Design of peptides that form amyloid-like fibrils capturing amyloid beta1-42 peptides

Amyloid beta-peptide (Abeta) plays a critical role in Alzheimer's disease (AD). The monomeric state of Abeta can self-assemble into oligomers, protofibrils, and amyloid fibrils. Since the fibrils and soluble oligomers are believed to be responsible for AD, the construction of molecules capable of capturing these species could prove valuable as a means of detecting these potentially toxic species and of providing information pertinent for designing drugs effective against AD. To this aim, we have designed short peptides with various hydrophobicities based on the sequence of Abeta14-23, which is a critical region for amyloid fibril formation. The binding of the designed peptides to Abeta and the amplification of the formation of peptide amyloid-like fibrils coassembled with Abeta are elucidated. A fluorescence assay utilizing thioflavin T, known to bind specifically to amyloid fibrils, revealed that two designed peptides (LF and VF, with the leucine and valine residues, respectively, in the hydrophobic core region) could form amyloid-like fibrils effectively by using mature Abeta1-42 fibrils as nuclei. Peptide LF also coassembled with soluble Abeta oligomers into peptide fibrils. Various analyses, including immunostaining with gold nanoparticles, enzyme-linked immunosorbent assays, and size-exclusion chromatography, confirmed that the LF and VF peptides formed amyloid-like fibrils by capturing and incorporating Abeta1-42 aggregates into their peptide fibrils. In this system, small amounts of mature Abeta1-42 fibrils or soluble oligomers could be transformed into peptide fibrils and detected by amplifying the amyloid-like fibrils with the designed peptides.     

Annular structures as intermediates in fibril formation of Alzheimer Abeta17-42

We report all-atom molecular dynamics simulations of annular beta-amyloid (17-42) structures, single- and double-layered, in solution. We assess the structural stability and association force of Abeta annular oligomers associated through different interfaces, with a mutated sequence (M35A), and with the oxidation state (M35O). Simulation results show that single-layered annular models display inherent structural instability: one is broken down into linear-like oligomers, and the other collapses. On the other hand, a double-layered annular structure where the two layers interact through their C-termini to form an NC-CN interface (where N and C are the N and C termini, respectively) exhibits high structural stability over the simulation time due to strong hydrophobic interactions and geometrical constraints induced by the closed circular shape. The observed dimensions and molecular weight of the oligomers from atomic force microscopy (AFM) experiments are found to correspond well to our stable double-layered model with the NC-CN interface. Comparison with K3 annular structures derived from the beta 2-microglobulin suggests that the driving force for amyloid formation is sequence specific, strongly dependent on side-chain packing arrangements, structural morphologies, sequence composition, and residue positions. Combined with our previous simulations of linear-like Abeta, K3 peptide, and sup35-derived GNNQQNY peptide, the annular structures provide useful insight into oligomeric structures and driving forces that are critical in amyloid fibril formation.     

Conformational restriction via cyclization in beta-amyloid peptide Abeta(1-28) leads to an inhibitor of Abeta(1-28) amyloidogenesis and cytotoxicity

The aggregation process of beta-amyloid peptide Abeta into amyloid is strongly associated with the pathology of Alzheimer's disease (AD). Aggregation may involve a transition of an alpha helix in Abeta(1-28) into beta sheets and interactions between residues 18-20 of the "Abeta amyloid core." We applied an i, i+4 cyclic conformational constraint to the Abeta amyloid core and devised side chain-to-side chain lactam-bridged cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28). In contrast to Abeta(1-28) and [Lys(17), Asp(21)]Abeta(1-28), cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was not able to form beta sheets and cytotoxic amyloid aggregates. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was able to interact with Abeta(1-28) and to inhibit amyloid formation and cytotoxicity. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) also interacted with Abeta(1-40) and interfered with its amyloidogenesis. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) or similarly constrained Abeta sequences may find therapeutic and diagnostic applications in AD.     

