Monomeric Amyloid Beta Research Articles

Investigating How Peptide Length and a Pathogenic Mutation Modify the Structural Ensemble of Amyloid Beta Monomer

The aggregation of amyloid beta (Aβ) peptides plays an important role in the development of Alzheimer's disease. Despite extensive effort, it has been difficult to characterize the secondary and tertiary structure of the Aβ monomer, the starting point for aggregation, due to its hydrophobicity and high aggregation propensity. Here, we employ extensive molecular dynamics simulations with atomistic protein and water models to determine structural ensembles for Aβ42, Aβ40, and Aβ42-E22K (the Italian mutant) monomers in solution. Sampling of a total of >700 microseconds in all-atom detail with explicit solvent enables us to observe the effects of peptide length and a pathogenic mutation on the disordered Aβ monomer structural ensemble. Aβ42 and Aβ40 have crudely similar characteristics but reducing the peptide length from 42 to 40 residues reduces β-hairpin formation near the C-terminus. The pathogenic Italian E22K mutation induces helix formation in the region of residues 20–24. This structural alteration may increase helix-helix interactions between monomers, resulting in altered mechanism and kinetics of Aβ oligomerization.

The Relationship Between the Number of Residues in the Dynamics and Stability of the A-Beta Amyloid, a Molecular Dynamics Study

Alzheimer's disease has been identified as a progressive, neurodegenerative disorder associated with protein misfolding due to the aggregation of monomeric beta-amyloid proteins (A-beta) to form fibrillar plaques. Experimental attempts to purify and chemically analyze the structure of A-beta protofibrils and to elucidate the mechanism of fibril formation have yet to reveal much about the molecular etiology of AD, due to the low solubility and non-crystalline nature of A-beta. Experimentally, it is shown that the 43-residue strand of A-beta has higher cytotoxicity than the 40 or 42 strands. To better understand the relationship between the number of residue and the fibril-forming propensity of the peptide we have ran several all atom explicit water molecular dynamics simulations of A-beta strands of 40, 42, and 43 residues starting from their NMR structures. Overall, our simulations shows a transition of existing alpha helices to 3(10)- or pi-helices in agreement with experimental data. Our simulation of A-beta 40 and 42 residue strands confirms the substantial shift of secondary structure formation due to the additional two residues. However, it is interesting that the addition of the 43rd residue (threonine) only cause a small secondary structure change in the A-beta strand of 42 residues. Therefore it seems that the residues 41 and 42 are more important in secondary structure formation than the residue 43.

Wheat germ agglutinin enhanced cerebral uptake of anti-Aβ antibody after intranasal administration in 5XFAD mice

Alzheimer's disease (AD) is the 6th leading cause of death in United States afflicting >5million Americans. This number is estimated to triple by the middle of the century if effective treatments are not discovered. Current therapy for AD is mainly symptomatic. Effective disease-modifying treatments are needed that would eliminate the cause rather than the symptoms of the disease. Polymerization of monomeric beta-amyloid peptide (Aβ) into dimers, soluble oligomers and insoluble fibrils is considered the prime causative factor in triggering AD pathogenesis. Based on these facts, removal/reduction of Aβ has gained importance as a primary therapeutic target in treating the cause of the disease. In that regard, passive immunotherapy with direct delivery of anti-Aβ antibodies to the brain has shown great promise, but awaits the challenge of overcoming greater influx of anti-Aβ antibody into the brain. This investigation was undertaken to maximize direct delivery of immunotherapeutics to the brain by using wheat germ agglutinin (WGA) as a novel axonal transporter-carrier to be conjugated with anti-Aβ antibody (6E10) raised against EFRHDS 3–8 amino acid (aa) epitopes of Aβ known to react with 1–16 aa residues of mono-/di-/oligomeric Aβ. This is the first report showing the use of WGA as an efficient axonal transporter carrier that not only enhanced the influx of anti-Aβ antibody directly into the brain but also resulted in greater reduction of cerebral Aβ compared to the unconjugated anti-Aβ antibody delivered intranasally in Alzheimer's 5XFAD model.

