Mapping abeta amyloid fibril secondary structure using scanning proline mutagenesis

Although the amyloid fibrils formed from the Alzheimer's disease amyloid peptide Abeta are rich in cross-beta sheet, the peptide likely also exhibits turn and unstructured regions when it becomes incorporated into amyloid. We generated a series of single-proline replacement mutants of Abeta(1-40) and determined the thermodynamic stabilities of amyloid fibrils formed from these mutants to characterize the susceptibility of different residue positions of the Abeta sequence to proline substitution. The results suggest that the Abeta peptide, when engaged in the amyloid fibril, folds into a conformation containing three highly structured segments, consisting of contiguous sequence elements 15-21, 24-28, and 31-36, that are sensitive to proline replacement and likely to include the beta-sheet portions of the fibrils. Residues relatively insensitive to proline replacement fall into two groups: (a) residues 1-14 and 37-40 are likely to exist in relatively unstructured, flexible elements extruded from the beta-sheet-rich amyloid core; (b) residues 22, 23, 29 and 30 are likely to occupy turn positions between these three structured elements. Although destabilized, fibrils formed from Abeta(1-40) proline mutants are very similar in structure to wild-type fibrils, as indicated by hydrogen-deuterium exchange and other analysis. Interestingly, however, some proline mutations destabilize fibrils while at the same time increasing the number of amide protons protected from hydrogen exchange. This suggests that the stability of amyloid fibrils, rather than being driven exclusively by the formation of H-bonded beta-sheet, is achieved, as in globular proteins, through a balance of stabilizing and destabilizing forces. The proline scanning data are most compatible with a model for amyloid protofilament structure loosely resembling the parallel beta-helix folding motif, such that each Abeta(15-36) core region occupies a single layer of a prismatic, H-bonded stack of peptides.     

Seeding specificity in amyloid growth induced by heterologous fibrils

Over residues 15-36, which comprise the H-bonded core of the amyloid fibrils it forms, the Alzheimer's disease plaque peptide amyloid beta (Abeta) possesses a very similar sequence to that of another short, amyloidogenic peptide, islet amyloid polypeptide (IAPP). Using elongation rates to quantify seeding efficiency, we inquired into the relationship between primary sequence similarity and seeding efficiency between Abeta-(1-40) and amyloid fibrils produced from IAPP as well as other proteins. In both a solution phase and a microtiter plate elongation assay, IAPP fibrils are poor seeds for Abeta-(1-40) elongation, exhibiting weight-normalized efficiencies of only 1-2% compared with Abeta-(1-40) fibrils. Amyloid fibrils of peptides with sequences completely unrelated to Abeta also exhibit poor to negligible seeding ability for Abeta elongation. Fibrils from a number of point mutants of Abeta-(1-40) exhibit intermediate seeding abilities for wild-type Abeta elongation, with differing efficiencies depending on whether or not the mutation is in the amyloid core region. The results suggest that amyloid fibrils from different proteins exhibit structural differences that control seeding efficiencies. Preliminary results also suggest that identical sequences can grow into different conformations of amyloid fibrils as detected by seeding efficiencies. The results have a number of implications for amyloid structure and biology.     

An atomic model for the pleated beta-sheet structure of Abeta amyloid protofilaments.

Synchrotron x-ray studies on amyloid fibrils have suggested that the stacked pleated beta-sheets are twisted so that a repeating unit of 24 beta-strands forms a helical turn around the fibril axis (. J. Mol. Biol. 273:729-739). Based on this morphological study, we have constructed an atomic model for the twisted pleated beta-sheet of human Abeta amyloid protofilament. In the model, 48 monomers of Abeta 12-42 stack (four per layer) to form a helical turn of beta-sheet. Each monomer is in an antiparallel beta-sheet conformation with a turn located at residues 25-28. Residues 17-21 and 31-36 form a hydrophobic core along the fibril axis. The hydrophobic core should play a critical role in initializing Abeta aggregation and in stabilizing the aggregates. The model was tested using molecular dynamics simulations in explicit aqueous solution, with the particle mesh Ewald (PME) method employed to accommodate long-range electrostatic forces. Based on the molecular dynamics simulations, we hypothesize that an isolated protofilament, if it exists, may not be twisted, as it appears to be when in the fibril environment. The twisted nature of the protofilaments in amyloid fibrils is likely the result of stabilizing packing interactions of the protofilaments. The model also provides a binding mode for Congo red on Abeta amyloid fibrils. The model may be useful for the design of Abeta aggregation inhibitors.     

