A comparative study of amyloid fibril formation by residues 15-19 of the human calcitonin hormone: a single beta-sheet model with a small hydrophobic core

Experimentally, the human calcitonin hormone (hCT) can form highly stable amyloid protofibrils. Further, a peptide consisting of hCT residues 15-19, DFNKF, was shown to create highly ordered fibrils, similar to those formed by the entire hormone sequence. However, there are limited experimental data regarding the detailed 3D arrangement of either of these fibrils. We have modeled the DFNKF protofibril, using molecular dynamics simulations. We tested the stabilities of single sheet and of various multi sheet models. Remarkably, our most ordered and stable model consists of a parallel-stranded, single beta-sheet with a relatively insignificant hydrophobic core. We investigate the chemical and physical interactions responsible for the high structural organization of this single beta-sheet amyloid fibril. We observe that the most important chemical interactions contributing to the stability of the DFNKF organization are electrostatic, specifically between the Lys and the C terminus, between the Asp and N terminus, and a hydrogen bond network between the Asn side-chains of adjacent strands. Additionally, we observe hydrophobic and aromatic pi stacking interactions. We further simulated truncated filaments, FNKF and DFNK. Our tetra-peptide mutant simulations assume models similar to the penta-peptide. Experimentally, the FNKF does not create fibrils while DFNK does, albeit short and less ordered than DFNKF. In the simulations, the FNKF system was less stable than the DFNK and DFNKF. DFNK also lost many of its original interactions becoming less organized, however, many contacts were maintained. Thus, our results emphasize the role played by specific amino acid interactions. To further study specific interactions, we have mutated the penta-peptide, simulating DANKF, DFNKA and EFNKF. Here we describe the model, its relationship to experiment and its implications to amyloid organization.     

Sequence dependence of amyloid fibril formation: insights from molecular dynamics simulations

The clarification of the physico-chemical determinants underlying amyloid deposition is critical for our understanding of misfolding diseases. With this purpose we have performed a systematic all-atom molecular dynamics (MD) study of a series of single point mutants of the de novo designed amyloidogenic peptide STVIIE. Sixteen different 50ns long simulations using explicit solvent have been carried out starting from four different conformations of a polymeric six-stranded beta-sheet. The simulations have provided evidence for the influence of a small number of site-specific hydrophobic interactions on the packing and stabilization of nascent aggregates, as well as the interplay between side-chain interactions and the net charge of the molecule on the strand arrangement of polymeric beta-sheets. This MD analysis has also shed light into the origin of the position dependence on mutation of beta-sheet polymerization that was found experimentally for this model system. Our results suggest that MD can be applied to detect critical positions for beta-sheet aggregation within a given amyloidogenic stretch. Studies similar to the one presented here can guide site-directed mutations or the design of drugs that specifically disrupt the key stabilizing interactions of beta-sheet aggregates.     

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.     

The stability of cylindrin β‐barrel amyloid oligomer models—A molecular dynamics study

Small‐soluble amyloid oligomers are believed to play a significant role in the pathology of amyloid diseases. Recently, the atomic structure of a toxic oligomer formed by an 11 residue and its tandem repeat was found to have an out‐off register antiparallel β‐strands in the shape of a β‐barrel. In the present article we investigate the effect of mutations in the hydrophobic cores on the structure and dynamic of the β‐barrels using all atom multiple molecular dynamics simulations with an explicit solvent. Extending previous experiments with molecular dynamics simulations we systematically test how stability and formation of cylindrin depends on the interplay between hydrophobicity and steric effects of the core residues. We find that strong hydrophobic interactions between geometrically fitting residues keep the strands (both in register and out‐off‐register interface) in close proximity, which in turn stabilizes the side‐chain and main‐chain hydrogen bonds, and the salt bridges on the outer surface along the weak out‐of‐register interface. Our simulations also indicate presence of water molecules in the hydrophobic interior of the cylindrin β‐barrel.Proteins 2013. © 2013 Wiley Periodicals, Inc.     

