Ethanol induced the formation of β‐sheet and amyloid‐like fibrils by surfactant‐like peptide A6K

Self‐assembly of natural or designed peptides into fibrillar structures based on β‐sheet conformation is a ubiquitous and important phenomenon. Recently, organic solvents have been reported to play inductive roles in the process of conformational change and fibrillization of some proteins and peptides. In this study, we report the change of secondary structure and self‐assembling behavior of the surfactant‐like peptide A6K at different ethanol concentrations in water. Circular dichroism indicated that ethanol could induce a gradual conformational change of A6K from unordered secondary structure to β‐sheet depending upon the ethanol concentration. Dynamic light scattering and atomic force microscopy revealed that with an increase of ethanol concentration the nanostructure formed by A6K was transformed from nanosphere/string‐of‐beads to long and smooth fibrils. Furthermore, Congo red staining/binding and thioflavin‐T binding experiments showed that with increased ethanol concentration, the fibrils formed by A6K exhibited stronger amyloid fibril features. These results reveal the ability of ethanol to promote β‐sheet conformation and fibrillization of the surfactant‐like peptide, a fact that may be useful for both designing self‐assembling peptide nanomaterials and clarifying the molecular mechanism behind the formation of amyloid fibrils. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Solvent effects on self-assembly of beta-amyloid peptide.

beta-amyloid peptide (A beta) is the primary protein component of senile plaques in Alzheimer's disease patients. Synthetic A beta spontaneously assembles into amyloid fibrils and is neurotoxic to cortical cultures. Neurotoxicity has been associated with the degree of peptide aggregation, yet the mechanism of assembly of A beta into amyloid fibrils is poorly understood. In this work, A beta was dissolved in several different solvents commonly used in neurotoxicity assays. In pure dimethylsulfoxide (DMSO), A beta had no detectable beta-sheet content; in 0.1% trifluoroacetate, the peptide contained one-third beta-sheet; and in 35% acetonitrile/0.1% trifluoroacetate, A beta was two-thirds beta-sheet, equivalent to the fibrillar peptide in physiological buffer. Stock solutions of peptide were diluted into phosphate-buffered saline, and fibril growth was followed by static and dynamic light scattering. The growth rate was substantially faster when the peptide was predissolved in 35% acetonitrile/0.1% trifluoroacetate than in 0.1% trifluoroacetate, 10% DMSO, or 100% DMSO. Differences in growth rate were attributed to changes in the secondary structure of the peptide in the stock solvent. These results suggest that formation of an intermediate with a high beta-sheet content is a controlling step in A beta self-assembly.     

The Alzheimer's peptide a beta adopts a collapsed coil structure in water

The self-assembly of the soluble peptide Abeta into Alzheimer's disease amyloid is believed to involve a conformational change. Hence the solution conformation of Abeta is of significant interest. In contrast to studies in other solvents, in water Abeta is collapsed into a compact series of loops, strands, and turns and has no alpha-helical or beta-sheet structure. Conformational stabilization is primarily attributed to van der Waals and electrostatic forces. A large conspicuous uninterrupted hydrophobic patch covers approximately 25% of the surface. The compact coil structure appears meta-stable, and because fibrillization leads to formation of intermolecular beta-sheet secondary structure, a global conformational rearrangement is highly likely. A molecular hypothesis for amyloidosis includes at least two primary driving forces, changes in solvation thermodynamics during formation of amyloid deposits and relief of internal conformational stress within the soluble precursor during formation of lower-energy amyloid fibrils.     

Solution conformation and amyloid-like fibril formation of a polar peptide derived from a beta-hairpin in the OspA single-layer beta-sheet