Surface-induced aggregation of beta amyloid peptide by co-substituted alkanethiol monolayers supported on gold

The primary pathological characteristic of Alzheimer's disease is the presence in the brain of self-assembled beta amyloid (Abeta) protein fibrils, consisting of 35-43 amino acid residues. The toxicity of the aggregated protein structures has previously been proposed to be related to the interaction of Abeta fibrils with neuronal membranes (phospholipid bilayers). Here, surfaces consisting of self-assembled alkanethiol monolayers with different end groups--supported on Au--are used to test the effect of surface chemistry on the structure and morphology of aggregates formed from an active fragment (Abeta10-35) of the Abeta peptide. The influence of monolayer nature (end group) on the aggregation of Abeta10-35 was examined using reflection-absorption infrared spectroscopy (RAIRS) and scanning force microscopy (SFM). Evaluation of the SFM and RAIRS data reveals the presence of Abeta10-35 protein on the various monolayer surfaces, with the surface protein possessing predominantly beta-sheet and random-coil conformations. Time-dependent studies of the extent of Abeta10-35 aggregation and deposition on the various surfaces and the effect of the monolayers on seeding of Abeta10-35 aggregates in solution are also discussed.     

Structure and dynamics of the Abeta(21-30) peptide from the interplay of NMR experiments and molecular simulations

We combine molecular dynamics simulations and new high-field NMR experiments to describe the solution structure of the Abeta(21-30) peptide fragment that may be relevant for understanding structural mechanisms related to Alzheimer's disease. By using two different empirical force-field combinations, we provide predictions of the three-bond scalar coupling constants ((3)J(H(N)H(alpha))), chemical-shift values, (13)C relaxation parameters, and rotating-frame nuclear Overhauser effect spectroscopy (ROESY) crosspeaks that can then be compared directly to the same observables measured in the corresponding NMR experiment of Abeta(21-30). We find robust prediction of the (13)C relaxation parameters and medium-range ROESY crosspeaks by using new generation TIP4P-Ew water and Amber ff99SB protein force fields, in which the NMR validates that the simulation yields both a structurally and dynamically correct ensemble over the entire Abeta(21-30) peptide. Analysis of the simulated ensemble shows that all medium-range ROE restraints are not satisfied simultaneously and demonstrates the structural diversity of the Abeta(21-30) conformations more completely than when determined from the experimental medium-range ROE restraints alone. We find that the structural ensemble of the Abeta(21-30) peptide involves a majority population (approximately 60%) of unstructured conformers, lacking any secondary structure or persistent hydrogen-bonding networks. However, the remaining minority population contains a substantial percentage of conformers with a beta-turn centered at Val24 and Gly25, as well as evidence of the Asp23 to Lys28 salt bridge important to the fibril structure. This study sets the stage for robust theoretical work on Abeta(1-40) and Abeta(1-42), for which collection of detailed NMR data on the monomer will be more challenging because of aggregation and fibril formation on experimental timescales at physiological conditions. In addition, we believe that the interplay of modern molecular simulation and high-quality NMR experiments has reached a fruitful stage for characterizing structural ensembles of disordered peptides and proteins in general.     

Energy landscapes of the monomer and dimer of the Alzheimer's peptide Abeta(1-28)

The cytotoxicity of Alzheimer's disease has been linked to the self-assembly of the 4042 amino acid of the amyloid-beta (Abeta) peptide into oligomers. To understand the assembly process, it is important to characterize the very first steps of aggregation at an atomic level of detail. Here, we focus on the N-terminal fragment 1-28, known to form fibrils in vitro. Circular dichroism and NMR experiments indicate that the monomer of Abeta(1-28) is alpha-helical in a membranelike environment and random coil in aqueous solution. Using the activation-relaxation technique coupled with the OPEP coarse grained force field, we determine the structures of the monomer and of the dimer of Abeta(1-28). In agreement with experiments, we find that the monomer is predominantly random coil in character, but displays a non-negligible beta-strand probability in the N-terminal region. Dimerization impacts the structure of each chain and leads to an ensemble of intertwined conformations with little beta-strand content in the region Leu17-Ala21. All these structural characteristics are inconsistent with the amyloid fibril structure and indicate that the dimer has to undergo significant rearrangement en route to fibril formation.     

