Search

Oxidative stress and amyloid-β (Aβ) formation are important processes that occur in Alzheimer's disease (AD). Amyloid formation is associated with the aggregation and precipitation of the Aβ peptide, while oxidative stress results from an imbalance in pro-oxidant/antioxidant homeostasis that produces harmful reactive oxygen species. The methionine-35 (Met35) residue of the Aβ peptide plays an important role in AD oxidative stress events and the associated neurotoxicity. We and other research groups previously demonstrated that in vitro oxidation of the Met35 side-chain to the sulfoxide (Met35red → Met35ox) impedes assembly and aggregation of monomeric Aβ peptide into protofibrils, the latter being the immediate precursors of amyloid plaques. Here, we report that Met35 oxidation state affects the stability of preexisting amyloid fibrils and plaques, where the Met35red → Met35ox process leads to changes in the morphology of filaments, protofibrils, mature fibrils, and loss of Congo red birefringence in senile plaques isolated from the brains of AD patients. The most notable differences were in fibril flexibility, as evidenced by changes from straight fibrils to irregularly shaped, rope-like fibrils. These findings suggest that the Met35 oxidation state and amyloid plaque formation may be intimately linked.

Cerebral deposition of amyloid β protein (Aβ) is an invariant feature of Alzheimer disease (AD), and epidemiological evidence suggests that moderate consumption of foods enriched with phenolic compounds reduce the incidence of AD. We reported previously that the phenolic compounds myricetin (Myr) and rosmarinic acid (RA) inhibited Aβ aggregation in vitro and in vivo. To elucidate a mechanistic basis for these results, we analyzed the effects of five phenolic compounds in the Aβ aggregation process and in oligomer-induced synaptic toxicities.Wenow report that the phenolic compounds blocked Aβ oligomerization, and Myr promoted significantNMRchemical shift changes of monomeric Aβ. Both Myr and RA reduced cellular toxicity and synaptic dysfunction of the Aβ oligomers. These results suggest that Myr and RA may play key roles in blocking the toxicity and early assembly processes associated with Aβ through different binding. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Formation of alpha-synuclein aggregates is proposed to be a crucial event in the pathogenesis of Parkinson's disease. Large soluble oligomeric species are observed as probable intermediates during fibril formation and these, or related aggregates, may constitute the toxic element that triggers neurodegeneration. Unfortunately, there is a paucity of information regarding the structure and composition of these oligomers. Here, the morphology and the conformational characteristics of the oligomers and filaments are investigated by a combined atomic force microscopy (AFM) and Raman microscopic approach on a common mica surface. AFM showed that in vitro early stage oligomers were globular with variable heights, while prolonged incubation caused the oligomers to become elongated as protofilaments. The height of the subsequently formed alpha-synuclein filaments was similar to that of the protofilaments. Analysis of the Raman amide I band profiles of the different alpha-synuclein oligomers establishes that the spheroidal oligomers contain a significant amount of alpha-helical secondary structure (47%), which decreases to about 37% in protofilaments. At the same time, when protofilaments form, beta-sheet structure increases to about 54% from the approximately 29% observed in spheroidal oligomers. Upon filament formation, the major conformation is beta-sheet (66%), confirmed by narrowing of the amide I band and the profile maximum shifting to 1667 cm(-1). The accumulation of spheroidal oligomers of increasing size but unchanged vibrational spectra during the fibrillization process suggests that a cooperative conformational change may contribute to the kinetic control of fibrillization.