A 23-residue peptide termed BH(9-10) was designed based on a beta-hairpin segment of the single-layer beta-sheet region of Borrelia OspA protein. The peptide contains a large number of charged amino acid residues, and it does not follow the amphipathic pattern that is commonly found in natural beta-sheets. In aqueous solution, the peptide was highly soluble and flexible, with a propensity to form a non-native beta-turn. Trifluoroethanol (TFE) stabilized a native-like beta-turn in BH(9-10). TFE also decreased the level of solubility of the peptide, resulting in peptide precipitation. The precipitation process accompanied a conformational conversion to a beta-sheet structure, as judged with circular dichroism spectroscopy. The precipitate was found to be fibrils similar to those associated with human amyloid diseases. The fibrillization kinetics depended on peptide and TFE concentrations, and had a nucleation step followed by an assembly step. The fibrillization was reversible, and the dissociation reaction involved two phases. TFE appears to induce the fibrils by stabilizing a beta-sheet conformation of the peptide that optimally satisfies hydrogen bonding and electrostatic complementarity. This TFE-induced fibrillization is quite unusual, because most amyloidogenic peptides form fibrils in aqueous solution and TFE disrupts these fibrils. Nevertheless, the BH(9-10) fibrils have similar structure to other fibrils, supporting the emerging idea that polypeptides possess an intrinsic ability to form amyloid-like fibrils. The high level of solubility of BH(9-10), the ability to precisely control fibril formation and dissociation, and the high-resolution structure of the same sequence in the beta-hairpin conformation in the OspA protein provide a tractable experimental system for studying the fibril formation mechanism.     

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.     

Organic solvent mediated self-association of an amyloid forming peptide from beta2-microglobulin: an atomic force microscopy study

Human beta(2)-microglobulin (beta(2)m) forms amyloid fibrils in hemodialysis related amyloidosis. Peptides spanning the beta strands of beta(2)m have been shown to form amyloid fibrils in isolation. We have studied the self-association of a 13-residue peptide Ac-DWSFYLLYYTEFT-am (Pbeta(2)m) spanning one of the beta-strands of human beta(2)-microglobulin when dissolved in various organic solvents such as methanol (MeOH), trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), and dimethylsulfoxide. We have observed that Pbeta(2)m forms amyloid fibrils when diluted from organic solvents into aqueous buffer at pH 7.0 as judged by increase in thioflavin T fluorescence. Fibril formation was observed to depend on the solvents in which peptide stock solutions were prepared. Circular dichroism spectra indicated propensity for helical conformation in MeOH, TFE, and HFIP. In buffer, beta-structure was observed irrespective of the solvent in which the peptide stock solutions were prepared. Atomic force microscopy images obtained by drying the peptide on mica from organic solvents indicated the ability of Pbeta(2)m to self-associate to form nonfibrillar structures. Morphology of the structures was dependent on the solvent in which the peptide was dissolved. Peptides that have the ability to self-associate such as amyloid-forming peptides would be attractive candidates for the generation of self-assembled structures with varying morphologies by appropriate choice of surfaces and solvents for dissolution.     

Morphology of self‐assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac‐VQIVYK‐amide (AcPHF6) and Ac‐QIVYK‐amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self‐association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly β‐conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for β‐structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and β‐structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Aggregation and conformational studies on a pentapeptide derivative

Most of the disease causing proteins such as beta amyloid, amylin, and huntingtin protein, which are natively disordered, readily form fibrils consisting of beta-sheet polymers. Though all amyloid fibrils are made up of beta-sheet polymers, not all peptides with predominant beta-sheet content in the native state develop into amyloid fibrils. We hypothesize that stable amyloid like fibril formation may require mixture of different conformational states in the peptide. We have tested this hypothesis on amyloid forming peptide namely HCl(Ile)(5)NH(CH(2)CH(2)O)(3)CH(3) (I). We show peptide I, has propensity to form self-assembled structures of beta-sheets in aqueous solutions. When incubated over a period of time in aqueous buffer, I self assembled into beta sheet like structures with diameters ranging from 30 to 60 A that bind with amyloidophilic dyes like Congo red and Thioflavin T. Interestingly peptide I developed into unstable fibrils after prolonged aging at higher concentration in contrast with the general mature fibril-forming propensity of various amyloid petides known to date.     

Synthesis and mass spectrometric characterization of a metal-affinity decapeptide: copper-induced conformational changes

A decapeptide with high affinity toward heavy metal ions (RCHQYHHNRE) has been prepared by Fmoc strategy using TGR resin as solid support. The model peptide provides a simple system that can be used for a systematic study of the impact of different metal ions on peptide secondary structure on a molecular level; histidine residues were incorporated into the peptide in a sequence similar to beta-amyloid peptide (Abeta1-40) to generate possible complexation sites for Cu (2+) ions. The peptide secondary structure, as investigated by circular dichroism, and self-assembled nanostructures were observed to depend strongly on the presence of copper and sodium dodecyl sulfate (SDS). Atomic force microscopy (AFM) revealed also that copper and SDS affected slightly the Abeta1-40 nanostructures. An explanation for the effect of metal ions and SDS on the self-assembly of peptides was proposed. The extensive beta-sheet formation may further promote peptide self-assembly into longer fibers.     

