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We have described a novel C-to-T mutation in the APP gene that corresponds to an alanine to valine substitution at position 673 in APP (A673V), or position 2 of the amyloid-β (Aβ) sequence. This mutation is associated with the early onset of AD-type dementia in homozygous individuals, whereas it has a protective effect in the heterozygous state. Correspondingly, we observed differences in the aggregation properties of the wild-type and mutated Aβ peptides and their mixture. We have carried out neutron diffraction (ND) and x-ray diffraction (XRD) experiments on magnetically-oriented fibers of Aβ1-28WT and its variant Aβ1-28A2V. The orientation propensity was higher for Aβ1-28A2V suggesting that it promotes the formation of fibrillar assemblies. The diffraction patterns by Aβ1-28WT and Aβ1-28A2V assemblies differed in shape and position of the equatorial reflections, suggesting that the two peptides adopt distinct lateral packing of the diffracting units. The diffraction patterns from a mixture of the two peptides differed from those of the single components, indicating the presence of structural interference during assembly and orientation. The lowest orientation propensity was observed for a mixture of Aβ1-28WT and a short N-terminal fragment, Aβ1-6A2V, which supports a role of Aβ's N-terminal domain in amyloid fibril formation.

Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis-trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aβ binding to g3p. NMR studies show that g3p binding to Aβ fibers is predominantly through middle and C-terminal residues of the Aβ subunit, indicating β strand-g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aβ fibers (fAβ) (KD=9.4nM); (2); blocks fAβ assembly (IC50~50nM) and (3) dissociates fAβ (EC50=40-100nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The sequence KLVFFAE (A beta 16-22) in Alzheimer's beta-amyloid is thought to be a core beta-structure that could act as a template for folding other parts of the polypeptide or molecules into fibrillar assemblies rich in beta-sheet. To elucidate the mechanism of the initial folding process, we undertook combined X-ray fiber/powder diffraction and infrared (IR) spectroscopy to analyze lyophilized A beta 16-22 and solubilized/dried peptide containing nitrile probes at F19 and/or F20. Solubilized/dried wild-type (WT) A beta 16-22 and the peptide containing cyanophenylalanine at F19 (19CN) or at F20 (20CN) gave fiber patterns consistent with slab-like beta-crystallites that were cylindrically averaged around the axis parallel to the polypeptide chain direction. The WT and 19CN assemblies showed 30-A period arrays arising from the stacking of the slabs along the peptide chain direction, whereas the 20CN assemblies lacked any such stacking. The electron density projection along the peptide chain direction indicated similar side-chain dispositions for WT and 20CN, but not for 19CN. These X-ray results and modeling imply that in the assembly of WT A beta 16-22 the F19 side chain is localized within the intersheet space and is involved in hydrophobic contact with amino acids across the intersheet space, whereas the F20 side chain localized near the slab surface is less important for the intersheet interaction, but involved in slab stacking. IR observations for the same peptides in dilute solution showed a greater degree of hydrogen bonding for the nitrile groups in 20CN than in 19CN, supporting this interpretation., ((c) 2010 Wiley-Liss, Inc.)