Inhibition of islet amyloid polypeptide fibril formation: a potential role for heteroaromatic interactions

The formation of amyloid fibril is associated with major human diseases, including Alzheimer's disease, prion diseases, and type 2 diabetes. Methods for efficient inhibition of amyloid fibril formation are therefore highly clinically important. A principal approach for the inhibition of amyloid formation is based on the use of modified molecular recognition elements. Here, we demonstrate efficient inhibition of amyloid formation of the type 2 diabetes-related human islet amyloid polypeptide (hIAPP) by a modified aromatic peptide fragment and a small aromatic polyphenol molecule. A molecular recognition assay using peptide array analysis suggested that molecular recognition between hIAPP and its core amyloidogenic module is mediated by aromatic rather than hydrophobic interactions. To study the possible effect of aromatic interactions on inhibition of hIAPP fibril formation, we have used peptide and small molecule inhibitors. The addition of a nonamyloidogenic peptide analogue of the core module NFGAILSS, in which phenylalanine was substituted with tyrosine (NYGAILSS), resulted in substantial inhibition of fibril formation by hIAPP. The inhibition was significantly stronger than the one achieved using a beta-sheet breaker-conjugated peptide NFGAILPP. On the basis of the molecular arrangement of the tyrosine-phenylalanine interaction, we suggest that the inhibition stems from the geometrical constrains of the heteroaromatic benzene-phenol interaction. In line with this notion, we demonstrate remarkable inhibition of hIAPP fibril formation and cytotoxicity toward pancreatic beta-cells by a small polyphenol molecule, the nontoxic phenol red compound. Taken together, our results provide further experimental support for the potential role of aromatic interactions in amyloid formation and establish a novel approach for its inhibition.     

The role of Phe in the formation of well-ordered oligomers of amyloidogenic hexapeptide (NFGAIL) observed in molecular dynamics simulations with explicit solvent

We observed fast aggregation of partially ordered oligomers in an earlier simulation study of an amyloidogenic hexapeptide NFGAIL. In this work, the nucleation of highly ordered oligomers was further investigated by a combined total of 960 ns molecular dynamics simulations with explicit solvent on NFGAIL and its nonamyloidogenic mutant NAGAIL. In these simulations, four dimer subunits that each was constrained by harmonic forces as a two-strand beta-sheet were used to enhance the rate of formation. It was found that a critical role played by the aromatic residue Phe was to direct the stacking of beta-sheets to form ordered multilayer aggregates. We also found that many molecular arrangements of the peptide satisfied the "cross-beta-structure", a hallmark of amyloid fibrils. The tendency for the peptide to form either parallel or antiparallel beta-sheet was comparable, as was the tendency for the beta-sheets to stack either in parallel or antiparallel orientation. Overall, approximately 85% of the native hexapeptide formed octamers. The fact that only 8% of the octamers were well-ordered species suggests that the dissociation of the disordered oligomers be the rate-limiting step in the formation of highly ordered oligomers. Among the well-ordered subunit pairs, about half was formed by the beta-sheet extension along the main-chain hydrogen-bond direction, whereas the other half was formed by the beta-sheet stacking. Hence, a delicate balance between intersheet and intrasheet interactions appeared to be crucial in the formation of a highly ordered nucleus of amyloid fibrils. The disordered oligomers were mainly stabilized by nonspecific hydrophobic interactions, whereas the well-ordered oligomers were further stabilized by cross-strand hydrogen bonds and favorable side-chain stacking.     