Role of water in protein aggregation and amyloid polymorphism.

A variety of neurodegenerative diseases are associated with amyloid plaques, which begin as soluble protein oligomers but develop into amyloid fibrils. Our incomplete understanding of this process underscores the need to decipher the principles governing protein aggregation. Mechanisms of in vivo amyloid formation involve a number of coconspirators and complex interactions with membranes. Nevertheless, understanding the biophysical basis of simpler in vitro amyloid formation is considered important for discovering ligands that preferentially bind regions harboring amyloidogenic tendencies. The determination of the fibril structure of many peptides has set the stage for probing the dynamics of oligomer formation and amyloid growth through computer simulations. Most experimental and simulation studies, however, have been interpreted largely from the perspective of proteins: the role of solvent has been relatively overlooked in oligomer formation and assembly to protofilaments and amyloid fibrils. In this Account, we provide a perspective on how interactions with water affect folding landscapes of amyloid beta (Aβ) monomers, oligomer formation in the Aβ16-22 fragment, and protofilament formation in a peptide from yeast prion Sup35. Explicit molecular dynamics simulations illustrate how water controls the self-assembly of higher order structures, providing a structural basis for understanding the kinetics of oligomer and fibril growth. Simulations show that monomers of Aβ peptides sample a number of compact conformations. The formation of aggregation-prone structures (N*) with a salt bridge, strikingly similar to the structure in the fibril, requires overcoming a high desolvation barrier. In general, sequences for which N* structures are not significantly populated are unlikely to aggregate. Oligomers and fibrils generally form in two steps. First, water is expelled from the region between peptides rich in hydrophobic residues (for example, Aβ16-22), resulting in disordered oligomers. Then the peptides align along a preferred axis to form ordered structures with anti-parallel β-strand arrangement. The rate-limiting step in the ordered assembly is the rearrangement of the peptides within a confining volume. The mechanism of protofilament formation in a polar peptide fragment from the yeast prion, in which the two sheets are packed against each other and create a dry interface, illustrates that water dramatically slows self-assembly. As the sheets approach each other, two perfectly ordered one-dimensional water wires form. They are stabilized by hydrogen bonds to the amide groups of the polar side chains, resulting in the formation of long-lived metastable structures. Release of trapped water from the pore creates a helically twisted protofilament with a dry interface. Similarly, the driving force for addition of a solvated monomer to a preformed fibril is water release; the entropy gain and favorable interpeptide hydrogen bond formation compensate for entropy loss in the peptides. We conclude by offering evidence that a two-step model, similar to that postulated for protein crystallization, must also hold for higher order amyloid structure formation starting from N*. Distinct water-laden polymorphic structures result from multiple N* structures. Water plays multifarious roles in all of these protein aggregations. In predominantly hydrophobic sequences, water accelerates fibril formation. In contrast, water-stabilized metastable intermediates dramatically slow fibril growth rates in hydrophilic sequences.

Simulation of the Oligomerization Pathway for Different Alloforms of the Amyloid Beta Protein Related to Alzheimer's Disease

Numerous recent experimental studies suggest that oligomers of amyloid beta protein (Aβ) are the principal neurotoxic agents in Alzheimer's disease. In fact, these oligomers interact with receptors, metal-ions, cell membrane and synapses causing neuronal dysfunction or even death. They also have the ability to form pores disrupting neurons homeostasis. Details of these mechanisms remain largely unknown at the molecular level and clearly structural information on these oligomers would provide crucial breakthrough in the understanding of their neurotoxicity. Yet, it is difficult to isolate specific Aβ oligomers and characterize their morphologies experimentally since they are prone to aggregation and they exist in equilibrium with monomers, fibrils and other orders of oligomers. Thus, to complement experimental results, numerical simulations are used to get insights on the molecular mechanisms of Aβ aggregation. We present our results on the amyloid beta monomer and dimer using an efficient simulation protocol that is introduced in details. We study specifically three alloforms of Aβ: Aβ1-40, which is less prone to aggregation; Aβ1-42, which is more toxic; and Aβ1-40(D23N), which is a single point mutation causing early onset Alzheimer's disease. Our results, which are in good agreement with experiment and help understand a number of observations, show that small variation in the sequence results in important morphological changes related to the different oligomerization pathways of these alloforms.