Targeting the initial formation of amyloid assemblies is a preferred approach to therapeutic intervention in amyloidoses, which include such diseases as Alzheimer's, Parkinson's, Huntington's, etc., as the early-stage, oligomers that form before the development of beta-conformation-rich fibers are thought to be toxic. X-ray patterns from amyloid assemblies always show two common intensity maxima: one at 4.7 A corresponding to the hydrogen-bonding spacing between the beta-chains, and the other at approximately 10 A corresponding to the spacing between beta-pleated sheets. We report here the application of fiber x-ray diffraction to monitor these structural indicators of amyloid fiber assembly in the presence of small, aromatic molecules, some of which have been assessed by other techniques as being inhibitory. The compounds included butylated hydroxytoluene, chloramphenicol, cotinine, curcumin, diphenylalanine (FF), ethyl 3-aminobenzoate methane sulfonate, hexachlorophene, melatonin, methylpyrrolidine, morin, nicotine, phenolphthalaine, PTI-00703 (Cat's claw), pyridine, quinine, sulfadiazine, tannic acid, tetracaine, tetrachlorosalicylanilide, and tetracycline. Their effects on the aggregation of Abeta1-40, Abeta11-25, Abeta12-28, Abeta17-28, Abeta16-22, and Abeta16-22[methylated] analogues were characterized in terms of the integral widths and integrated intensities of the two characteristic reflections. Peptide Abeta11-25 with or without small molecules showed varying relative intensities but similar coherent lengths of 28-49 A in the intersheet and 171-221 A in the H-bonding directions. PTI-00703, however, abolished the H-bonding reflection. Among previously reported aromatic inhibitors for Abeta11-25, PTI-00703, tannic acid, and quinine were more effective than curcumin, morin, and melatonin based on the criterion of crystallite volume. For the N-methylated and control samples, there were no substantial differences in spacings and coherent lengths; however, the relative volumes of the beta-crystallites, which were calculated from the magnitude of the intensities, decreased with increase in concentration of Abeta16-22Me. This may be accounted for by the binding of Abeta16-22Me to the monomer or preamyloid oligomer of Abeta16-22. The fiber diffraction approach, which can help to specify whether an amyloidophilic compound acts by impeding hydrogen-bonding or by altering intersheet interactions, may help provide a rationale basis for the development of other therapeutic reagents.

We consider here the chemistry of Congo red (CR), its binding equilibrium to Alzheimer's beta-amyloid, and the kinetics of beta-amyloid formation. Spectroscopic UV/V is measurements for the pH- and time-dependence binding of CR to Abeta analogues are analysed by Scatchard binding and the theory of nucleation-dependent fibril formation. CR likely binds electrostatically to the imidazolium sidechains of histidine residues that are exposed at the surface of amyloid fibrils. As revealed by atomic models of the Abeta protofilament, such as the nanotube beta-helix and parallel beta-sheet, the regular arrangement of histidines likely acts as a template for the end-to-end J-aggregation of CR molecules, which produces a red shift in UV/V is absorption.

Alzheimer's beta amyloid protein (A beta) is a 39 to 43 amino acid peptide that is a major component in the neuritic plaques of Alzheimer's disease (AD). The assemblies constituted from residues 25-35 (A beta 25-35), which is a sequence homologous to the tachykinin or neurokinin class of neuropeptides, are neurotoxic. We used X-ray diffraction and electron microscopy to investigate the structure of the assemblies formed by A beta 25-35 peptides and of various length sequences therein, and of tachykinin-like analogues. Most solubilized peptides after subsequent drying produced diffraction patterns characteristic of beta-sheet structure. Moreover, the peptides A beta 31-35 (Ile-Ile-Gly-Leu-Met) and tachykinin analogue A beta(Phe(31))31-35 (Phe-Ile-Gly-Leu-Met) gave powder diffraction patterns to 2.8A Bragg spacing. The observed reflections were indexed by an orthogonal unit cell having dimensions of a=9.36 A, b=15.83 A, and c=20.10 A for the native A beta 31-35 peptide, and a=9.46 A, b=16.22 A, and c=11.06 A for the peptide having the Ile31Phe substitution. The initial model was a beta strand where the hydrogen bonding, chain, and intersheet directions were placed along the a, b, and c axes. An atomic model was fit to the electron density distribution, and subsequent refinement resulted in R factors of 0.27 and 0.26, respectively. Both peptides showed a reverse turn at Gly33 which results in intramolecular hydrogen bonding between the antiparallel chains. Based on previous reports that antagonists for the tachykinin substance P require a reverse turn, and that A beta is cytotoxic when it is oligomeric or fibrillar, we propose that the tachykinin-like A beta 31-35 domain is a turn exposed at the A beta oligomer surface where it could interact with the ligand-binding site of the tachykinin G-protein-coupled receptor.