A cylinder-shaped double ribbon structure formed by an amyloid hairpin peptide derived from the beta-sheet of murine PrP: an X-ray and molecular dynamics simulation study

A structural model of the murine PrP small beta-sheet was obtained by synthesizing the RGYMLGSADPNGNQVYYRG peptide comprising the two beta-strands 127-133 and 159-164 linked by a four-residue sequence of high turn propensity. The DPNG turn sequence is a "short circuit" replacing the original protein sequence between the two strands. This 19-residue peptide spontaneously forms very long single fibrils as observed by electron microscopy. The X-ray diffraction patterns of a partially oriented sample reveals an average arrangement of the hairpin peptides into a structure which can be geometrically approximated by an empty-core cylinder. The hairpins are oriented perpendicular to the cylinder axis and a 130 A helix period is observed. Based on X-ray diffraction constraints and on more indirect general protein structure considerations, a precise and consistent fibril model was built. The structure consists of two beta-sheet ribbons wound around a cylinder and assembled into a single fibril with a hairpin orientation perpendicular to the fibril axis. Subsequent implicit and explicit solvent molecular dynamics simulations provided the final structure at atomic resolution and further insights into the stabilizing interactions. Particularly important are the zipper-like network of polar interactions between the edges of the two ribbons, including the partially buried water molecules. The hydrophobic core is not optimally compact explaining the low density of this region seen by X-ray diffraction. The present findings provide also a simple model for further investigating the sequence-stability relationship using a mutational approach with a quasi-independent consideration of the polar and apolar interactions.     

The stability and dynamics of the human calcitonin amyloid peptide DFNKF

The stability and dynamics of the human calcitonin-derived peptide DFNKF (hCT(15-19)) are studied using molecular dynamics (MD) simulations. Experimentally, this peptide is highly amyloidogenic and forms fibrils similar to the full length calcitonin. Previous comparative MD studies have found that the parallel beta-stranded sheet is a stable organization of the DFNKF protofibril. Here, we probe the stability and dynamics of the small parallel DFNKF oligomers. The results show that even small DFNKF oligomers, such as trimers and tetramers, are stable for a sufficient time in the MD simulations, indicating that the crucial nucleus seed size for amyloid formation can be quite small. The simulations also show that the stability of DFNKF oligomers increases with their sizes. The small but stable seed may reflect the experimental rapid formation of the DFNKF fibrils. Further, a noncooperative process of parallel beta-sheet formation from the out-of-register trimer is observed in the simulations. In general, the residues of DFNKF peptides near the N-/C-termini are more flexible, whereas the interior residues are more stable. Simulations of mutants and capped peptides show that both interstrand hydrophobic and electrostatic interactions play important roles in stabilizing the DFNKF parallel oligomers. This study provides insights into amyloid formation.     

The sequence dependence of fiber organization. A comparative molecular dynamics study of the islet amyloid polypeptide segments 22-27 and 22-29

Amyloid fiber formation and the possible polymorphism of molecular arrangements depend on the polypeptide length and composition. Here, we seek the chemical clues underlying these processes. Our starting point is based on the experimental observation that some short peptide segments are able to develop fibers that are very similar to those of their original parent proteins. We focus our study on the NFGAILSS peptide, derived from the human islet amyloid polypeptide (residues 22-29). This peptide turned out to be a perfect example, illustrating the fact that the amyloid microscopic organization is highly complex, rather than simply involving hydrogen bond formation. Furthermore, obtaining a reliable molecular model has allowed us to analyze the differences between the amyloid structure we have obtained for this peptide and that obtained for the previously studied, two residues shorter, segment (residues 22-27, NFGAIL). This comparative study yields some clues about chemical events that govern the aggregation of proteins into oriented fibers, such as molecular packing between sheets and the degree of interaction specificity. We characterize the important role played by the hydrophobic and aromatic residues in the inter-sheet association and present new approaches toward the understanding of the nature of events that are likely to take place during fibril formation. These include analysis of interaction patterns derived from specific sheet-associated packing.     

