Oligomerization of amyloid Abeta16-22 peptides using hydrogen bonds and hydrophobicity forces

The 16-22 amino-acid fragment of the beta-amyloid peptide associated with the Alzheimer's disease, Abeta, is capable of forming amyloid fibrils. Here we study the aggregation mechanism of Abeta16-22 peptides by unbiased thermodynamic simulations at the atomic level for systems of one, three, and six Abeta16-22 peptides. We find that the isolated Abeta16-22 peptide is mainly a random coil in the sense that both the alpha-helix and beta-strand contents are low, whereas the three- and six-chain systems form aggregated structures with a high beta-sheet content. Furthermore, in agreement with experiments on Abeta16-22 fibrils, we find that large parallel beta-sheets are unlikely to form. For the six-chain system, the aggregated structures can have many different shapes, but certain particularly stable shapes can be identified.     

The structure of the Alzheimer amyloid beta 10-35 peptide probed through replica-exchange molecular dynamics simulations in explicit solvent

The conformational states sampled by the Alzheimer amyloid beta (10-35) (Abeta 10-35) peptide were probed using replica-exchange molecular dynamics (REMD) simulations in explicit solvent. The Abeta 10-35 peptide is a fragment of the full-length Abeta 40/42 peptide that possesses many of the amyloidogenic properties of its full-length counterpart. Under physiological temperature and pressure, our simulations reveal that the Abeta 10-35 peptide does not possess a single unique folded state. Rather, this peptide exists as a mixture of collapsed globular states that remain in rapid dynamic equilibrium with each other. This conformational ensemble is dominated by random coil and bend structures with insignificant presence of an alpha-helical or beta-sheet structure. The 3D structure of Abeta 10-35 is seen to be defined by a salt bridge formed between the side-chains of K28 and D23. This salt bridge is also observed in Abeta fibrils and our simulations suggest that monomeric conformations of Abeta 10-35 contain pre-folded structural motifs that promote rapid aggregation of this peptide.     

Dissociation of Abeta(16-22) amyloid fibrils probed by molecular dynamics

The mechanisms of deposition and dissociation are implicated in the assembly of amyloid fibrils. To investigate the kinetics of unbinding of Abeta(16-22) monomers from preformed fibrils, we use molecular dynamics (MD) simulations and the structures for Abeta(16-22) amyloid fibrils. Consistent with experimental studies, the dissociation of Abeta(16-22) peptides involves two main stages, locked and docked, after which peptides unbind. The lifetime of the locked state, in which a peptide retains fibril-like structure and interactions, extends up to 0.5 micros under normal physiological conditions. Upon cooperative rupture of all fibril-like hydrogen bonds (HBs) with the fibril, a peptide enters a docked state. This state is populated by disordered random coil conformations and its lifetime ranges from approximately 10 to 200 ns. The docked state is stabilized by hydrophobic side chain interactions, while the contribution from HBs is small. Our simulations also suggest that the peptides located on fibril edges may form stable beta-strand conformations distinct from the fibril "bulk". We propose that such edge peptides can act as fibril caps, which impede fibril elongation. Our results indicate that the interactions between unbinding peptides constitute the molecular basis for cooperativity of peptide dissociation. The kinetics of fibril growth is reconstructed from unbinding assuming the reversibility of deposition/dissociation pathways. The relation of in silica dissociation kinetics to experimental observations is discussed.     

Time-dependent DNA condensation induced by amyloid beta-peptide

The major protein component of the amyloid deposition in Alzheimer's disease is a 39-43 residue peptide, amyloid beta (Abeta). Abeta is toxic to neurons, although the mechanism of neurodegeneration is uncertain. Evidence exists for non-B DNA conformation in the hippocampus of Alzheimer's disease brains, and Abeta was reportedly able to transform DNA conformation in vitro. In this study, we found that DNA conformation was altered in the presence of Abeta, and Abeta induced DNA condensation in a time-dependent manner. Furthermore, Abeta sheets, serving as condensation nuclei, were crucial for DNA condensation, and Cu(2+) and Zn(2+) ions inhibited Abeta sheet-induced DNA condensation. Our results suggest DNA condensation as a mechanism of Abeta toxicity.     