"Click peptide" based on the "o-acyl isopeptide method": control of A beta1-42 production from a photo-triggered A beta1-42 analogue

A clear understanding of the dynamic events of amyloid beta peptide (Abeta) 1-42, such as the folding, self-assembly, and aggregation processes, would be of great significance in Alzheimer's disease (AD) research. However, elucidation of these Abeta1-42 dynamic events is a difficult issue due to uncontrolled polymerization, which also poses a significant obstacle for establishing an experimental system that clarifies the pathological function of Abeta1-42. On the basis of the O-acyl isopeptide method, we herein developed a novel photo-triggered "click peptide" of Abeta1-42, for example, 26-N-Nvoc-26-AIAbeta42, in which the photocleavable 6-nitroveratryloxycarbonyl (Nvoc) group was introduced at the alpha-amino group of Ser26 in 26-O-acyl isoAbeta1-42 (26-AIAbeta42). From the results, (1) the click peptide did not exhibit the self-assembling nature under physiological conditions due to one single modified ester; (2) photoirradiation of the click peptide and subsequent O-N intramolecular acyl migration afforded the intact Abeta1-42 with a quick and one-way conversion reaction (so-called "click"), while the click peptide was stable under nonphotolytic or storage conditions. In addition, it is advantageous that no additional fibril inhibitory auxiliaries were released during conversion to Abeta1-42. This method provides a novel system useful for investigating the dynamic biological functions of Abeta1-42 in AD by inducible activation of Abeta1-42 self-assembly.     

Dynamics and fluidity of amyloid fibrils: a model of fibrous protein aggregates

A previous experimentally defined model for the fibril formed from the core residues of the beta-amyloid (Abeta) peptides of Alzheimer's disease, 10YEVHHQKLVFFAEDVGSNKGAIIGLM, Abeta(10-35) using spectroscopic and scattering analyses reports on the average structure, benefiting immensely from the homogeneous assembly of Abeta(10-35). However, the energetic constraints that contribute to fibril dynamics and stability remain poorly understood. Here we perform molecular dynamics simulations to extend the structural assignment by providing evidence for a dynamic average ensemble with transient backbone H-bonds and internal solvation contributing to the inherent stability of amyloid fibrils.     

The unique Alzheimer's β-amyloid triangular fibril has a cavity along the fibril axis under physiological conditions

Elucidating the structure of Aβ(1-40) fibrils is of interest in Alzheimer's disease research because it is required for designing therapeutics that target Aβ(1-40) fibril formation at an early stage of the disease. M35 is a crucial residue because of its potential oxidation and its strong interactions across β-strands and across β-sheets in Aβ fibrils. Experimentally, data for the three-fold symmetry structure of the Aβ(9-40) fibril suggest formation of tight hydrophobic core through M35 interactions across the fibril axis and strong I31-V39 interactions between different cross-β units. Herein, on the basis of experimental data, we probe conformers with three-fold symmetry of the full-length Aβ(1-40). Our all-atom molecular dynamics simulations in explicit solvent of conformers based on the ssNMR data reproduced experimental observations of M35-M35 and I31-V39 distances. Our interpretation of the experimental data suggests that the observed ～5-7 ? M35-M35 distance in the fibril three-fold symmetry structure is likely to relate to M35 interactions along the fibril axis, rather than across the fibril axis, since our measured M35-M35 distances across the fibril axis are consistently above 15 ?. Consequently, we revealed that the unique Aβ(1-40) triangular structure has a large cavity along the fibril axis and that the N-termini can assist in the stabilization of the fibril by interacting with the U-turn domains or with the C-termini domains. Our findings, together with the recent cyroEM characterization of the hollow core in Aβ(1-42) fibrils, point to the relevance of a cavity in Aβ(1-40/1-42) oligomers which should be considered when targeting oligomer toxicity.     