Phenol red interacts with the protofibril-like oligomers of an amyloidogenic hexapeptide NFGAIL through both hydrophobic and aromatic contacts

Amyloid-associated diseases affect millions of people worldwide. Phenol red exhibits modest inhibition toward fibril formation of human Islet amyloid polypeptide (hIAPP) and its toxicity, which is associated with type II diabetes mellitus. However, the molecular level mechanisms of interactions remain elusive. The binding of phenol red molecules to the protofibrils of an amyloidogenic fragment (NFGAIL) of hIAPP has been investigated by molecular dynamics simulations with explicit solvent. The phenol red molecules were observed to bind primarily along either beta-sheet stacking or beta-strand directions. Through its three aromatic rings, the phenol red molecule preferentially interacted with the hydrophobic side chains of Phe, Leu, and Ile; and the polar sulfone and hydroxyl groups were mainly exposed in solvent. Thus, phenol red improves the solubility of the early protofibrils and represses further growth. Interestingly, there was no obvious preference toward the aromatic Phe residue in comparison to the hydrophobic Leu or Ile residues. The lack of binding along the hydrogen bond direction indicates that phenol red does not directly block the beta-sheet extension. Further free energy analysis suggested that a phenol red analog may potentially improve the binding affinity.     

New insights into the mechanism of Alzheimer amyloid-beta fibrillogenesis inhibition by N-methylated peptides

Alzheimer's disease is a debilitating neurodegenerative disorder associated with the abnormal self-assembly of amyloid-beta (Abeta) peptides into fibrillar species. N-methylated peptides homologous to the central hydrophobic core of the Abeta peptide are potent inhibitors of this aggregation process. In this work, we use fully atomistic molecular dynamics simulations to study the interactions of the N-methylated peptide inhibitor Abeta16-20m (Ac-Lys(16)-(Me)Leu(17)-Val(18)-(Me)Phe(19)-Phe(20)-NH(2)) with a model protofilament consisting of Alzheimer Abeta16-22 peptides. Our simulations indicate that the inhibitor peptide can bind to the protofilament at four different sites: 1), at the edge of the protofilament; 2), on the exposed face of a protofilament layer; 3), between the protofilament layers; and 4), between the protofilament strands. The different binding scenarios suggest several mechanisms of fibrillogenesis inhibition: 1), fibril inhibition of longitudinal growth (in the direction of monomer deposition); 2), fibril inhibition of lateral growth (in the direction of protofilament assembly); and 3), fibril disassembly by strand removal and perturbation of the periodicity of the protofilament (disruption of fibril morphology). Our simulations suggest that the Abeta16-20m inhibitor can act on both prefibrillar species and mature fibers and that the specific mechanism of inhibition may depend on the structural nature of the Abeta aggregate. Disassembly of the fibril can be explained by a mechanism through which the inhibitor peptides bind to disaggregated or otherwise free Abeta16-22 peptides in solution, leading to a shift in the equilibrium from a fibrillar state to one dominated by inhibitor-bound Abeta16-22 peptides.     

Insights into the determinants of beta-sheet stability: 1H and 13C NMR conformational investigation of three-stranded antiparallel beta-sheet-forming peptides.

In a previous study we designed a 20-residue peptide able to adopt a significant population of a three-stranded antiparallel beta-sheet in aqueous solution (de Alba et al. [1999]Protein Sci.8, 854-865). In order to better understand the factors contributing to beta-sheet folding and stability we designed and prepared nine variants of the parent peptide by substituting residues at selected positions in its strands. The ability of these peptides to form the target motif was assessed on the basis of NMR parameters, in particular NOE data and 13Calpha conformational shifts. The populations of the target beta-sheet motif were lower in the variants than in the parent peptide. Comparative analysis of the conformational behavior of the peptides showed that, as expected, strand residues with low intrinsic beta-sheet propensities greatly disfavor beta-sheet folding and that, as already found in other beta-sheet models, specific cross-strand side chain-side chain interactions contribute to beta-sheet stability. More interestingly, the performed analysis indicated that the destabilization effect of the unfavorable strand residues depends on their location at inner or edge strands, being larger at the latter. Moreover, in all the cases examined, favorable cross-strand side chain-side chain interactions were not strong enough to counterbalance the disfavoring effect of a poor beta-sheet-forming residue, such as Gly.     