The ABri is a 34 residue peptide that is the major component of amyloid deposits in familial British dementia. In the amyloid deposits, the ABri peptide adopts aggregated beta-pleated sheet structures, similar to those formed by the Abeta peptide of Alzheimer's disease and other amyloid forming proteins. As a first step toward elucidating the molecular mechanisms of the beta-amyloidosis, we explored the ability of the environmental variables (pH and peptide concentration) to promote beta-sheet fibril structures for synthetic ABri peptides. The secondary structures and fibril morphology were characterized in parallel using circular dichroism, atomic force microscopy, negative stain electron microscopy, Congo red, and thioflavin-T fluorescence spectroscopic techniques. As seen with other amyloid proteins, the ABri fibrils had characteristic binding with Congo red and thioflavin-T, and the relative amounts of beta-sheet and amyloid fibril-like structures are influenced strongly by pH. In the acidic pH range 3.1-4.3, the ABri peptide adopts almost exclusively random structure and a predominantly monomeric aggregation state, on the basis of analytical ultracentrifugation measurements. At neutral pH, 7.1-7.3, the ABri peptide had limited solubility and produced spherical and amorphous aggregates with predominantly beta-sheet secondary structure, whereas at slightly acidic pH, 4.9, spherical aggregates, intermediate-sized protofibrils, and larger-sized mature amyloid fibrils were detected by atomic force microscopy. With aging at pH 4.9, the protofibrils underwent further association and eventually formed mature fibrils. The presence of small amounts of aggregated peptide material or seeds encourage fibril formation at neutral pH, suggesting that generation of such seeds in vivo could promote amyloid formation. At slightly basic pH, 9.0, scrambling of the Cys5-Cys22 disulfide bond occurred, which could lead to the formation of covalently linked aggregates. The presence of the protofibrils and the enhanced aggregation at slightly acidic pH is consistent with the behavior of other amyloid-forming proteins, which supports the premise that a common mechanism may be involved in protein misfolding and beta-amyloidosis.

Inheritance of apoE4 is a strong risk factor for the development of late-onset sporadic Alzheimer's disease (AD). Several lines of evidence suggest that apoE4 binds to the Alzheimer Abeta protein and, under certain experimental conditions, promotes formation of beta-sheet structures and amyloid fibrils. Deposition of amyloid fibrils is a critical step in the development of AD. We report here that addition of melatonin to Abeta in the presence of apoE resulted in a potent isoform-specific inhibition of fibril formation, the extent of which was far greater than that of the inhibition produced by melatonin alone. This effect was structure-dependent and unrelated to the antioxidant properties of melatonin, since it could be reproduced neither with the structurally related indole N-acetyl-5-hydroxytryptamine nor with the antioxidants ascorbate, alpha-tocophenol, and PBN. The enhanced inhibitory effects of melatonin and apoE were lost when bovine serum albumin was substituted for apoE. In addition, Abeta in combination with apoE was highly neurotoxic (apoE4 > apoE3) to neuronal cells in culture, and this activity was also prevented by melatonin. These findings suggest that reductions in brain melatonin, which occur during aging, may contribute to a proamyloidogenic microenvironment in the aging brain.

The aggregation of the β-peptide into amyloid is a key pathological event in Alzheimer's disease. This process (β-amyloidosis) involves the conversion of soluble random coil, α-helical or β-sheet conformations into insoluble, aggregated β-pleated sheet structures. The pH is a significant extrinsic factor that influences β-amyloidosis, which must be related to the presence of ionizable groups in the β-peptide. To further evaluate this effect, we determined the dissociation constants (pKa) of the side chains for the aspartic acid (Asp), glutamic acid (Glu), histidine (His), and tyrosine (Tyr) amino acid residues using NMR spectroscopy. The measurements were performed under different solution conditions, where the predominant conformation is either random coil or α-helix. We have used a peptide fragment that comprises residues 1−28 [β-(1−28)] of the natural β-(1−40) or β-(1−42) peptides, which is an appropriate model since the remaining 29−40 or 29−42 regions are devoid of polar and charged amino acid residu...