Spectroscopic investigation on gel-forming beta-sheet assemblage of peptide derivatives

The conformational studies of peptide derivatives A and B in a gel state were studied by using circular dichroism (CD), Fourier transformed infrared (FTIR), and fluorescence spectroscopic techniques. Birefringence and electron microscopic studies were carried out to characterize the morphological aspects of the fibrils in the gel. The FTIR spectra of the peptides show the absence of free NH in the gel state, implying that the intermolecular hydrogen-bond formation is the driving force for the aggregation. The CD spectrum of the peptide gels shows the presence of antiparallel and parallel beta-sheet conformation for peptide derivatives A and B, respectively. Electron microscopic studies (EM) of the peptide derivatives A and B reveal that peptide A formed rigid, rod-like structures without cross-linking and peptide B formed loose fibrils organized into highly noncovalently cross-linked mesh-like structural aggregates. Peptide A was much more soluble in alcoholic solvents than peptide B, and no birefringence was observed with Congo red (CR) staining in the temperature range of 0-80 degrees C. The spectroscopic studies indicate that peptide B consists of domains having a significant amount of beta-sheet structure and exhibiting golden yellow birefringence between 53 and 56 degrees C when stained with Congo red. On the other hand, peptide A gives no evidence of birefringence under polarized light. Fluorescence probe binding studies with pyrene in gel state with peptides A and B indicates the polarity in the interior of the aggregates. The data presented in the present work indicate that peptide B forms fibrils, which is similar to amyloid aggregates that are present in biological systems.     

Conformational polymorphism of the amyloidogenic peptide homologous to residues 113-127 of the prion protein

Conformational transitions are thought to be the prime mechanism of amyloid formation in prion diseases. The prion proteins are known to exhibit polymorphic behavior that explains their ability of "conformation switching" facilitated by structured "seeds" consisting of transformed proteins. Oligopeptides containing prion sequences showing the polymorphism are not known even though amyloid formation is observed in these fragments. In this work, we have observed polymorphism in a 15-residue peptide PrP (113-127) that is known to form amyloid fibrils on aging. To see the polymorphic behavior of this peptide in different solvent environments, circular dichroism (CD) spectroscopic studies on an aqueous solution of PrP (113-127) in different trifluoroethanol (TFE) concentrations were carried out. The results show that PrP (113-127) have sheet preference in lower TFE concentration whereas it has more helical conformation in higher TFE content (>40%). The structural transitions involved in TFE solvent were studied using interval-scan CD and FT-IR studies. It is interesting to note that the alpha-helical structure persists throughout the structural transition process involved in amyloid fibril formation implicating the involvement of both N- and C-terminal sequences. To unravel the role of the N-terminal region in the polymorphism of the PrP (113-127), CD studies on another synthetic peptide, PrP (113-120) were carried out. PrP(113-120) exhibits random coil conformation in 100% water and helical conformation in 100% TFE, indicating the importance of full-length sequence for beta-sheet formation. Besides, the influence of different chemico-physical conditions such as concentration, pH, ionic strength, and membrane like environment on the secondary structure of the peptide PrP (113-127) has been investigated. At higher concentration, PrP (113-127) shows features of sheet conformation even in 100% TFE suggesting aggregation. In the presence of 5% solution of sodium dodecyl sulfate, PrP (113-127) takes high alpha-helical propensity. The environment-dependent conformational polymorphism of PrP (113-127) and its marked tendency to form stable beta-sheet structure at acidic pH could account for its conformation switching behavior from alpha-helix to beta-sheet. This work emphasizes the coordinative involvement of N-terminal and C-terminal sequences in the self-assembly of PrP (113-127).     

Water-soluble beta-sheet models which self-assemble into fibrillar structures.