Kinetics of Amyloid-Beta Monomer to Oligomer Exchange by NMR Relaxation

Kinetics of Amyloid-Beta Monomer to Oligomer Exchange by NMR Relaxation

Electric Field Effects on Short Fibrils of Aβ Amyloid Peptides.

Amyloid fibrils are highly ordered protein aggregates, which are associated with many neurodegenerative diseases. The assembling dynamics of monomeric beta-amyloid peptides, Aβ, into small aggregates (and then into long fibrils) is still debated and has become a hot topic. In this study, we conducted molecular dynamics simulations in explicit water of small Aβ protofibrils (from monomer to pentamer) under the perturbation of an externally applied electric field with the aim of investigating the fundamental molecular interactions involved in the aggregation mechanism. Dynamics of small adducts of Aβ(16-42) in the presence of an electric field, which was shown before to accelerate the conformational change of a single molecule, indicate that the structural resilience increases with the number of molecules in the aggregate. In particular, for 50 ns, the pentamer shows an enhanced stability in secondary structure, number of hydrogen bonds, and number of salt bridges, even in the presence of the field perturbation. The resilience to the field perturbation is linked to the variation of the induced dipole moment of the aggregates that tends to level off very rapidly with the growing number of molecules, thereby reducing the energy available per molecule to produce structural changes. The results also show that in the presence of the field the stability of the hydrophobic second β-sheet (β2, residues 31-42) is higher than that of the first one (β1, residues 18-26). In particular, we identify Gly33, Gly37, and Met35 as the most important residues that stabilize the intermolecular packing and may act as nucleation sites for fibrillization. Furthermore, dynamics of the full-length Aβ(1-42) pentameric aggregate, which include the highly charged random coil residues 1-15, confirmed the key role of the second hydrophobic core in the protofibril structure.

Analysis of monomeric Aβ (1–40) peptide by capillary electrophoresis

A method was developed to characterize and quantify preparations of monomeric beta-amyloid (Abeta) peptide using capillary electrophoresis (CE) with UV absorbance detection. The detection limit for Abeta monomer using this method was 0.5 microM (19 pg). The self-assembly of Abeta to form amyloid fibrils is closely linked to Alzheimer's disease and is the subject of intense investigations. Consistent preparation of Abeta monomer samples at known concentrations and free of aggregates is a significant challenge for researchers studying the mechanism of Abeta fibril formation and searching for small molecules that inhibit Abeta fibril formation. Samples of Abeta monomer are known to sometimes contain pre-existing aggregates that can affect the kinetics and structure of amyloid fibrils. The CE method presented here showed that some of the monomeric Abeta samples prepared for this study contained a species producing a second peak (in addition to the major monomer peak). The aggregation was monitored using a thioflavin T fluorescence assay, and the resulting fibrils were characterized by transmission electron microscopy. Monomer samples containing the additional peak based on CE analysis were shown to aggregate more rapidly than monomer samples that were free of this putative Abeta aggregate peak.

A sensitive assay measuring the binding of amyloid-beta monomers to aggregates reveals the potential of ELND005 (scylloinositol) to reduce and reverse amyloid accumulation

A sensitive assay measuring the binding of amyloid-beta monomers to aggregates reveals the potential of ELND005 (scylloinositol) to reduce and reverse amyloid accumulation

Comparative Molecular Dynamics Studies of Wild-Type and Oxidized Forms of Full-Length Alzheimer Amyloid β-Peptides Aβ(1−40) and Aβ(1−42)

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

Benchmarking implicit solvent folding simulations of the amyloid beta(10-35) fragment.