Detecting the inter-peptide arrangement and maturation process of transthyretin (105-115) amyloid fibril using a FRET pair with short Förster distance

Transthyretin (TTR) is an amyloidogenic protein involved in many mental diseases. The peptide derived from TTR (105-115) has been widely studied as a model peptide for understanding the mechanism of amyloid fibril formation. However, the detailed arrangement of this peptide in amyloid fibril is still unclear. We have studied the amyloid fibril formation process of TTR (105-115) by introducing a pair of FRET probes into the peptide with a dansyl group at the N-terminal and a tryptophan residue at the C-terminal. Our experiment demonstrated that the strands of TTR (105-115) in the same beta-sheet may be parallel and the mating sheets may be anti-parallel to each other in the amyloid fibril. The kinetics followed by FRET and EM indicated for a possible intermediate state and the distance between sheets became shorter when the intermediate amyloid fibril turns into a more matured form.     

Modulation of fibrillation of hIAPP core fragments by chemical modification of the peptide backbone

The well-ordered cross β-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form β-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. β-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6-10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe-Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the -NH- groups has been replaced by -CH(2)-) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone.     

Molecular interactions of Alzheimer's Aβ protofilaments with lipid membranes

Amyloid fibrils and peptide oligomers play central roles in the pathology of Alzheimer's disease, type 2 diabetes, Parkinson's disease, Huntington's disease, and prion-related disease. Here, we investigate the molecular interactions between preformed amyloid β (Aβ) molecular protofilaments and lipid bilayer membranes, in the presence of explicit water molecules, using computational models and all-atom molecular dynamics. These interactions play an important role in the stability and function of both Aβ fibrils and the adjacent cellular membrane. Taking advantage of the symmetry-related and directional properties of the protofilaments, we build models that cover several relative protofilament-membrane orientations. Our molecular dynamics simulations reveal the relative contributions of different structural elements to the dynamics and stability of Aβ protofilament segments near membranes, and the first steps in the mechanism of fibril-membrane interactions. During this process, we observe a significant alteration of the side-chain contact pattern in protofilaments, although a fraction of the characteristic β-sheet content is preserved. As a major driving force, we identify the electrostatic interactions between Aβ charged side chains, including E22, D23, and K28, and lipid headgroups. Together with hydrogen bonding with atoms from lipid headgroups, these interactions can facilitate the penetration of hydrophobic C-terminal amino acids through the lipid headgroup region, which can finally lead both to further loss of the initial fibril structure and to local membrane-thinning effects. Our results may guide new experiments that could test the extent to which the structural features of water-formed amyloid fibrils are preserved, lost, or reshaped by membrane-mediated interactions.     

Structural features of the Abeta amyloid fibril elucidated by limited proteolysis

Although the gross morphology of amyloid fibrils is fairly well understood, very little is known about how the constituent polypeptides fold within the amyloid folding motif. In the experiments reported here, we used trypsin and chymotrypsin to conduct limited proteolysis studies on synthetic amyloid fibrils composed of the Alzheimer's disease peptide Abeta(1-40). In both reactions, the extreme N-terminal proteolytic fragment is released from fibrils as rapidly as it is from the Abeta monomer, while other proteolytic fragments are generated much more slowly. Furthermore, aggregated material isolated by centrifugation of intermediate digestion time points from both proteases contains, in addition to full-length material, peptides that possess mature C-termini but truncated N-termini. These data strongly suggest that the N-terminal region of Abeta is not involved in the beta-sheet network of the amyloid fibril, while the C-terminus is essentially completely engaged in protective-presumably beta-sheet-structure. In both digests, release of the extreme N-terminal fragments of Abeta(1-40) reaches plateau values corresponding to about 80% of the total available Abeta. This suggests that there are two classes of peptides in the fibril: while the majority of Abeta molecules have an exposed N-terminus, about 20% of the peptides have an N-terminus that is protected from proteolysis within the fibril structure. The most likely cause of this heterogeneity is the lateral association of protofilaments into the fibril structure, which would be expected to generate a unique environment for those Abeta N-termini located at protofilament packing interfaces and/or in the interior core region between the packed protofilaments. This suggests that the N-terminal region of Abeta, while not directly involved in the beta-sheet network of the fibril, may contribute to fibril stability by participating in protofilament packing.     