Thermodynamics of beta-amyloid fibril formation

Amyloid fibers are aggregates of proteins. They are built out of a peptide called beta-amyloid (Abeta) containing between 41 and 43 residues, produced by the action of an enzyme which cleaves a much larger protein known as the amyloid precursor protein (APP). X-ray diffraction experiments have shown that these fibrils are rich in beta-structures, whereas the shape of the peptide displays an alpha-helix structure within the APP in its biologically active conformation. A realistic model of fibril formation is developed based on the 17 residues Abeta12-28 amyloid peptide, which has been shown to form fibrils structurally similar to those of the whole Abeta peptide. With the help of physical arguments and in keeping with experimental findings, the Abeta12-28 monomer is assumed to be in four possible states (i.e., native helix conformation, beta-hairpin, globular low-energy state, and unfolded state). Making use of these monomeric states, oligomers (dimers, tertramers, and octamers) were constructed. With the help of short, detailed molecular dynamics calculations of the three monomers and of a variety of oligomers, energies for these structures were obtained. Making use of these results within the framework of a simple yet realistic model to describe the entropic terms associated with the variety of amyloid conformations, a phase diagram can be calculated of the whole many-body system, leading to a thermodynamical picture in overall agreement with the experimental findings. In particular, the existence of micellar metastable states seem to be a key issue to determine the thermodynamical properties of the system.     

A strand-loop-strand structure is a possible intermediate in fibril elongation: long time simulations of amyloid-beta peptide (10-35)

A total of 6.2 micros molecular dynamics simulations of amyloid-beta (10-35) (Abeta) were performed in explicit water solvent. The results reveal that the collapsed-coil (cc) structure determined by experiments is stable at pH 5.6 for hundreds of nanoseconds, but it can exchange with a strand-loop-strand (SLS) structure on the microsecond time scale. The SLS structure has D23-K28 as a reverse loop and the central hydrophobic core and the C-terminal in hydrophobic contact. This SLS structure topologically resembles the proposed monomer conformation in fibrils. Since it has been suggested that a special conformation of Abeta is needed when the monomer binds to fibril ends to elongate fibrils, we propose that the SLS structure may be an important intermediate binding structure for Abeta fibril growth. Simulations at pH 2.0, which is used to mimic the mutation of E22Q and D23N, and at high temperature (400 K) indicate that the SLS structure is considerably populated under these conditions while the cc structure is disrupted. These results imply that the SLS structures may also be a binding intermediate in other conditions such as E22Q and/or D23N mutations and high temperature, which have been proved to promote fibril formation previously.     

Comparative molecular dynamics studies of wild-type and oxidized forms of full-length Alzheimer amyloid beta-peptides Abeta(1-40) and Abeta(1-42)

In this study, all-atom 50 ns molecular dynamics simulations are performed on the full-length amyloid beta (Abeta) monomers (WT-Abeta(1-40) and WT-Abeta(1-42)) and their oxidized forms (Met35(O)-Abeta(1-40) and Met35(O)-Abeta(1-42)) in aqueous solution. The effects of the oxidation state of Met35 and the presence of dipeptide (Ile41-Ala42) on the secondary structures of the three distinct regions (the central hydrophobic core region 17-21 (LVFFA), the loop 23-28 (DVGSNK), and the second hydrophobic domain 29-35 (GAIIGLM)) of all monomers have been analyzed in detail, and results are compared with the available experimental information. Our simulations indicate that the WT-Abeta(1-40) monomer adopts an overall beta-hairpin-like structure, which is promoted by the turn region (24-27). This turn region is stabilized through salt-bridge formation between the Asp23 and Lys28 residues. In contrast, the overall structure of the oxidized (Met35(O)-Abeta(1-40)) monomer can be divided into three well-defined bend regions separated by coil segments. These structural differences may be critical for the measured decrease in the rate of aggregation of Met35(O)-Abeta(1-40) peptide. In the WT-Abeta(1-42) monomer, in comparison to the WT-Abeta(1-40), the Asp23-Lys28 salt bridge is absent, and consequently, the turn in the middle (24-27) region has a smaller curvature. The observed difference in the aggregation rates of these two peptides may be related to the opening of the turn (24-27) stabilized by the Asp23-Lys28 salt bridge. For WT-Abeta(1-42), in the absence of this salt bridge, the unfolding and aggregation events may be more favorable than for WT-Abeta(1-40).     