A pathogenetic feature of Alzhemier disease is the aggregation of monomeric beta-amyloid proteins (Abeta) to form oligomers. Usually these oligomers of long peptides aggregate on time scales of microseconds or longer, making computational studies using atomistic molecular dynamics models prohibitively expensive and making it essential to develop computational models that are cheaper and at the same time faithful to physical features of the process. We benchmark the ability of our implicit solvent model to describe equilibrium and dynamic properties of monomeric Abeta(10-35) using all-atom Langevin dynamics (LD) simulations, since Alphabeta(10-35) is the only fragment whose monomeric properties have been measured. The accuracy of the implicit solvent model is tested by comparing its predictions with experiment and with those from a new explicit water MD simulation, (performed using CHARMM and the TIP3P water model) which is approximately 200 times slower than the implicit water simulations. The dependence on force field is investigated by running multiple trajectories for Alphabeta(10-35) using the CHARMM, OPLS-aal, and GS-AMBER94 force fields, whereas the convergence to equilibrium is tested for each force field by beginning separate trajectories from the native NMR structure, a completely stretched structure, and from unfolded initial structures. The NMR order parameter, S2, is computed for each trajectory and is compared with experimental data to assess the best choice for treating aggregates of Alphabeta. The computed order parameters vary significantly with force field. Explicit and implicit solvent simulations using the CHARMM force fields display excellent agreement with each other and once again support the accuracy of the implicit solvent model. Alphabeta(10-35) exhibits great flexibility, consistent with experiment data for the monomer in solution, while maintaining a general strand-loop-strand motif with a solvent-exposed hydrophobic patch that is believed to be important for aggregation. Finally, equilibration of the peptide structure requires an implicit solvent LD simulation as long as 30 ns.

Inhaled Anesthetic Modulation of Amyloid β 1-40 Assembly and Growth

Anesthesia and surgery have been reported to produce long-term cognitive problems, and to accelerate neurodegenerative disorders in the elderly. In previous work, we found that inhaled anesthetics enhance fibril formation and cytotoxicity of amyloid beta peptide. In this work we show that the inhaled anesthetics halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) and isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether) also favor intermediate oligomers of amyloid beta(1-40), and reduce solubility of amyloid beta(1-40) monomer. Size-exclusion chromatography, analytical ultracentrifugation and photo-induced cross-linking experiments indicate halothane enhancement of oligomeric species having molecular weight approximately 44-100 kDa. Bis-ANS fluorescence experiments revealed that halothane stabilizes a population of diffusible oligomers relative to the monomer or the mature fibril. These data show that inhaled anesthetics lower the amyloid beta(1-40) concentration necessary to initiate oligomer formation, probably by preferential binding to intermediate oligomers en route to fibril formation.

Small Molecule Inhibitors of Aggregation Indicate That Amyloid β Oligomerization and Fibrillization Pathways Are Independent and Distinct

Alzheimer disease is characterized by the abnormal aggregation of amyloid beta peptide into extracellular fibrillar deposits known as amyloid plaques. Soluble oligomers have been observed at early time points preceding fibril formation, and these oligomers have been implicated as the primary pathological species rather than the mature fibrils. A significant issue that remains to be resolved is whether amyloid oligomers are an obligate intermediate on the pathway to fibril formation or represent an alternate assembly pathway that may or may not lead to fiber formation. To determine whether amyloid beta oligomers are obligate intermediates in the fibrillization pathway, we characterized the mechanism of action of amyloid beta aggregation inhibitors in terms of oligomer and fibril formation. Based on their effects, the small molecules segregated into three distinct classes: compounds that inhibit oligomerization but not fibrillization, compounds that inhibit fibrillization but not oligomerization, and compounds that inhibit both. Several compounds selectively inhibited oligomerization at substoichiometric concentrations relative to amyloid beta monomer, with some active in the low nanomolar range. These results indicate that oligomers are not an obligate intermediate in the fibril formation pathway. In addition, these data suggest that small molecule inhibitors are useful for clarifying the mechanisms underlying protein aggregation and may represent potential therapeutic agents that target fundamental disease mechanisms.