The amyloid beta-peptide is the major protein constituent of neuritic plaques in Alzheimer's disease. The beta-peptide varies slightly in length and exists in two predominant forms: (1) the shorter, 40 residue beta-(1-40), found mainly in cerebrovascular amyloid; and (2) the longer, 42 residue beta-(1-42), which is the major component in amyloid plaque core deposits. We report here that the sodium dodecyl sulphate (SDS) micelle, a membrane-mimicking system for biophysical studies, prevents aggregation of the beta-(1-40) and the beta-(1-42) into the neurotoxic amyloid-like, beta-pleated sheet structure, and instead encourages folding into predominantly alpha-helical structures at pH 7.2. Analysis of the nuclear Overhauser enhancement (NOE) and the alphaH NMR chemical shift data revealed no significant structural differences between the beta-(1-40) and the beta-(1-42). The NMR-derived, three-dimensional structure of the beta-(1-42) consists of an extended chain (Asp1-Gly9), two alpha-helices (Tyr10-Val24 and Lys28-Ala42), and a looped region (Gly25-Ser26-Asn27). The most stable alpha-helical regions reside at Gln15-Val24 and Lys28-Val36. The majority of the amide (NH) temperature coefficients were less than 5, indicative of predominately strong NH backbone bonding. The lack of a persistent region with consistently low NH coefficients, together with the rapid NH exchange rates in deuterated water and spin-labeled studies, suggests that the beta-peptide is located at the lipid-water interface of the micelle and does not become inbedded within the hydrophobic interior. This result has implications for the circulation of membrane-bound beta-peptide in biological fluids, and may also facilitate the design of amyloid inhibitors to prevent an alpha-helix-->beta-sheet conversion in Alzheimer's disease.

The major protein constituent of amyloid deposits in Alzheimer's disease is the β-peptide, which in solution can fold as a random coil, monomeric α-helix, or oligomeric β-sheet structure, the latte...

It is generally postulated that the amyloid beta protein (Abeta) plays a central role in the progressive neurodegeneration observed in Alzheimer's disease. Important pathologic properties of this protein, such as neurotoxicity and resistance to proteolytic degradation, depend on the ability of Abeta to form beta-sheet structures or amyloid fibrils. We report that melatonin, a hormone recently found to protect neurons against Abeta toxicity, interacts with Abeta1-40 and Abeta1-42 and inhibits the progressive formation of beta-sheets and amyloid fibrils. These interactions between melatonin and the amyloid peptides were demonstrated by circular dichroism and electron microscopy for Abeta1-40 and Abeta1-42 and by nuclear magnetic resonance spectroscopy for Abeta1-40. Inhibition of beta-sheets and fibrils could not be accomplished in control experiments when a free radical scavenger or a melatonin analog were substituted for melatonin under otherwise identical conditions. In sharp contrast with conventional anti-oxidants and available anti-amyloidogenic compounds, melatonin crosses the blood-brain barrier, is relatively devoid of toxicity, and constitutes a potential new therapeutic agent in Alzheimer's disease.

Major problems exist with pharmacological and biophysical studies of the synthetic β(1-42) peptide, in that the results often lack reproducibility. The starting aggregation states and structures of the various synthetic commercial lots appear to vary, resulting in signijicant lot-to-lot variability. We describe here an easy, efficient trifluoroacetic acid (TFA) pretreatment method that renders the Aβ easily soluble both in aqueous, buffered solutions and in organic solvents such as hexafluoroisopropanol (HFIP) or dimethylsulfoxide (DMSO). The TFA treated Aβ exhibits the properties of monomeric, random coil structures and lacks pre-aggregated material that can act as seeds in fibrilization assays, thus reducing the batch to batch variation. In addition, nuclear magnetic resonance (NMR) spectra recorded in deuterated TFA allow measurement of the peptide purity that is not obtainable with other analytical methods.

Beta-peptide is a major component of amyloid deposits in Alzheimer's disease. We report here a proton nuclear magnetic resonance (NMR) spectroscopic investigation of a synthetic peptide that is homologous to residues 1-28 of beta-peptide [abbreviated as beta-(1-28)]. The beta-(1-28) peptide produces insoluble beta-pleated sheet structures in vitro, similar to the beta-pleated sheet structures of beta-peptide in amyloid deposits in vivo. For peptide solutions in the millimolar range, in aqueous solution at pH 1-4 the beta-(1-28) peptide adopts a monomeric random coil structure, and at pH 4-7 the peptide rapidly precipitates from solution as an oligomeric beta-sheet structure, analogous to amyloid deposition in vivo. The NMR work shown here demonstrates that the beta-(1-28) peptide can adopt a monomeric alpha-helical conformation in aqueous trifluoroethanol solution at pH 1-4. Assignment of the complete proton NMR spectrum and the determination of the secondary structure were arrived at from interpretation of two-dimensional (2D) NMR data, primarily (1) nuclear Overhauser enhancement (NOE), (2) vicinal coupling constants between the amide (NH) and alpha H protons, and (3) temperature coefficients of the NH chemical shifts. The results show that at pH 1.0 and 10 degrees C the beta-(1-28) peptide adopts an alpha-helical structure that spans the entire primary sequence. With increasing temperature and pH, the alpha-helix unfolds to produce two alpha-helical segments from Ala2 to Asp7 and Tyr10 to Asn27. Further increases in temperature to 35 degrees C cause the Ala2-Asp7 section to become random coil, while the His13-Phe20 section stays alpha-helical. A mechanism involving unfavorable interactions between charged groups and the alpha-helix macrodipole is proposed for the alpha-helix----beta-sheet conversion observed at midrange pH.