The binding mechanism of Congo red (CR) to Alzheimer's disease (AD) amyloid fibrils (A beta) in terms of binding affinity and number of sites was quantitated from absorption spectroscopy (at 200-700 nm) by measuring the concentration of CR bound (CR-B) to AD A beta assemblies as a function of CR concentration and pH in 80% ethanol. The rationale for the use of this high concentration of ethanol derives from its use in histological screens for amyloid in tissue sections. Moreover, free CR can be separated from bound CR by filtration in ethanolic but not aqueous medium. The A beta analogs studied here included: (1) peptides having different lengths: A beta1-40, A beta11-28, A beta13-28, A beta19-28, A beta11-25; (2) wildtype, control sequences of A beta1-40 and sequences having different natural amino acid substitutions: primate Pr1-40, rodent Ro1-40, hereditary cerebral haemorrhage with amyloidosis, Dutch type (HCHWA-D) Du1-40, primate reverse sequence Pr40-1; and (3) A beta11-25 sequences having different substitutions: H13D, H14D, and D23K. Negative-staining showed that A beta1-40 fibrils in buffer were indistinguishable from those in buffered ethanolic medium. For all amyloid analogs except A beta19-28, which has no histidine residues and showed no CR binding over the entire pH range 4.0-9.5, CR-B decreased as a function of increasing pH. The decrease was steepest at about pH 5 and became zero above pH 7. For analogs having the same number of histidines, CR-B fell on the same binding curve, indicating that histidine residues are the likely binding sites for CR in this medium. The pH titration of the binding was parameterized by the stoichiometry of dye to the sites, the number of histidines per molecule, the binding dissociation constant Kd, and the apparent proton dissociation constant pK of the histidine; and the calculated pH-titration curves were found to fit the observed ones. For the peptides having 1-3 histidines the average pK was 5.0-5.5, which was similar to the expected pK of histidine in low dielectric medium (80% ethanol), and the Kd's were 2.8-5.9 microM. That histidine residues underlie CR binding in A beta amyloid is consistent with previous findings that A beta peptides sediment as fibrillar assemblies at pH-3-7 and bind Congo red over the same pH range in aqueous medium. Further, the conformation near the binding motif His13-His14-Gln15-Lys16 in A beta assemblies is not greatly altered in 80% ethanol.

Using Scatchard analysis, we have formulated as a function of time and pH the relationship between the binding of Congo red to Alzheimer's beta-amyloid and the aggregation number (i.e., monomer concentration within fibrils) as defined by nucleation-dependent self-assembly. This provides a basis on which to determine the kinetic parameters for fibril formation from the observed concentration of bound Congo red., (Copyright 2000 Academic Press.)

Detailed structural studies of amyloid fibrils can elucidate the way in which their constituent polypeptides are folded and self-assemble, and exert their neurotoxic effects in Alzheimer's disease (AD). We have previously reported that when aqueous solutions of the N-terminal hydrophilic peptides of AD beta-amyloid (A beta) are gradually dried in a 2-Tesla magnetic field, they form highly oriented fibrils that are well suited to x-ray fiber diffraction. The longer, more physiologically relevant sequences such as A beta(1-40) have not been amenable to such analysis, owing to their strong propensity to polymerize and aggregate before orientation is achieved. In seeking an efficient and inexpensive method for rapid screening of conditions that could lead to improved orientation of fibrils assembled from the longer peptides, we report here that the birefringence of a small drop of peptide solution can supply information related to the cooperative packing of amyloid fibers and their capacity for magnetic orientation. The samples were examined by electron microscopy (negative and positive staining) and x-ray diffraction. Negative staining showed a mixture of straight and twisted fibers. The average width of both types was approximately 70 A, and the helical pitch of the latter was approximately 460 A. Cross sections of plastic-embedded samples showed a approximately 60-A-wide tubular structure. X-ray diffraction from these samples indicated a cross-beta fiber pattern, characterized by a strong meridional reflection at 4.74 A and a broad equatorial reflection at 8.9 A. Modeling studies suggested that tilted arrays of beta-strands constitute tubular, 30-A-diameter protofilaments, and that three to five of these protofilaments constitute the A beta fiber. This type of structure--a multimeric array of protofilaments organized as a tubular fibril--resembles that formed by the shorter A beta fragments (e.g., A beta(6-25), A beta(11-25), A beta(1-28)), suggesting a common structural motif in AD amyloid fibril organization.