Isotope-edited vibrational circular dichroism study reveals a flexible N-terminal structure of islet amyloid peptide (NFGAIL) in amyloid fibril form: A site-specific local structural analysis

The short peptide fragment NFGAIL (IAPf) is a well-known amyloidogenic peptide (22–27), derived from human islet amyloid polypeptide(hIAPP), whose fibrillar structure is often used to better understand the wild-type hIAPP amyloid fibrils, associated with type II diabetes. Despite an extensive study, the fibrillar structure of IAPf at the amino acid residue level is still unclear. Herein, the vibrational circular dichroism(VCD) spectroscopic technique coupled with isotope labelling strategy has been used to study the site-specific local structure of IAPf amyloid fibrils. Two ¹³C labeled IAPfs were designed and used along with unlabelled IAPf to achieve this. The ¹³C labelled (on -C=O) glycine(IAPf-G) and phenylalanine (IAPf-F) residues were introduced into the IAPf sequence separately by replacing natural glycine (residue 24) and phenylalanine (residue 23), respectively. VCD spectral analysis on IAPf-G suggests that IAPf fibrils adopt parallel β-sheet conformation with glycine residues are part of β-sheet and in-register. Unlike IAPf-G, VCD analysis on IAPf-F reveals that phenylalanine residues exist in the turn/hairpin conformation rather than β-sheet region. Both VCD results thus suggest that IAPf amyloid fibril consists of a mixture of β-sheet as a major conformation involving GAIL and turn/hairpin as a minor conformation involving NF rather than an idealized β-sheet involving all the amino acids. While previous studies speculated that the full NFGAIL sequence could participate in the β-sheet formation, the present site-specific structural analysis of IAPf amyloid fibrils at residue level using isotope-edited VCD has gained significant attention. Such residue level information has important implications for understanding the role of NFGAIL sequence in the amyloid fibrillation of hIAPP.     

Molecular dynamics simulation of amyloid beta dimer formation

Recent experiments with amyloid beta (Abeta) peptide indicate that formation of toxic oligomers may be an important contribution to the onset of Alzheimer's disease. The toxicity of Abeta oligomers depends on their structure, which is governed by assembly dynamics. Due to limitations of current experimental techniques, a detailed knowledge of oligomer structure at the atomic level is missing. We introduce a molecular dynamics approach to study Abeta dimer formation. 1), We use discrete molecular dynamics simulations of a coarse-grained model to identify a variety of dimer conformations; and 2), we employ all-atom molecular mechanics simulations to estimate thermodynamic stability of all dimer conformations. Our simulations of a coarse-grained Abeta peptide model predicts 10 different planar beta-strand dimer conformations. We then estimate the free energies of all dimer conformations in all-atom molecular mechanics simulations with explicit water. We compare the free energies of Abeta(1-42) and Abeta(1-40) dimers. We find that 1), dimer conformations have higher free energies compared to their corresponding monomeric states; and 2), the free-energy difference between the Abeta(1-42) and the corresponding Abeta(1-40) dimer conformation is not significant. Our results suggest that Abeta oligomerization is not accompanied by the formation of thermodynamically stable planar beta-strand dimers.     

Molecular dynamics studies of hexamers of amyloid-beta peptide (16-35) and its mutants: influence of charge states on amyloid formation

To study the early stage of amyloid-beta peptide (Abeta) aggregation, hexamers of the wild-type (WT) Abeta(16-35) and its mutants with amyloid-like conformations have been studied by molecular dynamics simulations in explicit water for a total time of 1.7 micros. We found that the amyloid-like structures in the WT oligomers are destabilized by the solvation of ionic D23/K28 residues, which are buried in the fibrils. This means that the desolvation of D23/K28 residues may contribute to the kinetic barrier of aggregation in the early stage. In the E22Q/D23N, D23N/K28Q, and E22Q/D23N/K28Q mutants, hydration becomes much less significant because the mutated residues have neutral amide side-chains. These amide side-chains can form linear cross-strand hydrogen bond chains, or "polar zippers", if dehydrated. These "polar zippers" increase the stability of the amyloid-like conformation, reducing the barrier for the early-stage oligomerization. This is in accord with experimental observations that both the D23/K28 lactamization and the E22Q/D23N mutation promote aggregation. We also found that the E22Q/D23N mutant prefers an amyloid-like conformation that differs from the one found for WT Abeta. This suggests that different amyloid structures may be formed under different conditions.     