Accumulating evidence suggests that neurons prone to degeneration in Alzheimer's Disease (AD) exhibit evidence of re-entry into an aberrant mitotic cell cycle. Our laboratory recently demonstrated that, in a genomic amyloid precursor protein (APP) mouse model of AD (R1.40), neuronal cell cycle events (CCEs) occur in the absence of beta-amyloid (Aβ) deposition and are still dependent upon the amyloidogenic processing of the amyloid precursor protein (APP). These data suggested that soluble Aβ species might play a direct role in the induction of neuronal CCEs. Here, we show that exposure of non-transgenic primary cortical neurons to Aβ oligomers, but not monomers or fibrils, results in the retraction of neuronal processes, and induction of CCEs in a concentration dependent manner. Retraction of neuronal processes correlated with the induction of CCEs and the Aβ monomer or Aβ fibrils showed only minimal effects. In addition, we provide evidence that induction of neuronal CCEs are autonomous to primary neurons cultured from the R1.40 mice. Finally, our results also demonstrate that Aβ oligomer treated neurons exhibit elevated levels of activated Akt and mTOR (mammalian Target Of Rapamycin) and that PI3K, Akt or mTOR inhibitors blocked Aβ oligomer-induced neuronal CCEs. Taken together, these results demonstrate that Aβ oligomer-based induction of neuronal CCEs involve the PI3K-Akt-mTOR pathway.

Pardaxin is a mucosal secretion of the Pacific sole Pardachirus pavoninus that exhibits unusual shark repellent and surfactant properties [Thompson, S. A., Tachibana, K., Nakanishi, K., & Kubota, I. (1986) Science 233, 341-343]. This 33 amino acid polypeptide folds into ordered structures in trifluoroethanol-water solution and in micelles but adopts a random-coiled structure in water solution. The complete proton NMR spectrum of pardaxin P-2 has been assigned in CF3CD2OD/H2O (1:1) solution, and the three-dimensional structure has been elucidated with distance restrained molecular dynamics calculations. It is demonstrated that peptide segments within the 7-11 and 14-26 residue stretches are helical while residues at the C- and N-terminus exist predominantly in extended conformations in solution. The dipeptide 12-13 segment connecting the two helices exists as a bend or a hinge allowing the two helices to be oriented in a L-shaped configuration. These studies establish that pardaxin P-2 adopts a novel amphiphilic helix (7-11)-bend (12-13)-helix (14-26) motif with Pro-13 forming the focal point of the turn or bend between the two helices.

Amyloid plaque deposition involves the aggregation of normally soluble proteins into insoluble amyloid fibrils (fibrillization) and proceeds through intermediates with distinct morphologies, including spherical aggregates, protofibrils, and mature fibrils. Recently, a novel annular protofibril-like intermediate with unique pore-like properties was produced by alpha-synuclein, A beta-Arctic and amylin, which are proteins associated with Parkinson's disease, Alzheimer's disease, and type-II diabetes. The observation of annular structures coupled with size selective channel-like activity by these proteins suggests that these structures may be responsible for vesicle permeability by ion-channel formation. Using atomic force spectroscopy, we report here that the ABri peptide associated with familial British dementia produces similar annular and ring-like protofibril structures during the following sequence of events: spherical aggregates (0.4-1.5 nm height)-->chain-like protofibrils (1.5-2.3 nm height)-->ring-like protofibrils and annular protofibrils (1.5-2.3 nm height). This suggests that ABri fibrillization occurs in a similar fashion to other amyloidogenic proteins and that the annular protofibrillar structures may represent a common amyloid intermediate.