Self-assembly of beta-sheet domains resulting in the formation of pathogenic, fibrillar protein aggregates (amyloids) is a characteristic feature of various medical disorders. These include neurodegenerative diseases, such as Alzheimer's, Huntington's, and Creutzfeldt-Jacob's. A significant problem in studying such aggregation processes is the poor solubility of these beta-sheet complexes. The present work describes water-soluble de novo beta-sheet peptides which self-assemble into fibrillar structures. The model peptides enable studies of the relationship between beta-sheet stability and association behavior. The peptides [DPKGDPKG-(VT)n-GKGDPKPD-NH2, n = 3-8] are composed of a central beta-sheet-forming domain (VT-sequence), and N- and C-terminal nonstructured octapeptide sequences which promote water solubility. Conformational analyses by circular dichroism and Fourier transform infrared spectroscopy indicate the influence of peptide length, D-amino acid substitution, and concentration on the ability of the peptides to form stable beta-sheet structures. The association behavior investigated by analytical ultracentrifugation and dynamic light scattering was found to correlate strongly with the stability of a beta-sheet conformation. Model peptides with n >/= 6 form stable, water-soluble beta-sheet complexes with molecular masses of more than 2000 kDa, which are organized in fibrillar structures. The fibrils examined by Congo Red staining and electron microscopy show some similarities with naturally occurring amyloid fibrils.     

Formation of low-dimensional crystalline nucleus region during insulin amyloidogenesis process

Insulin, as other amyloid proteins, can form amyloid fibrils at certain conditions. The self-assembled aggregation process of insulin can result in a variety of conformations, starting from small oligomers, going through various types of protofibrils, and finishing with bundles of fibrils. One of the most common consensuses among the various self-assembly processes that are suggested in the literature is the formation of an early stage nucleus conformation. Here we present an additional insight for the self-assembly process of insulin. We show that at the early lag phase of the process (prior to fibril formation) the insulin monomers self-assemble into ordered nanostructures. The most notable feature of this early self-assembly process is the formation of nanocrystalline nucleus regions with a strongly bound electron-hole confinement, which also change the secondary structure of the protein. Each step in the self-assembly process is characterized by an optical spectroscopic signature, and possesses a narrow size distribution. By following the spectroscopic signature we can measure the potency of amyloid fibrils inhibitors already at the lag phase. We further demonstrate it by the use of epigallocatechin gallate, a known inhibitor for insulin fibrils. The findings can result in a spectroscopic-based application for the analysis of amyloid fibrils inhibitors.     

The N-terminal propeptide of lung surfactant protein C is necessary for biosynthesis and prevents unfolding of a metastable alpha-helix

The lung surfactant-associated protein C (SP-C) consists mainly of a polyvaline alpha-helix, which is stable in a lipid membrane. However, in agreement with the predicted beta-strand conformation of a polyvaline segment, helical SP-C unfolds and transforms into beta-sheet aggregates and amyloid fibrils within a few days in aqueous organic solvents. SP-C fibril formation and aggregation have been associated with lung disease. Here, we show that in a recently isolated biosynthetic precursor of SP-C (SP-Ci), a 12 residue N-terminal propeptide locks the metastable polyvaline part in a helical conformation. The SP-Ci helix does not aggregate or unfold during several weeks of incubation, as judged by hydrogen/deuterium exchange and mass spectrometry. Hydrogen/deuterium exchange experiments further indicate that the propeptide reduces exchange in parts corresponding to mature SP-C. Finally, in an acidic environment, SP-Ci unfolds and aggregates into amyloid fibrils like SP-C. These data suggest a direct role of the N-terminal propeptide in SP-C biosynthesis.     

The dependence of the distribution of ionophoric macrotetrolide antibiotics on the composition of their immiscible phases

Influence of various solvents on the content of macrotetrolides during their prolonged storage in solution was shown. Under the conditions of room temperature 90 per cent acetone induced hydrolysis of polyether antibiotics. 90 per cent ethanol and dry chloroform induced respectively slight and intensive cyclization of linear precursors (oxyacids). The study on distribution of macrotetrolides between immiscible phases of various composition revealed that addition of ethanol or acetone to the water-chloroform system of organic solvents induced the antibiotic liberation into the water phase which increased with increasing concentration of the substances being added. Inorganic cations intensified the process during either the oxyacid cyclization or the antibiotic hydrolysis and liberated the reaction products from the equilibrium mixture. Under such conditions the value of the ion radius or selectivity of the macrotetrolides to definite cations was in principle of no importance.     

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.     