Amyloid-like formation by self-assembly of peptidolipids in two dimensions

The accumulation of beta-amyloid peptide (Abeta) in the human brain is known to be the major cause that drives Alzheimer's disease pathogenesis. Abeta, a 39-42 amino acid peptide, is the cleavage product of amyloid precursor protein in the hydrophobic transmembrane region. The present study employs a two-dimensional (2D) approach. Two synthetic peptidolipids, C18-IIGLM-OH and C18-IIGLM-NH2, are selected based on the fragment 31-35 of Abeta which is recognized as one of the determining segments that induces formation of amyloid fibril plaques. The aliphatic hydrocarbon chain C18 is attached to the N-terminal of the fragment 31-35 to facilitate the 2D study at the air-water interface. The aggregation process is observed by two measurements: (1) surface pressure-area and surface dipole moment-area isotherms and (2) epifluorescence microscopy of the Langmuir films to investigate the topography of the amyloid-like formation.     

Absolute structural constraints on amyloid fibrils from solid-state NMR spectroscopy of partially oriented samples

We demonstrate that absolute, molecular-level structural information can be obtained from solid-state NMR measurements on partially oriented amyloid fibrils. Specifically, we show that the direction of the fibril axis relative to a carbonyl 13C chemical shift anisotropy (CSA) tensor can be determined from magic-angle spinning (MAS) sideband patterns in 13C NMR spectra of fibrils deposited on planar substrates. Deposition of fibrils on a planar substrate creates a highly anisotropic distribution of fibril orientations (hence, CSA tensor orientations) with most fibrils lying in the substrate plane. The anisotropic orientational distribution gives rise to distorted spinning sideband patterns in MAS spectra from which the fibril axis direction can be inferred. The experimentally determined fibril axis direction relative to the carbonyl CSA tensor of Val12 in fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta1-40) agrees well with the predictions of a recent structural model (Petkova et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16742-16747) in which Val12 is contained in a parallel beta-sheet in the cross-beta motif characteristic of amyloid fibrils.     

Spectroscopic investigation of Ginkgo biloba terpene trilactones and their interaction with amyloid peptide Abeta(25-35)

The beneficial effects of Ginkgo biloba extract in the "treatment" of dementia are attributed to its terpene trilactone (TTL) constituents. The interactions between TTLs and amyloid peptide are believed to be responsible in preventing the aggregation of peptide. These interactions have been investigated using infrared vibrational absorption (VA) and circular dichroism (VCD) spectra. Four TTLs, namely ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC) and bilobalide (BB) and amyloid Abeta(25-35) peptide, as a model for the full length peptide, are used in this study. GA-monoether and GA-diether have also been synthesized and investigated to help understand the role of individual carbonyl groups in these interactions. The precipitation and solubility issues encountered with the mixture of ginkgolide+Abeta peptide for VA and VCD studies were overcome using binary ethanol-D(2)O solvent mixture. The experimental VA and VCD spectra of GA, GB, GC and BB, GA-monoether and GA-diether have been analyzed using the corresponding spectra predicted with density functional theory. The time-dependent experimental VA and VCD spectra of Abeta(25-35) peptide and the corresponding experimental spectra in the presence of TTLs indicated that the effect of the TTLs in modulating the aggregation of Abeta(25-35) peptide is relatively small. Such small effects might indicate the absence of a specific interaction between the TTLs and Abeta(25-35) peptide as a major force leading to the reduced aggregation of amyloid peptides. It is possible that the therapeutic effect of G. biloba extract does not originate from direct interactions between TTLs and the Abeta(25-35) peptide and is more complex.     

Micellization of surfactin and its effect on the aggregate conformation of amyloid beta(1-40)

The aggregation of amyloid beta-peptide (Abeta(1-40)) into fibrils is a key pathological process associated with Alzheimer's disease. This work has investigated the micellization process of biosurfactant surfactin and its effect on the aggregation behavior of Abeta(1-40). The results show that surfactin has strong self-assembly ability to form micelles and the micelles tend to form larger aggregates. Surfactin adopts a beta-turn conformation at low micelle concentration but a beta-sheet conformation at high micelle concentration. The effect of surfactin on the Abeta(1-40) aggregation behavior exhibits a strong concentration-dependent fashion. Below the critical micelle concentration of surfactin, the electrostatic binding of surfactin monomers on Abeta(1-40) causes Abeta(1-40) molecules to unfold. Assisted by the hydrophobic interaction among surfactin monomers on the Abeta(1-40) chain, the conformation of Abeta(1-40) transfers to the beta-sheet structure, which promotes the formation of fibrils. At low surfactin micelle concentration, besides the electrostatic force and hydrophobic interaction, hydrogen bonds formed between surfactin micelles and adjacent Abeta(1-40) peptide chains may promote the ordered organization of these Abeta(1-40) peptide chains, thus leading to the formation of beta-sheets and fibrils to a great extent. At high surfactin micelle concentration, the separating of Abeta(1-40) chains by the excessive surfactin micelles and the aggregation of the complexes of Abeta(1-40) with surfactin micelles inhibit the formation of beta-sheets and fibrils.     