Amyloid‐beta Induced Endothelial‐Monocyte Interactions Involved in Alzheimer’s Disease

Cerebral amyloid angiopathy (CAA) associated with Alzheimer’s disease (AD) is characterized by cerebrovascular deposition of the fibrillar form of the amyloid-beta protein, assembled from amyloid-beta monomer. Vascular amyloid-beta accumulation initiates a cascade of events culminating in weakening of the vessel wall and increased risk of hemorrhagic stroke. Among these events is an increase in vessel-associated monocyte/macrophage cells, which could promote vascular damage and may contribute to elevated immune responses observed in AD brain. We have investigated the ability of interactions between amyloid-beta and endothelial monolayers to enhance adhesion and subsequent transmigration of monocyte cells. Treatment of endothelial monolayers with amyloid-beta aggregation mixtures, containing mature fibrils, protofibril intermediates, and unassembled monomer, enhances monocyte adhesion and promotes subsequent transmigration. In contrast, treatment with either amyloid-beta fibril or monomer alone has no effect. These results demonstrate that the presence of aggregated amyloid-beta is insufficient to stimulate endothelium. Monomeric amyloid-beta and protofibrils were purified on a SEC Superdex 75 column. Aggregation was monitored by thioflavin T fluorescence and dynamic light scattering. Support by a grant from the American Heart Association.

The conformations of the amyloid-β (21–30) fragment can be described by three families in solution

Alzheimer's disease has been linked to the self-assembly of the amyloid-beta protein of 40 and 42 residues. Although monomers are in equilibrium with higher-order species ranging from dimers to heptamers, structural knowledge of the monomeric amyloid-beta (Abeta) peptides is an important issue. Recent experimental data have shown that the fragment (21-30) is protease-resistant within full-length Abeta peptides and displays two structural families in solution. Because the details of the Abeta(21-30) structures found using distinct force fields and protocols differ at various degrees from those of the NMR structures, we revisit the conformational space of this peptide using the activation-relaxation technique (ART nouveau) coupled with a coarse-grained force field (OPEP v.3.0). We find that although Abeta(21-30) does not have a secondary structure, it dominantly populates three structural families, with a loop spanning residues Val24-Lys28. The first two families, which differ in the nature of the electrostatic interactions, satisfy the five interproton rotating frame nuclear Overhauser effect spectroscopy (ROESY) distances and superpose well onto the NMR structures. The third family, which cannot be seen by ROESY NMR experiments, displays a more open structure. This numeric study complements the experimental results by providing a much more detailed description of the dominant structures. Moreover, it provides further evidence of the capability of ART OPEP in providing a reliable conformational picture of peptides in solution.

P4-249: Insulin-degrading enzyme gene therapy reduces amyloid deposits in male and female APP/PS1 mice