Cross-strand pairing and amyloid assembly

Amino acid cross-strand pairing interactions along a beta-sheet surface have been implicated in protein beta-structural assembly and stability, yet the relative contributions have been difficult to evaluate directly. Here we develop the central core sequence of the Abeta peptide associated with Alzheimer's disease, Abeta(16-22), as an experimental system for evaluating these interactions. The peptide allows for internal comparisons between electrostatic and steric interactions within the beta-sheet and an evaluation of these cross-strand pair contributions to beta-sheet registry. A morphological transition from fibers to hollow nanotubes arises from changes in beta-sheet surface complementarity and provides a convenient indicator of the beta-strand strand registry. The intrinsic beta-sequence and pair correlations are critical to regulate secondary assembly. These studies provide evidence for a critical desolvation step that is not present in most models of the nucleation-dependent pathway for amyloid assembly.     

Stability and structure of oligomers of the Alzheimer peptide Abeta16-22: from the dimer to the 32-mer

Several neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases are associated with amyloid fibrils formed by different polypeptides. We probe the structure and stability of oligomers of different sizes of the fragment Abeta(16-22) of the Alzheimer beta-amyloid peptide using atomic-detail molecular dynamics simulations with explicit solvent. We find that only large oligomers form a stable beta-sheet aggregate, the minimum nucleus size being of the order of 8-16 peptides. This effect is attributed to better hydrophobic contacts and a better shielding of backbone-backbone hydrogen bonds from the solvent in bigger assemblies. Moreover, the observed stability of beta-sheet aggregates with a different number of layers can be explained on the basis of their solvent-accessible surface area. Depending on the stacking interface between the sheets, we observe straight or twisted structures, which could be linked to the experimentally observed polymorphism of amyloid fibrils. To compare our 32-mer structure to experimental data, we calculate its x-ray diffraction pattern. Good agreement is found between experimentally and theoretically determined reflections, suggesting that our model indeed closely resembles the structures found in vitro.     

Structural analysis of amyloid beta peptide fragment (25-35) in different microenvironments

Amyloid beta (Abeta) peptides are one of the classes of amphiphilic molecules that on dissolution in aqueous solvents undergo interesting conformational transitions. These conformational changes are known to be associated with their neuronal toxicity. The mechanism of structural transition involved in the monomeric Abeta to toxic assemblage is yet to be understood at the molecular level. Early results indicate that oriented molecular crowding has a profound effect on their assemblage formation. In this work, we have studied how different microenvironments affect the conformational transitions of one of the active amyloid beta-peptide fragments (Abeta(25-35)). Spectroscopic techniques such as CD and Fourier transform infrared spectroscopy were used. It was observed that a stored peptide concentrates on dissolution in methanol adopts a minor alpha-helical conformation along with unordered structures. On changing the methanol concentration in the solvated film form, the conformation switches to the antiparallel beta-sheet structure on the hydrophilic surface, whereas the peptide shows transition from a mixture of helix and unordered structure into predominantly a beta-sheet with minor contribution of helix structure on the hydrophobic surface. Our present investigations indicate that the conformations induced by the different surfaces dictate the gross conformational preference of the peptide concentrate.     

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.     

Dissecting the assembly of Abeta16-22 amyloid peptides into antiparallel beta sheets

Multiple long molecular dynamics simulations are used to probe the oligomerization mechanism of Abeta(16-22) (KLVFFAE) peptides. The peptides, in the monomeric form, adopt either compact random-coil or extended beta strand-like structures. The assembly of the low-energy oligomers, in which the peptides form antiparallel beta sheets, occurs by multiple pathways with the formation of an obligatory alpha-helical intermediate. This observation and the experimental results on fibrillogenesis of Abeta(1-40) and Abeta(1-42) peptides suggest that the assembly mechanism (random coil --> alpha helix --> beta strand) is universal for this class of peptides. In Abeta(16-22) oligomers both interpeptide hydrophobic and electrostatic interactions are critical in the formation of the antiparallel beta sheet structure. Mutations of either hydrophobic or charged residues destabilize the oligomer, which implies that the 16-22 fragments of Arctic (E22G), Dutch (E22Q), and Italian (E22K) mutants are unlikely to form ordered fibrils.     