An intersheet packing interaction in A beta fibrils mapped by disulfide cross-linking

Most models for the central cross-beta folding unit in amyloid fibrils of the Alzheimer's plaque protein Abeta align the peptides in register in H-bonded, parallel beta-sheet structure. Some models require the Abeta peptide to undergo a chain reversal when folding into the amyloid core, while other models feature very long extended chains, or zigzag chains, traversing the protofilament. In this paper we introduce the use of disulfide bond cross-linking to probe the fold within the core and the packing interactions between beta-sheets. In one approach, amyloid fibrils grown under reducing conditions from each of three double cysteine mutants (17/34, 17/35, and 17/36) of the Abeta(1-40) sequence were subjected to oxidizing conditions. Of these three mutants, only the Leu17Cys/Leu34Cys peptide could be cross-linked efficiently while resident in fibrils. In another approach, double Cys mutants were cross-linked as monomers before aggregation, and the resulting fibrils were assessed for stability, antibody binding, dye binding, and cross-seeding efficiency. Here too, fibrils from the 17/34 double Cys mutant most closely resemble wild-type Abeta(1-40) fibrils. These data support models of the Abeta fibril in which the Leu17 and Leu34 side chains of the same peptide pack against each other at the beta-sheet interface within the amyloid core. Related cross-linking strategies may reveal longer range spatial relationships. The ability of the cross-linked 17/35 double Cys mutant Abeta to also make amyloid fibrils illustrates a remarkable plasticity of the amyloid structure and suggests a structural mechanism for the generation of conformational variants of amyloid.     

Alanine scanning mutagenesis of Abeta(1-40) amyloid fibril stability

We describe here an alanine scanning mutational analysis of the Abeta(1-40) amyloid fibril monitored by fibril elongation thermodynamics derived from critical concentration values for fibril growth. Alanine replacement of most residues in the amyloid core region, residues 15-36, leads to destabilization of the elongation step, compared to wild-type, by about 1kcal/mol, consistent with a major role for hydrophobic packing in Abeta(1-40) fibril assembly. Where comparisons are possible, the destabilizing effects of Ala replacements are generally in very good agreement with the effects of Ala replacements of the same amino acid residues in an element of parallel beta-sheet in the small, globular protein Gbeta1. We utilize these Ala-WT DeltaDeltaG values to filter previously described Pro-WT DeltaDeltaG values, creating Pro-Ala DeltaDeltaG values that specifically assess the sensitivity of a sequence position, in the structural context of the Abeta fibril, to replacement by proline. The results provide a conservative view of the energetics of Abeta(1-40) fibril structure, indicating that positions 18-21, 25-26, and 32-33 within amyloid structure are particularly sensitive to the main-chain disrupting effects of Pro replacements. In contrast, residues 14-17, 22, 24, 27-31, and 34-39 are relatively insensitive to Pro replacements; most N-terminal residues were not tested. The results are discussed in terms of amyloid fibril structure and folding energetics, in particular focusing on how the data compare to those from other structural studies of Abeta(1-40) amyloid fibrils grown in phosphate-buffered saline at 37 degrees C under unstirred ("quiescent") conditions.     

Supramolecular structural constraints on Alzheimer's beta-amyloid fibrils from electron microscopy and solid-state nuclear magnetic resonance