Exploring the binding mechanism of thioflavin-T to the β-amyloid peptide by blind docking method

Using blind dock method,we find that thioflavin-T(ThT) can bind to both monomers and fibrils of the full-length β-amyloid peptide(Aβ1-42) and has a higher binding affinity to the fibrils.It is shown that the hydrophobic interaction between the ligand(ThT) and substrate(Aβ1-42) are stronger than hydrogen bonds.Furthermore,ThT tends to be located near the C-terminus of Aβ monomer through hydrophobic and electrostatic interactions,while it tends to contact the residues Met35 and Gly27 of the fibril surface mainly through hydrophobic interaction.Finally,according to the docking results and ThT fluorescence assay,a kinetic equation is proposed to deduce the aggregation rate coefficient of Aβ1-42.     

Effect of PEG crystallization on the self-assembly of PEG/peptide copolymers containing amyloid peptide fragments

The effect of poly(ethylene glycol) PEG crystallization on beta-sheet fibril formation is studied for a series of three peptide/PEG conjugates containing fragments modified from the amyloid beta peptide, specifically KLVFF, FFKLVFF, and AAKLVFF. These are conjugated to PEG with M n = 3300 g mol (-1). It is found, via small-angle X-ray scattering, X-ray diffraction, atomic force microscopy, and polarized optical microscopy, that PEG crystallinity in dried samples can disturb fibrillization, in particular cross-beta amyloid structure formation, for the conjugate containing the weak fibrillizer KLVFF, whereas this is retained for the conjugates containing the stronger fibrillizers AAKLVFF and FFKLVFF. For these two samples, the alignment of peptide fibrils also drives the orientation of the attached PEG chains. Our results highlight the importance of the antagonistic effects of PEG crystallization and peptide fibril formation in PEG/peptide conjugates.     

Structural model of the amyloid fibril formed by beta(2)-microglobulin #21-31 fragment based on vibrational spectroscopy

A structural model of amyloid fibril formed by the #21-31 fragment of beta2-microglobulin is proposed from microscope IR measurements on specifically 13C-labeled peptide fibrils and Raman spectra of the dispersed fibril solution. The 13C-shifted amide frequency indicated the secondary structure of the labeled residues. The IR spectra have demonstrated that the region between F22 and V27 forms the core part with the extended beta-sheet structure. Raman spectra indicated the formation of a dimer with a disulfide bridge between C25 residues.     

Mechanism of copper(II) inhibiting Alzheimer's amyloid beta-peptide from aggregation: a molecular dynamics investigation

The aggregation of an amyloid beta peptide (Abeta) into fibrils is a key pathological event in Alzheimer's disease (AD). Under certain conditions, Cu2+ markedly inhibits Abeta from aggregation and is considered as a potential factor in the normal brain preventing Abeta from aggregation. The possible mechanism of the inhibitory effect of Cu2+ was investigated for the first time by molecular dynamics (MD) simulations. On the basis of the radial distribution function analysis of the MD data, a novel strategy, the Q function, was proposed to explore the binding sites of Cu2+ by evaluating the coordination priority of atoms in Abeta, and the [6-5-5] tri-ring 4N binding mode of the Cu2+-Abeta complexes was found. The mechanism of the conformational transition of Abeta from the beta conformation to distorted beta conformations, which destabilizes the aggregation of Abeta into fibrils, was also revealed. All the results provide helpful clues for an improved understanding of the role of Cu2+ in the pathogenesis of AD and contribute to the development of an anti-amyloid therapeutic strategy.