of GAV-9 peptide on mica surface

Self-assembly of peptides into fibrils and other morphologies has attracted much attention inmany fields,especially in nanofabrication,pathology and biochemistry.In this paper,self-assembly of GAV-9peptide in organic solvents,ethanol and acetone,was investigated using atomic force microscopy(AFM)and attenuated total reflectance-Fourier transform infrared spectroscopy(ATR-FTIR).The results indicatedthat GAV-9 self-assembled into various nanostructures in both solvents after deposited and evaporated onmica.Fibrils with β-sheet conformation were observed in both solvents when the peptide concentrationwas higher than 280 μM.However,ordered fibrils with β-sheet conformation were formed in ethanol,butnot in acetone,with a peptide concentration ranging from 7 μM to 28 μM.We attribute the formation ofvarious nanostructures to the different physicochemical properties of the polar organic solvents on the self-assembly of GAV-9 peptide.     

Characterization of the PEG layer of sterically stabilized liposomes: a SAXS study

Synchrotron small-angle X-ray scattering analysis of the bilayer structure of a pharmacologically relevant sterically stabilized liposome system is presented. Describing the electron density profile of the bilayer with the superposition of Gaussian functions, the contribution of the poly(ethylene glycol) (PEG) layers to the total electron density was identified. The changes in the thickness of the PEG layer as well as the distribution of the PEG chains among the outer and inner leaflets of the bilayers were followed by changing the molar ratio of the PEG-lipid and the molar weight of the PEG molecule.