Background: Dysfunctional amyloid catabolism may be causal in AD, and reversing levels of toxic amyloid may be therapeutically effective. Insulin degrading enzyme (IDE) can catabolize monomeric amyloid beta and potentially shift equilibrium away from deposition, and compromised IDE function may contribute to AD. Objective(s): Assess the efficacy of IDE gene therapy for reducing levels of deposited brain amyloid, including possible gender differences. Methods: Male and female double transgenic APP/PS1 mice (Jackson Laboratories) approximately 200 days old received unilateral hippocampal injections of 2.4e10 genome particles (gp) of IDE-AAV2/8 or 2.5e10 gp of pGFP-AAV2/82.8x10. Vectors for human IDE or control green fluorescent protein (GFP) expression consisted of coding sequences, chicken beta actin/cytomegalovirus hybrid promoter, between AAV2 inverted repeat sequences, packaged in AAV8 capsids. One month later the mice were sacrificed and the brains were processed for thioflavin S labeling. “Soluble” oligomeric amyloid beta was immunolabeled with primary antibody A11 (Chemicon 9234) and secondary antibody conjugated to quantum dots. Synaptic and potential neuronal degeneration was labeled with FluoroJadeC. Epifluorescence microscopy was used to generate digital video images for treatment-blinded stereological estimation of the fractional area occupation of thioflavin S labeling. Results: Extensive amyloid deposits were present throughout the neocortex and hippocampus. GFP fluorescence and immunoreactivity, and HAtagged IDE immunoreactivity, revealed transgene expression throughout much of the hippocampus. Oligomer labeling, observed in most neurons, showed no obvious relationship with either vector injections or plaques, but some spherical clusters of dystrophic neurites labeled strongly. FluoroJade C-labeled dystrophic neurites were found in some but not all plaques. Side differences in the thioflavin S-labeled hippocampal but not neocortical tissue area fraction were greater after IDE gene transfer than GFP (p 0.04). Gender was not significant. Conclusions: Localized overexpression of IDE via AAV vector-mediated gene transfer may be effective in reducing brain amyloid accumulation. This material is the result of work supported by AG10485 from NIA and with resources and the use of facilities at the NF/SG VA Medical Center, Gainesville, FL.

Treating Alzheimers Disease by Inactivating Bioactive Amyloid β Peptide

Treating Alzheimer's disease (AD) is one of today's biggest unmet medical needs. The drugs currently available transiently relieve some symptoms but have no significant effects on the progression of the disease. Progress in the past decade suggests that the amyloidogenesis of the inactive monomeric amyloid beta peptide (Abeta) into a subset of toxic Abeta polymers is responsible for neurodegeneration in AD. Not all forms of Abeta aggregates are damaging, for there are patients whose brains accumulated large amounts of Abeta in the form of plaques, but they had no obvious neurodegeneration and symptoms of dementia. Since Abeta can polymerize into many types of polymers or aggregates, the form of Abeta that induces neurodegeneration in AD, defined here as bioactive Abeta, is not clear. Preventing the formation of bioactive Abeta or inactivating previously formed bioactive Abeta is a promising approach for treating AD. This review describes our efforts to develop a cell-based assay for detecting bioactive Abeta, to verify the concept of bioactive Abeta in an animal model of AD and in post mortem brain tissue from AD patients, and to use this assay to screen for drugs that can inactivate bioactive Abeta. These studies show the proof in principle that inactivating bioactive Abeta is a promising approach to treat AD. Several promising compounds that can inactivate bioactive Abeta species are also described.

Mitochondrial Oxidative Damage in Aging and Alzheimer′s Disease: Implications for Mitochondrially Targeted Antioxidant Therapeutics

The overall aim of this article is to review current therapeutic strategies for treating AD, with a focus on mitochondrially targeted antioxidant treatments. Recent advances in molecular, cellular, and animal model studies of AD have revealed that amyloid precursor protein derivatives, including amyloid beta (Aβ) monomers and oligomers, are likely key factors in tau hyperphosphorylation, mitochondrial oxidative damage, inflammatory changes, and synaptic failure in the brain tissue of AD patients. Several therapeutic strategies have been developed to treat AD, including anti-inflammatory, antioxidant, and antiamyloid approaches. Among these, mitochondrial antioxidant therapy has been found to be the most efficacious in reducing pathological changes and in not producing adverse effects; thus, mitochondrial antioxidant therapy is promising as a treatment for AD patients. However, a major limitation in applying mitochondrial antioxidants to AD treatment has been the inability of researchers to enhance antioxidant levels in mitochondria. Recently, however, there has been a breakthrough. Researchers have recently been able to promote the entry of certain antioxidants—including MitoQ, MitoVitE, MitoPBN, MitoPeroxidase, and amino acid and peptide-based SS tetrapeptides—into mitochondria, several hundred-fold more than do natural antioxidants. Once in the mitochondria, they rapidly neutralize free radicals and decrease mitochondrial toxicity. Thus, mitochondrially targeted antioxidants are promising candidates for treating AD patients.