We describe electron microscopy (EM), scanning transmission electron microscopy (STEM), and solid-state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the 42-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1)(-)(42)) and by residues 10-35 of the full-length peptide (Abeta(10)(-)(35)). These measurements place constraints on the supramolecular structure of the amyloid fibrils, especially the type of beta-sheets present in the characteristic amyloid cross-beta structural motif and the assembly of these beta-sheets into a fibril. EM images of negatively stained Abeta(10)(-)(35) fibrils and measurements of fibril mass per length (MPL) by STEM show a strong dependence of fibril morphology and MPL on pH. Abeta(10)(-)(35) fibrils formed at pH 3.7 are single "protofilaments" with MPL equal to twice the value expected for a single cross-beta layer. Abeta(10)(-)(35) fibrils formed at pH 7.4 are apparently pairs of protofilaments or higher order bundles. EM and STEM data for Abeta(1)(-)(42) fibrils indicate that protofilaments with MPL equal to twice the value expected for a single cross-beta layer are also formed by Abeta(1)(-)(42) and that these protofilaments exist singly and in pairs at pH 7.4. Solid-state NMR measurements of intermolecular distances in Abeta(10)(-)(35) fibrils, using multiple-quantum (13)C NMR, (13)C-(13)C dipolar recoupling, and (15)N-(13)C dipolar recoupling techniques, support the in-register parallel beta-sheet organization previously established by Lynn, Meredith, Botto, and co-workers [Benzinger et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13407-13412; Benzinger et al. (2000) Biochemistry 39, 3491-3499] and show that this beta-sheet organization is present at pH 3.7 as well as pH 7.4 despite the differences in fibril morphology and MPL. Solid-state NMR measurements of intermolecular distances in Abeta(1)(-)(42) fibrils, which represent the first NMR data on Abeta(1)(-)(42) fibrils, also indicate an in-register parallel beta-sheet organization. These results, along with previously reported data on Abeta(1)(-)(40) fibrils, suggest that the supramolecular structures of Abeta(10)(-)(35), Abeta(1)(-)(40), and Abeta(1)(-)(42) fibrils are quite similar. A schematic structural model of these fibrils, consistent with known experimental EM, STEM, and solid-state NMR data, is presented.     

Structure and orientation of peptide inhibitors bound to beta-amyloid fibrils

Polymerization of the soluble beta-amyloid peptide into highly ordered fibrils is hypothesized to be a causative event in the development of Alzheimer's disease. Understanding the interactions of Abeta with inhibitors on an atomic level is fundamental for the development of diagnostics and therapeutic approaches, and can provide, in addition, important indirect information of the amyloid fibril structure. We have shown recently that trRDCs can be measured in solution state NMR for peptide ligands binding weakly to amyloid fibrils. We present here the structures for two inhibitor peptides, LPFFD and DPFFL, and their structural models bound to fibrillar Abeta(14-23) and Abeta(1-40) based on transferred nuclear Overhauser effect (trNOE) and transferred residual dipolar coupling (trRDC) data. In a first step, the inhibitor peptide structure is calculated on the basis of trNOE data; the trRDC data are then validated on the basis of the trNOE-derived structure using the program PALES. The orientation of the peptide inhibitors with respect to Abeta fibrils is obtained from trRDC data, assuming that Abeta fibrils orient such that the fibril axis is aligned in parallel with the magnetic field. The trRDC-derived alignment tensor of the peptide ligand is then used as a restraint for molecular dynamics docking studies. We find that the structure with the lowest rmsd value is in agreement with a model in which the inhibitor peptide binds to the long side of an amyloid fibril. Especially, we detect interactions involving the hydrophobic core, residues K16 and E22/D23 of the Abeta sequence. Structural differences are observed for binding of the inhibitor peptide to Abeta14-23 and Abeta1-40 fibrils, respectively, indicating different fibril structure. We expect this approach to be useful in the rational design of amyloid ligands with improved binding characteristics.     

Stabilization of discordant helices in amyloid fibril-forming proteins

Several proteins and peptides that can convert from alpha-helical to beta-sheet conformation and form amyloid fibrils, including the amyloid beta-peptide (Abeta) and the prion protein, contain a discordant alpha-helix that is composed of residues that strongly favor beta-strand formation. In their native states, 37 of 38 discordant helices are now found to interact with other protein segments or with lipid membranes, but Abeta apparently lacks such interactions. The helical propensity of the Abeta discordant region (K16LVFFAED23) is increased by introducing V18A/F19A/F20A replacements, and this is associated with reduced fibril formation. Addition of the tripeptide KAD or phospho-L-serine likewise increases the alpha-helical content of Abeta(12-28) and reduces aggregation and fibril formation of Abeta(1-40), Abeta(12-28), Abeta(12-24), and Abeta(14-23). In contrast, tripeptides with all-neutral, all-acidic or all-basic side chains, as well as phosphoethanolamine, phosphocholine, and phosphoglycerol have no significant effects on Abeta secondary structure or fibril formation. These data suggest that in free Abeta, the discordant alpha-helix lacks stabilizing interactions (likely as a consequence of proteolytic removal from a membrane-associated precursor protein) and that stabilization of this helix can reduce fibril formation.     