From the wide-angle, equatorial X-ray data of a beta-amyloid analogue, we previously calculated the electron density of the constituent beta-crystallite, which assembles as multimers (four to six crystallites) in building the amyloid fibre. In the scattering region where the spacing d < approximately 10 A, the observed reflections were indexed by an orthogonal lattice with a unit cell having a = 9.44 A, b = 6.92 A and c = 10.76 A. The phases were initially derived from the atomic coordinates of the beta-keratin backbone and were optimized by including new peaks (as point atom or sphere) in the subsequent Fourier iteration. The R-factor between the observed and calculated amplitudes was refined to 35%. In further developing our analysis, we have now applied an alternative constraint to the optimization by eliminating the negative electron densities, and found that the R-factor decreased to 19% after three iterations. The refined electron density map fits phenylalanine, indicating that the amyloid core likely comes from the hydrophobic Leu-Val-Phe-Phe residues. We have applied the same type of optimization, using beta-silk as an initial phase model, to the hydrophobic H1 domain of the prion protein for which the monoclinic unit cell constants are a = 9.51 A, b = 7.06 A, c = 15.94 A and beta = 88.4 degrees. The R-factor decreased to 11% from 64% after two iterations. The electron density map shows a silk-like quarter-staggered arrangement of beta-sheets which, in the intersheet direction, have circular peaks in one beta-sheet and elongated peaks in the alternating beta-sheet. These peaks were interpreted as arising from the C-terminal alanine-rich domain and N-terminal hydrophobic residues. Skeletal atomic models for these core regions support this interpretation.

To elucidate the relation between amyloid fibril formation in Alzheimer disease and the primary structure of the beta/A4 protein, which is the major component of the amyloid, we have been investigating the ability of peptides sharing sequences with beta/A4 to form fibrils in vitro. In previous studies we focused on the macroscopic morphology of the assemblies formed by synthetic peptides corresponding in sequence to different regions of this protein. In the present study we analyze the x-ray diffraction patterns obtained from these assemblies. All specimens showed wide angle reflections that could be indexed by an orthogonal lattice of beta-crystallites having unit cell dimensions a = 9.4 A, b = 7 A, and c = 10 A, where a refers to hydrogen bonding direction, b to polypeptide chain direction, and c to intersheet direction. Given the amino acid sequence of beta/A4 as NH2-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT-COOH, we found that, based on their orientation and assembly, the analogues could be classified into three groups: Group A, residues 19-28, 13-28, 12-28, 11-28, 9-28, 1-28, 1-38, 1-40, 6-25, 11-25 and 34-42; Group B, residues 18-28, 17-28, and 15-28; and Group C, residues 22-35 and 26-33. For Groups A and C, the sharpest reflections were (h00), indicating that the assemblies were fibrillar, i.e., elongated in a single direction. Lateral alignment of the crystallites in Group A account for its cross-beta pattern, in which the hydrogen bonding (H-bonding) direction is the fiber (rotation) axis. By comparison, the beta-crystallites of Group C had no preferential orientation, thus giving circular scattering. For Group B, the sharpest reflections were (h0l) on the meridian, indicating that the assemblies were plate-like, i.e., extended in two directions. A series of equatorial Bragg reflections having a 40 A period indicated regular stacking of the plates, and the rotation axis was normal to the surface of the plates. Of the Group A peptides, the analogues 11-28 and 6-25 showed intensity maxima on the equator as well as on higher layer lines, indicating that the beta-crystallites are highly ordered relative to one another in the axial, H-bonding direction. This sampling of the layer lines by a larger period (60 A) suggests that the beta-crystallites are arrayed either in cylindrical or small restricted crystalline lattices. Consistent with its electron microscopic images, we modeled the structure as a tube with five or six f,-crystallites constituting the wall and with the individual crystallite, which either rotates freely or is restricted, made of five or fewer beta-pleated sheets. For the Group B peptides, the electron density projection along the b-axis was calculated from the observed intensities using phase combinations from fl-keratin.Amino acid side-chain positions were apparent and, when refined as 4-A-diameter spheres, led to a substantial decrease in the R-factors.For peptide 18-28 the electron density peaks, which are thought to correspond to side chains, were centered 3.3 A from the peptide backbone, whereas for peptides 17-28 and 15-28, these peaks were centered 1 A or more further from the backbone. Peaks having high electron density faced peaks having lower density, suggesting a favorable stereochemical arrangement of the residues. Thus, our analysis of the fiber x-ray patterns from beta/A4 peptides shows the organization of the beta-crystallites that form the wall of the amyloid fibrils as well as possible side-chain interactions.