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.

The pathogenesis of Alzheimer's disease is characterized by the aggregation and fibrillation of the 40-residue A beta(1-40) and 42-residue A beta(1-42) peptides into amyloid plaques. The structural changes associated with the conversion of monomeric A beta peptide building blocks into multimeric fibrillar beta-strand aggregates remain unknown. Recently, we established that oxidation of the methionine-35 side chain to the sulfoxide (Met35(red) --Met35(ox)) significantly impedes the rate of aggregation and fibrillation of the A beta peptide. To explore this effect at greater resolution, we carefully compared the (1)H, (15)N, and (13)C NMR chemical shifts of four A beta peptides that had the Met35 reduced or oxidized (A beta(1-40)Met35(red), A beta(1-40)Met35(ox), A beta(1-42)Met35(red), and A beta(1-42)Met35(ox)). With the use of a special disaggregation protocol, the highly aggregation prone A beta peptides could be studied at higher, millimolar concentrations (as required by NMR) in aqueous solution at neutral pH, remaining largely monomeric at 5 degrees C as determined by sedimentation equilibrium studies. The NOE, amide-NH temperature coefficients, and chemical shift indices of the (1)H alpha, (13)C alpha, and (13)C beta established that the four peptides are largely random, extended chain structures, with the Met35(ox) reducing the propensity for beta-strand structure at two hydrophobic regions (Leu17-Ala21 and Ile31-Val36), and turn- or bendlike structures at Asp7-Glu11 and Phe20-Ser26. Additional NMR studies monitoring changes that occur during aging at 37 degrees C established that, along with a gradual loss of signal/noise, the Met35(ox) significantly hindered upfield chemical shift movements of the 2H NMR signals for the His6, His13, and His14 side chains. Taken together, the present NMR studies demonstrate that the Met35(red) --Met35(ox) conversion prevents aggregation by reducing both hydrophobic and electrostatic association and that the A beta(1-40)Met35(red), A beta(1-40)Met35(ox), A beta(1-42)Met35(red), and A beta(1-42)Met35(ox) peptides may associate differently, through specific, sharp changes in structure during the initial stages of aggregation.