Mechanical manipulation of Alzheimer's amyloid beta1-42 fibrils

The 39- to 42-residue-long amyloid beta-peptide (Abeta-peptide) forms filamentous structures in the neuritic plaques found in the neuropil of Alzheimer's disease patients. The assembly and deposition of Abeta-fibrils is one of the most important factors in the pathogenesis of this neurodegenerative disease. Although the structural analysis of amyloid fibrils is difficult, single-molecule methods may provide unique insights into their characteristics. In the present work, we explored the nanomechanical properties of amyloid fibrils formed from the full-length, most neurotoxic Abeta1-42 peptide, by manipulating individual fibrils with an atomic force microscope. We show that Abeta-subunit sheets can be mechanically unzipped from the fibril surface with constant forces in a reversible transition. The fundamental unzipping force (approximately 23 pN) was significantly lower than that observed earlier for fibrils formed from the Abeta1-40 peptide (approximately 33 pN), suggesting that the presence of the two extra residues (Ile and Ala) at the peptide's C-terminus result in a mechanical destabilization of the fibril. Deviations from the constant force transition may arise as a result of geometrical constraints within the fibril caused by its left-handed helical structure. The nanomechanical fingerprint of the Abeta1-42 is further influenced by the structural dynamics of intrafibrillar interactions.     

Direct observation of Abeta amyloid fibril growth and inhibition

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.     

A molecular model of Alzheimer amyloid beta-peptide fibril formation

Polymerization of the amyloid beta (Abeta) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer's disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected Abeta-fragments containing the Abeta16-20 sequence, previously shown essential for Abeta-Abeta binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was Abeta14-23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Abeta1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of Abeta14-23 oligomers in an antiparallel beta-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of Abeta-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.     

Beta-amyloid fibril formation is promoted by step edges of highly oriented pyrolytic graphite

The aggregation of the amyloid-beta-protein (Abeta) is an important step in the pathogenesis of Alzheimer's disease. As Abeta fibrils are not found in all brain regions, endogenous factors may influence Abeta fibril formation. In this study, atomic force microscopy was used to investigate the role of surface phenomena in directing amyloid aggregation. Abeta1-40 was applied to a surface of highly oriented pyrolytic graphite at a concentration of 0.5 microM. Steps formed by edge-plane surface defects on the graphite were found to act as a template to promote the assembly of Abeta into fibrils. Initially, after being deposited on the graphite surface, Abeta had a uniform beaded morphology. However, after incubating (aging) the Abeta on the surface for several hours, the Abeta assembled along step edges to form linear aggregates. After more prolonged incubation, the linear Abeta aggregates fused to form mature fibrils with a distinctive helical morphology. The results demonstrate that surface interactions can promote the aggregation of Abeta into amyloid fibrils and they suggest that similar interactions could promote amyloid aggregation in vivo.     

Structural differences in Abeta amyloid protofibrils and fibrils mapped by hydrogen exchange--mass spectrometry with on-line proteolytic fragmentation

We report here structural differences between Abeta(1-40) protofibrils and mature amyloid fibrils associated with Alzheimer's disease as determined using hydrogen-deuterium exchange-mass spectrometry (HX-MS) coupled with on-line proteolysis. Specifically, we have identified regions of the Abeta(1-40) peptide containing backbone amide hydrogen atoms that are protected from HX or exposed when this peptide is incorporated into protofibrils or amyloid fibrils formed in phosphate-buffered saline without stirring at 37 degrees C. Study of protofibrils was facilitated by use of the protofibril-stabilizing agent calmidazolium chloride. Our data clearly show that both the C-terminal segment 35-40 and the N-terminal segment 1-19 are highly exposed to HX in both fibrils and protofibrils. In contrast, the internal fragment 20-34 is highly protected from exchange in fibrils but much less so in protofibrils. The data suggest that the beta-sheet elements comprising the amyloid fibril are already present in protofibrils, but that they are expanded into some adjacent residues upon the formation of mature amyloid. The N-terminal approximately ten residues appear to be unstructured in both protofibrils and fibrils. The 20-30 segment of Abeta(1-40) is more ordered in fibrils than in protofibrils, suggesting that, if protofibrils are a mechanistic precursor of fibrils, the transition from protofibril to fibril involves substantial ordering of this region of the Abeta peptide.     