Modulation of fibrillation of hIAPP core fragments by chemical modification of the peptide backbone

The well-ordered cross β-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form β-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. β-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6-10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe-Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the − NH − groups has been replaced by − CH₂−) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone.     

Alzheimer Aβ peptide interactions with lipid membranes

Fibrillar aggregates of misfolded amyloid proteins are involved in a variety of diseases such as Alzheimer disease (AD), type 2 diabetes, Parkinson, Huntington and prion-related diseases. In the case of AD amyloid β (Aβ) peptides, the toxicity of amyloid oligomers and larger fibrillar aggregates is related to perturbing the biological function of the adjacent cellular membrane. We used atomistic molecular dynamics (MD) simulations of Aβ_9–40 fibrillar oligomers modeled as protofilament segments, including lipid bilayers and explicit water molecules, to probe the first steps in the mechanism of Aβ-membrane interactions. Our study identified the electrostatic interaction between charged peptide residues and the lipid headgroups as the principal driving force that can modulate the further penetration of the C-termini of amyloid fibrils or fibrillar oligomers into the hydrophobic region of lipid membranes. These findings advance our understanding of the detailed molecular mechanisms and the effects related to Aβ-membrane interactions, and suggest a polymorphic structural character of amyloid ion channels embedded in lipid bilayers. While inter-peptide hydrogen bonds leading to the formation of β-strands may still play a stabilizing role in amyloid channel structures, these may also present a significant helical content in peptide regions (e.g., termini) that are subject to direct interactions with lipids rather than with neighboring Aβ peptides.     

Structural elements regulating amyloidogenesis: a cholinesterase model system

Polymerization into amyloid fibrils is a crucial step in the pathogenesis of neurodegenerative syndromes. Amyloid assembly is governed by properties of the sequence backbone and specific side-chain interactions, since fibrils from unrelated sequences possess similar structures and morphologies. Therefore, characterization of the structural determinants driving amyloid aggregation is of fundamental importance. We investigated the forces involved in the amyloid assembly of a model peptide derived from the oligomerization domain of acetylcholinesterase (AChE), AChE(586-599), through the effect of single point mutations on beta-sheet propensity, conformation, fibrilization, surfactant activity, oligomerization and fibril morphology. AChE(586-599) was chosen due to its fibrilization tractability and AChE involvement in Alzheimer's disease. The results revealed how specific regions and residues can control AChE(586-599) assembly. Hydrophobic and/or aromatic residues were crucial for maintaining a high beta-strand propensity, for the conformational transition to beta-sheet, and for the first stage of aggregation. We also demonstrated that positively charged side-chains might be involved in electrostatic interactions, which could control the transition to beta-sheet, the oligomerization and assembly stability. Further interactions were also found to participate in the assembly. We showed that some residues were important for AChE(586-599) surfactant activity and that amyloid assembly might preferentially occur at an air-water interface. Consistently with the experimental observations and assembly models for other amyloid systems, we propose a model for AChE(586-599) assembly in which a steric-zipper formed through specific interactions (hydrophobic, electrostatic, cation-pi, SH-aromatic, metal chelation and polar-polar) would maintain the beta-sheets together. We also propose that the stacking between the strands in the beta-sheets along the fiber axis could be stabilized through pi-pi interactions and metal chelation. The dissection of the specific molecular recognition driving AChE(586-599) amyloid assembly has provided further knowledge on such poorly understood and complicated process, which could be applied to protein folding and the targeting of amyloid diseases.     

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

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