Protein misfolding and aggregation are now well-recognized processes that often lead to amyloid fibril formation (amyloidosis). Because these events are coupled with many types of human disease, the field of protein amyloidosis is under intense investigation. In the amyloid fibrils, the proteins adopt cross β-pleated sheet structures with distinct tinctorial and morphological properties, and typically consist of long, unbranched filaments that bind to diagnostic dyes such as Thioflavin-T and Congo Red (Temussi et al., 2003xFrom Alzheimer to Huntington: why is a structural understanding so difficult?. Temussi, P.A., Masino, L., and Pastore, A. EMBO J. 2003; 22: 355–361Crossref | PubMed | Scopus (120)See all ReferencesTemussi et al., 2003). There are currently 24 proteins that produce amyloid fibrils associated with human disease (Westermark et al., 2002xAmyloid fibril protein nomenclature. Westermark, P., Benson, M.D., Buxbaum, J.N., Cohen, A.S., Frangione, B., Ikeda, S., Masters, C.L., Merlini, G., Saraiva, M.J., and Sipe, J.D. Amyloid. 2002; 9: 197–200Crossref | PubMedSee all ReferencesWestermark et al., 2002), including the Aβ of Alzheimer’s disease, prion of transmissible spongiform encephalopathies, amylin of maturity-onset diabetes, transthyretin of familial amyloidosis, huntingtin of Huntington’s disease, and α-synuclein of Parkinson’s disease. Albeit these proteins have very different primary sequences, molecular sizes, and folded tertiary structures, they all produce amyloid fibrils. Amyloidosis may also be a general property for all proteins, since de novo designed peptides and other naturally occurring proteins not associated with human disease (such as myoglobin) (Fandrich et al., 2001xAmyloid fibrils from muscle myoglobin. Fandrich, M., Fletcher, M.A., and Dobson, C.M. Nature. 2001; 410: 165–166Crossref | PubMed | Scopus (568)See all ReferencesFandrich et al., 2001) can be encouraged to aggregate as amyloid fibrils.Despite the plethora of research, details about the molecular mechanisms of amyloidosis are still lacking. Most scientists agree that unraveling the chemical mechanisms is absolutely essential for the development of specific inhibitors to prevent amyloidosis in humans. The most obvious missing details are high-resolution structural data, particularly regarding the soluble β-sheet aggregates and the amyloid fibril structures. Given that amyloid fibrils are not amenable to standard x-ray crystallography (i.e., they do not form crystals), lower-resolution analytical techniques such as x-ray fibril diffraction, negative stain electron microscopy, and atomic force microscopy have been employed. Although these methods have provided valuable data about the fibril morphology, including the discovery of “toxic” intermediates such as the “protofibrils” (Caughey and Lansbury, 2003xProtofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Caughey, B. and Lansbury, P.T. Jr. Annu. Rev. Neurosci. 2003; 26: 267–298Crossref | PubMed | Scopus (1114)See all ReferencesCaughey and Lansbury, 2003), they cannot provide critical atomic level structural information, such as what amino acids are interacting during the aggregation processes and whether or not the fibrils adopt unique, folded structures. In this regard, the work presented by the Tycko and Meredith research groups in this issue makes an important contribution about the Aβ fibril structure of Alzheimer’s disease. These research groups have excellent track records with handling the Aβ peptide and were the first to demonstrate that the peptide adopts the rare parallel β-sheet organization in the amyloid fibrils.The recent finding that solid-state NMR spectroscopy is applicable to amyloid fibril structure determination represents a timely and significant breakthrough (Tycko, 2003xInsights into the amyloid folding problem from solid-state NMR. Tycko, R. Biochemistry. 2003; 42: 3151–3159Crossref | PubMed | Scopus (166)See all ReferencesTycko, 2003). For biomolecules, solid-state NMR has unique capabilities, in that it can be used with samples of limited solubility that often precipitate as noncrystalline solids. Solid-state NMR can provide accurate distances and torsion angles between site-specific 15N- and/or 13C-labeled atoms, and, under certain conditions, the NMR constraints can generate structural models on par with those obtained by solution NMR and x-ray. Solid-state NMR is likewise a rapidly growing field, and studies of uniformly or specifically labeled samples have provided important information about membrane-bound peptide channels and the active sites of other membrane proteins (Thompson, 2002xSolid-state NMR studies of the structure and mechanisms of proteins. Thompson, L.K. Curr. Opin. Struct. Biol. 2002; 12: 661–669Crossref | PubMed | Scopus (58)See all ReferencesThompson, 2002), both of which are systems not amenable to solution NMR or x-ray.The predominate forms of the Alzheimer’s Aβ peptide are the 40-residue Aβ(1–40) and 42-residue Aβ(1–42). The earliest solid-state NMR studies utilized peptide fragments composed of regions important for amyloidosis, such as the Aβ(34–42) and Aβ(16–22) peptides, in which both form the antiparallel β-sheet as the major structural motif. However, solid-state NMR studies of the longer 35-residue Aβ(10–35) and native Aβ(1–40) peptides found different results that were consistent with in-register parallel β-sheet structures. The overall conclusion was that accurate structural models must use constraints obtained from the longer, full-length Aβ peptides, and that despite the common cross β-pleated sheet motif, specific structural details can be sequence dependent. Nonetheless, the parallel versus antiparallel variation was a conundrum, since the shorter Aβ peptides still form classic amyloid fibrils based on Congo Red and electron microscopy.As described in this issue, Gordon et al. contemplated that peptide amphiphilicity may influence the parallel versus antiparallel orientation. Inspection of the Aβ primary sequence reveals that the Aβ(16–22) and Aβ(34–42) peptides are nonamphiphilic, whereas the Aβ(10–35) and Aβ(1–40) are amphiphilic. Additionally, the Aβ(1–40) peptide has surfactant properties and forms micelles. Amphiphilicity would stabilize the parallel β-sheet orientation and nonamphiphilicity the antiparallel, possibly by way of hydrogen-bonding or electrostatic interactions between oppositely charged side chains or termini.To test this hypothesis, the Aβ(16–22) peptide was made amphiphilic by attaching a long hydrophobic chain to one end of the molecule (Scheme 1Scheme 1). This objective was fulfilled by acylation of the N-terminus with octanoic acid (CH3(CH2)6COOH), which increased the peptide’s amphiphilicity as measured from its stability at the air-water interface, but did not reduce its ability to form amyloid fibrils. Based on electron microscopy and Congo Red, the modified octanoyl-Aβ(16–22) forms amyloid fibrils that are indistinguishable from those of unmodified Aβ(16–22). Without a doubt, the hypothesis was proven accurate by solid-state NMR with peptides containing 13C- and 15N-labels at strategic sites, where the octanoyl-Aβ(16–22) formed parallel β-sheets and the Aβ(16–22) antiparallel β-sheets. For consistency, the NMR methods employed were identical to those used previously by the same group for generating the Aβ(1–40) peptide model (Petkova et al., 2002xA structural model for Alzheimer’s {beta}-amyloid fibrils based on experimental constraints from solid state NMR. Petkova, A.T., Ishii, Y., Balbach, J.J., Antzutkin, O.N., Leapman, R.D., Delaglio, F., and Tycko, R. Proc. Natl. Acad. Sci. USA. 2002; 99: 16742–16747Crossref | PubMed | Scopus (1215)See all ReferencesPetkova et al., 2002), which included a combination of low-precision (linewidths and chemical shifts) and high-precision (distances and torsion angles) constraints. The labeling and dilution strategy utilized four different peptide mixtures and exemplifies the experience these groups have in studying the Aβ by solid-state NMR.Scheme 1View Large Image | View Hi-Res Image | Download PowerPoint SlideThe work of Gordon et al. in this issue has several important implications; notably, that peptide amphiphilicity is a critical parameter for controlling the β-sheet organization of amyloid fibrils. This provides a rationale for the proclivity toward parallel or antiparallel arrangements and, more importantly, that amyloid-forming proteins may each adopt unique fibril structures that are only visible by high-resolution techniques such as NMR. The unique fibril structures and the importance of amphiphilicity suggest that specific compounds could be targeted toward inhibiting amyloidosis of a single or select group of proteins, possibly by altering the brain microenvironment in a manner to prevent formation of the native parallel arrangement. Related approaches with modulating the peptide amphiphilicity has been used in the design of tertiary and supramolecular structures and may also be useful in the development of self-assembling, nanoscale materials.In closing, the article by Gordon et al. in this issue clearly shows that high-resolution structure determination is urgently needed in the amyloid research area, and that solid-state NMR has emerged as an indispensable tool in this endeavor.