Structural and dynamic features of Alzheimer's Abeta peptide in amyloid fibrils studied by site-directed spin labeling

Electron paramagnetic resonance spectroscopy analysis of 19 spin-labeled derivatives of the Alzheimer's amyloid beta (Abeta) peptide was used to reveal structural features of amyloid fibril formation. In the fibril, extensive regions of the peptide show an in-register, parallel arrangement. Based on the parallel arrangement and side chain mobility analysis we find the amyloid structure to be mostly ordered and specific, but we also identify more dynamic regions (N and C termini) and likely turn or bend regions (around residues 23-26). Despite their different aggregation properties and roles in disease, the two peptides, Abeta40 and Abeta42, homogeneously co-mix in amyloid fibrils suggesting that they possess the same structural architecture.     

Is Congo red an amyloid-specific dye?

Congo red (CR) binding, monitored by characteristic yellow-green birefringence under crossed polarization has been used as a diagnostic test for the presence of amyloid in tissue sections for several decades. This assay is also widely used for the characterization of in vitro amyloid fibrils. In order to probe the structural specificity of Congo red binding to amyloid fibrils we have used an induced circular dichroism (CD) assay. Amyloid fibrils from insulin and the variable domain of Ig light chain demonstrate induced CD spectra upon binding to Congo red. Surprisingly, the native conformations of insulin and Ig light chain also induced Congo red circular dichroism, but with different spectral shapes than those from fibrils. In fact, a wide variety of native proteins exhibited induced CR circular dichroism indicating that CR bound to representative proteins from different classes of secondary structure such as alpha (citrate synthase), alpha + beta (lysozyme), beta (concavalin A), and parallel beta-helical proteins (pectate lyase). Partially folded intermediates of apomyoglobin induced different Congo red CD bands than the corresponding native conformation, however, no induced CD bands were observed with unfolded protein. Congo red was also found to induce oligomerization of native proteins, as demonstrated by covalent cross-linking and small angle x-ray scattering. Our data suggest that Congo red is sandwiched between two protein molecules causing protein oligomerization. The fact that Congo red binds to native, partially folded conformations and amyloid fibrils of several proteins shows that it must be used with caution as a diagnostic test for the presence of amyloid fibrils in vitro.     

Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloid protein for solid-state NMR

Fibrillar protein aggregates contribute to the pathology of a number of disease states. To facilitate structural studies of these amyloid fibrils by solid-state NMR, efficient methods for the production of milligram quantities of isotopically labeled peptide are necessary. Bacterial expression of recombinant amyloid proteins and peptides allows uniform isotopic labeling, as well as other patterns of isotope incorporation. However, large-scale production of recombinant amyloidogenic peptides has proven particularly difficult, due to their inherent propensity for aggregation and the associated toxicity of fibrillar material. Yields of recombinant protein are further reduced by the small molecular weights of short amyloidogenic fragments. Here, we report high-yield expression and purification of a peptide comprising residues 11-26 of the Alzheimer's beta-amyloid protein (Abeta(11-26)), with homoserine lactone replacing serine at residue 26. Expression in inclusion bodies as a ketosteroid isomerase fusion protein and subsequent purification under denaturing conditions allows production of milligram quantities of uniformly labeled (13)C- and (15)N-labeled peptide, which forms amyloid fibrils suitable for solid-state NMR spectroscopy. Initial structural data obtained by atomic force microscopy, electron microscopy, and solid-state NMR measurements of Abeta(11-26) fibrils are also presented.     

Enhanced correction methods for hydrogen exchange-mass spectrometric studies of amyloid fibrils

We describe methods for minimization of and correction for artifactual forward and backward exchange occurring during hydrogen exchange-mass spectrometric (HX-MS) studies of amyloid fibrils of the Abeta(1-40) peptide. The quality of the corrected data obtained using published and new correction algorithms is evaluated quantitatively. Using the new correction methods, we have determined that 20.1 +/- 1.4 of the 39 backbone amide hydrogens in Abeta(1-40) exchange with deuteriums in 100 h when amyloid fibrils of this peptide are suspended in D(2)O. These data reinforce our previous conclusions based on uncorrected data that amyloid fibrils contain a rigid protective core structure that involves only about half of the Abeta backbone amides. The methods developed here should be of general value for HX-MS studies of amyloid fibrils and other protein aggregates.     