Deposition of extraneuronal fibrils that assemble from the 39-43 residue beta/A4 amyloid protein is one of the earliest histopathological features of Alzheimer's disease. We have used negative-stain electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and fiber X-ray diffraction to examine the structure and properties of synthetic peptides corresponding to residues 1-40 of the beta/A4 protein of primate [Pm(1-40); human and monkey], rodent [Ro(1-40); with Arg5-->Gly, Tyr10-->Phe, and His13-->Arg], and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) [Du(1-40); with Glu22-->Gln]. As controls, we examined a reverse primate sequence [Pm*(40-1)] and an extensively substituted primate peptide [C(1-40); with Glu3-->Arg, Arg5-->Glu, Asp7-->Val, His13-->Lys, Lys16-->His, Val18-->Asp, Phe19-->Ser, Phe20-->Tyr, Ser26-->Pro, Ala30-->Val, Ile31-->Ala, Met35-->norLeu, Gly38-->Ile, Val39-->Ala, and Val40-->Gly]. The assembly of these peptides was studied to understand the relationship between species-dependent amyloid formation and beta/A4 sequence and the effect of a naturally occurring point mutation of fibrillogenesis. The three N-terminal amino acid differences between Pm(1-40) and Ro(1-40) had virtually no effect on the morphology or organization of the fibrils formed by these peptides, indicating that the lack of amyloid deposits in rodent brain is not due directly to specific changes in its beta/A4 sequence. beta-Sheet and fibril formation, judged by FT-IR, was maximal within the pH range 5-8 for Pm(1-40), pH 5-10.5 for Du(1-40), and pH 2.5-8 for Ro(1-40).(ABSTRACT TRUNCATED AT 250 WORDS)

To elucidate the relationship between amyloid fibril formation in Alzheimer disease (AD) and the primary structure of the beta-amyloid protein (beta-AP), we investigated the ability of peptides sharing sequences with beta-AP to form fibrils in vitro and to recognize anti-beta-amyloid antisera. The peptides, which were synthesized using a FMOC solid phase procedure and purified by HPLC, consisted of residues 6-25 from the putative aqueous domain, residues 22-35, which overlaps the putative aqueous and transmembrane domains, and residues 1-38 and 1-40 representing nearly the full length of beta-AP. Electron microscopy of negative-stained or thin-sectioned preparations revealed that the peptides assembled into fibrils having different morphologies, some of which resembled in situ AD amyloid. Peptide 6-25 fibrils had diameters of 50-80 A and occasionally showed a central groove suggestive of constituent filaments. Cross sections of the fibril showed a penta- or hexameric arrangement of globular subunits with diameters of 25-30 A. Peptide 22-35 fibrils were helical, with a pitch of 1,100 A and a width of 120 A at its greatest and 50-60 A at its narrowest. The fibrils formed by peptides 1-38 and 1-40 were 70-90 A in diameter. When the peptide assemblies were singly oriented by sedimentation or doubly oriented in a magnetic field, their X-ray diffraction patterns all showed reflections typical of a cross-beta pleated sheet conformation. The patterns differed mainly in their small-angle equatorial intensity, which arises from the packing of fibrils having different widths. Antiserum raised to either native amyloid or to synthetic peptide beta-(1-28) was highly reactive in an inhibition-ELISA assay to beta-(6-25) and beta-(1-38), but not to beta-(22-35), and immunostained beta-(1-40) on Western blots. These studies show that the beta-(6-25), beta-(1-38) and beta-(1-40) peptides can assemble into cross-beta fibrils that retain epitopes characteristic of AD amyloid.