Preferred peptide backbone conformations in the unfolded state revealed by the structure analysis of alanine-based (AXA) tripeptides in aqueous solution.

We have combined Fourier transform IR, polarized Raman spectroscopy, and vibrational CD measurements of the amide I' band profile of alanyl-X-alanine tripeptides in (2)H(2)O to obtain the dihedral angles of their central amino acid residue. X represents glycine, valine, methionine, histidine, serine, proline, lysine, leucine, tryptophan, tyrosine, and phenylalanine. The experimental data were analyzed by means of a recently developed algorithm, which exploits the excitonic coupling between the amide modes of the two peptide groups. The results were checked by measuring the respective electronic CD spectra. The investigated peptides can be sorted into three classes. Valine, phenylalanine, tryptophan, histidine, and serine predominantly adopt an extended beta-strand conformation. Cationic lysine and proline prefer a polyproline II-like structure. Alanine, methionine, glycine, and leucine populate these two conformations with comparable probability. Our results are in variance with the prediction of the random-coil model, but supportive of Flory's isolated-pair hypothesis. We combined the obtained structural propensities of the investigated residues and similar information about other residues in the literature (i.e., glutamate, aspartate, isoleucine, and glutamine) to predict possible conformations of the monomeric amyloid beta peptide A beta(1-42) in aqueous solution, which reproduces results from most recent spectroscopic studies. Thus, it is demonstrated that the unfolded state of peptides can be understood in terms of the intrinsic structural propensities of their amino acid residues.

Interactions of Alzheimer amyloid-beta peptides with glycosaminoglycans effects on fibril nucleation and growth.

Proteoglycans and their constituent glycosaminoglycans are associated with all amyloid deposits and may be involved in the amyloidogenic pathway. In Alzheimer's disease, plaques are composed of the amyloid-beta peptide and are associated with at least four different proteoglycans. Using CD spectroscopy, fluorescence spectroscopy and electron microscopy, we examined glycosaminoglycan interaction with the amyloid-beta peptides 1-40 (Abeta40) and 1-42 (Abeta42) to determine the effects on peptide conformation and fibril formation. Monomeric amyloid-beta peptides in trifluoroethanol, when diluted in aqueous buffer, undergo a slow random to amyloidogenic beta sheet transition. In the presence of heparin, heparan sulfate, keratan sulfate or chondroitin sulfates, this transition was accelerated with Abeta42 rapidly adopting a beta-sheet conformation. This was accompanied by the appearance of well-defined amyloid fibrils indicating an enhanced nucleation of Abeta42. Incubation of preformed Abeta42 fibrils with glycosaminoglycans resulted in extensive lateral aggregation and precipitation of the fibrils. The glycosaminoglycans differed in their relative activities with the chondroitin sulfates producing the most pronounced effects. The less amyloidogenic Abeta40 isoform did not show an immediate structural transition that was dependent upon the shielding effect by the phosphate counter ion. Removal or substitution of phosphate resulted in similar glycosaminoglycan-induced conformational and aggregation changes. These findings clearly demonstrate that glycosaminoglycans act at the earliest stage of fibril formation, namely amyloid-beta nucleation, and are not simply involved in the lateral aggregation of preformed fibrils or nonspecific adhesion to plaques. The identification of a structure-activity relationship between amyloid-beta and the different glycosaminoglycans, as well as the condition dependence for glycosaminoglycan binding, are important for the successful development and evaluation of glycosaminoglycan-specific therapeutic interventions.