Structure of amyloid beta fragments in aqueous environments

Conformational studies on amyloid beta peptide (Abeta) in aqueous solution are complicated by its tendency to aggregate. In this study, we determined the atomic-level structure of Abeta(28-42) in an aqueous environment. We fused fragments of Abeta, residues 10-24 (Abeta(10-24)) or 28-42 (Abeta(28-42)), to three positions in the C-terminal region of ribonuclease HII from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). We then examined the structural properties in an aqueous environment. The host protein, Tk-RNase HII, is highly stable and the C-terminal region has relatively little interaction with other parts. CD spectroscopy and thermal denaturation experiments demonstrated that the guest amyloidogenic sequences did not affect the overall structure of the Tk-RNase HII. Crystal structure analysis of Tk-RNase HII(1-197)-Abeta(28-42) revealed that Abeta(28-42) forms a beta conformation, whereas the original structure in Tk-RNase HII(1-213) was alpha helix, suggesting beta-structure formation of Abeta(28-42) within full-length Abeta in aqueous solution. Abeta(28-42) enhanced aggregation of the host protein more strongly than Abeta(10-24). These results and other reports suggest that after proteolytic cleavage, the C-terminal region of Abeta adopts a beta conformation in an aqueous environment and induces aggregation, and that the central region of Abeta plays a critical role in fibril formation. This study also indicates that this fusion technique is useful for obtaining structural information with atomic resolution for amyloidogenic peptides in aqueous environments.     

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.     

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.     

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.     

Comparison of the structures of beta amyloid peptide (25-35) and substance P in trifluoroethanol/water solution

The three-dimensional structure of beta amyloid peptide (25-35) in aqueous solution with 50% (vol/vol) 2,2,2-trifluoroethanol was determined by NMR spectroscopy. Beta amyloid peptide(Abeta) is the major component of senile plaques found in the brain of patient of Alzheimer's disease. Abeta25-35 is biologically active fragment of Abeta and exhibits some sequence homology with the tachykinin family. In this study, we present the structural similarity between Abeta25-35 and substance P which is a member of tachykinin family in order to examine the possibility of sharing pathways mediated by tachykinin receptors. Both peptides have alpha-helical structures in their C-terminal regions and aromatic rings or hydrophobic side chains in the center of the helix protrude outside. These conformational features are expected to be the key for the interaction with the receptors.     

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.     

Probing the role of backbone hydrogen bonding in beta-amyloid fibrils with inhibitor peptides containing ester bonds at alternate positions

Protein-protein interactions are frequently mediated by stable, intermolecular beta-sheets. A number of cytokines and the HIV Protease, for example, dimerize through beta-sheet motifs. Evidence also suggests that the macromolecular assemblies of peptides and proteins in amyloid fibrils are stabilized by intermolecular beta-sheets. In this paper, we report that interfering with the backbone hydrogen bonding of an amyloidgenic peptide (Abeta16-20) by replacing amide bonds with ester bonds prevents the aggregation of the peptide. The ester bonds were incorporated in an alternating fashion so that the peptide presents two unique hydrogen bonding faces when arrayed in an extended, beta-strand conformation; one face of the peptide has normal hydrogen bonding capabilities, but the other face is missing amide protons and its ability to hydrogen bond is severely limited. Analytical ultracentrifugation experiments demonstrate that this ester peptide, Abeta16-20e, is predominantly monomeric under solution conditions, unlike the fibril-forming Abeta16-20 peptide. Abeta16-20e also inhibits the aggregation of the Abeta1-40 peptide and disassembles preformed Abeta1-40 fibrils. These results suggest that backbone hydrogen bonding is critical for the assembly of amyloid fibrils.