Photoaffinity cross-linking of Alzheimer's disease amyloid fibrils reveals interstrand contact regions between assembled beta-amyloid peptide subunits

The assembly of the beta-amyloid peptide (Abeta) into amyloid fibrils is essential to the pathogenesis of Alzheimer's disease. Detailed structural information about fibrillogenesis has remained elusive due to the highly insoluble, noncrystalline nature of the assembled peptide. X-ray fiber diffraction, infrared spectroscopy, and solid-state NMR studies performed on fibrils composed of Abeta peptides have led to conflicting models of the intermolecular alignment of beta-strands. We demonstrate here the use of photoaffinity cross-linking to determine high-resolution structural constraints on Abeta monomers within amyloid fibrils. A photoreactive Abeta(1-40) ligand was synthesized by substituting L-p-benzoylphenylalanine (Bpa) for phenylalanine at position 4 (Abeta(1-40) F4Bpa). This peptide was incorporated into synthetic amyloid fibrils and irradiated with near-UV light. SDS-PAGE of dissolved fibrils revealed the light-dependent formation of a covalent Abeta dimer. Enzymatic cleavage followed by mass spectrometric analysis demonstrated the presence of a dimer-specific ion at MH(+) = 1825.9, the predicted mass of a fragment composed of the N-terminal Abeta(1-5) F4Bpa tryptic peptide covalently attached to the C-terminal Abeta(29-40) tryptic peptide. MS/MS experiments and further chemical modifications of the cross-linked dimer led to the localization of the photo-cross-link between the ketone of the Bpa4 side chain and the delta-methyl group of the Met35 side chain. The Bpa4-Met35 intermolecular cross-link is consistent with an antiparallel alignment of Abeta peptides within amyloid fibrils.     

Reversible mechanical unzipping of amyloid beta-fibrils

Amyloid fibrils are self-associating filamentous structures, the deposition of which is considered to be one of the most important factors in the pathogenesis of Alzheimer's disease and various other disorders. Here we used single molecule manipulation methods to explore the mechanics and structural dynamics of amyloid fibrils. In mechanically manipulated amyloid fibrils, formed from either amyloid beta (Abeta) peptides 1-40 or 25-35, beta-sheets behave as elastic structures that can be "unzipped" from the fibril with constant forces. The unzipping forces were different for Abeta1-40 and Abeta25-35. Unzipping was fully reversible across a wide range of stretch rates provided that coupling, via the beta-sheet, between bound and dissociated states was maintained. The rapid, cooperative zipping together of beta-sheets could be an important mechanism behind the self-assembly of amyloid fibrils. The repetitive force patterns contribute to a mechanical fingerprint that could be utilized in the characterization of different amyloid fibrils.     

Prediction of amyloid fibril-forming proteins

In Alzheimer's disease and spongiform encephalopathies proteins transform from their native states into fibrils. We find that several amyloid-forming proteins harbor an alpha-helix in a polypeptide segment that should form a beta-strand according to secondary structure predictions. In 1324 nonredundant protein structures, 37 beta-strands with > or =7 residues were predicted in segments where the experimentally determined structures show helices. These discordances include the prion protein (helix 2, positions 179-191), the Alzheimer amyloid beta-peptide (Abeta, positions 16-23), and lung surfactant protein C (SP-C, positions 12-27). In addition, human coagulation factor XIII (positions 258-266), triacylglycerol lipase from Candida antarctica (positions 256-266), and d-alanyl-d-alanine transpeptidase from Streptomyces R61 (positions 92-106) contain a discordant helix. These proteins have not been reported to form fibrils but in this study were found to form fibrils in buffered saline at pH 7.4. By replacing valines in the discordant helical part of SP-C with leucines, an alpha-helix is found experimentally and by secondary structure predictions. This analogue does not form fibrils under conditions where SP-C forms abundant fibrils. Likewise, when Abeta residues 14-23 are removed or changed to a nondiscordant sequence, fibrils are no longer formed. We propose that alpha-helix/beta-strand-discordant stretches are associated with amyloid fibril formation.