Nanostructures by self-assembling peptide amphiphile as potential selective drug carriers

The self-assembling behavior, at physiological pH, of the amphiphile peptide (C18)(2)L5CCK8 in nanostructures is reported. Stable aggregates presenting a critical micellar concentration of 2 x 10(-6) mol kg(-1), and characterized by water exposed CCK8 peptide in beta-sheet conformation, are obtained. Small angle neutron scattering experiments are indicative for a 3D structure with dimensions > or =100 nm. AFM images confirm the presence of nanostructures. Fluorescence experiments indicating the sequestration of pyrene, chosen as drug model, and the anticancer Doxorubicin within the nanostructures are reported.     

Effect of organic solvents on the conformation and interaction of catalase and anticatalase antibodies

Effect of six organic solvents—methanol, ethanol, propanol, dimethyl sulphoxide (DMSO), N,N-dimethyl formamide (DMF), and glycerol on the conformation and interaction of catalase and anticatalase antibodies were studied with the aim of identifying the solvents in which antigen–antibody interactions are strong. The antigen binding activity of the antibodies in the various organic solvents increased in the following order: ethanol < methanol < no organic solvent < propanol < DMSO < DMF < glycerol. The structure of both the antibody and the antigen molecule was affected significantly in 40% concentration of the organic solvents used in this study. Catalase activity was inhibited in DMSO. However, the enzyme was activated in DMF upto about 50% of its concentration.     

The roles of turn formation and cross-strand interactions in fibrillization of peptides derived from the OspA single-layer beta-sheet

We previously demonstrated that a beta-hairpin peptide, termed BH(9-10), derived from a single-layer beta-sheet of Borrelia OspA protein, formed a native-like beta-turn in trifluoroethanol (TFE) solution, and it assembled into amyloid-like fibrils at higher TFE concentrations. This peptide is highly charged, and fibrillization of such a hydrophilic peptide is quite unusual. In this study, we designed a circularly permutated peptide of BH(9-10), termed BH(10-9). When folded into their respective beta-hairpin structures found in OspA, these peptides would have identical cross-strand interactions but different turns connecting the strands. NMR study revealed that BH(10-9) had little propensity to form a turn structure both in aqueous and TFE solutions. At higher TFE concentration, BH(10-9) precipitated with a concomitant alpha-to-beta conformational conversion, in a similar manner to the BH(9-10) fibrillization. However, the BH(10-9) precipitates were nonfibrillar aggregation. The precipitation kinetics of BH(10-9) was exponential, consistent with a first-order molecular assembly reaction, while the fibrillization of BH(9-10) showed sigmoidal kinetics, indicative of a two-step reaction consisting of nucleation and molecular assembly. The correlation between native-like turn formation and fibrillization of our peptide system strongly suggests that BH(9-10) adopts a native-like beta-hairpin conformation in the fibrils. Remarkably, seeding with the preformed BH(10-9) precipitates changed the two-step BH(9-10) fibrillization to a one-step molecular assembly reaction, and disrupted the BH(9-10) fibril structure, indicating interactions between the BH(10-9) aggregates and the BH(9-10) peptide. Our results suggest that, in these peptides, cross-strand interactions are the driving force for molecular assembly, and turn formation limits modes of peptide assembly.     

Conformational switching and fibrillogenesis in the amyloidogenic fragment of apolipoprotein a-I

The N-terminal portion of apolipoprotein A-I corresponding to the first 93 residues has been identified as the main component of apolipoprotein A-I fibrils in a form of systemic amyloidosis. We have been able to characterize the process of conformational switching and fibrillogenesis in this fragment of apolipoprotein A-I purified directly from ex vivo amyloid material. The peptide exists in an unstructured form in aqueous solution at neutral pH. The acidification of the solution provokes a collapse into a more compact, intermediate state and the transient appearance of a helical conformation that rapidly converts to a stable, mainly beta-structure in the fibrils. The transition from helical to sheet structure occurs concomitantly with peptide self-aggregation, and fibrils are detected after 72 h. The alpha-helical conformation is induced by the addition of trifluoroethanol and phospholipids. Interaction of the amyloidogenic polypeptide with phospholipids prevents the switching from helical to beta-sheet form and inhibits fibril formation. The secondary structure propensity of the apolipoprotein A-I fragment appears poised between helix and the beta-sheet. These findings reinforce the idea of a delicate balance between natively stabilizing interactions and fatally stabilizing interactions and stress the importance of cellular localization and environment in the maintenance of protein conformation.