The major component of amyloid plaques in Alzheimer's disease (AD) is Abeta, a small peptide that has high propensity to assemble as aggregated beta-sheet structures. Using three well established techniques for studying amyloid structure, namely circular dichroism, thioflavin-T fluorescence, and atomic force microscopy, we demonstrate that oxidation of the Met-35 side chain to a methionine sulfoxide (Met-35(ox)) significantly hinders the rate of fibril formation for the 42-residue Abeta-(1-42) at physiological pH. Met-35(ox) also alters the characteristic Abeta fibril morphology and prevents formation of the protofibril, which is a key intermediate in beta-amyloidosis and the associated neurotoxicity. The implications of these results for the biological function and role of Abeta with oxidative stress in AD are discussed.

Intramolecular quenching of tryptophan fluorescence by protein functional groups was studied in a series of rigid cyclic hexapeptides containing a single tryptophan. The solution structure of the canonical peptide c[D-PpYTFWF] (pY, phosphotyrosine) was determined in aqueous solution by 1D- and 2D-(1)H NMR techniques. The peptide backbone has a single predominant conformation. The tryptophan side chain has three chi(1) rotamers: a major chi(1) = -60 degrees rotamer with a population of 0.67, and two minor rotamers of equal population. The peptides have three fluorescence lifetimes of about 3.8, 1.8, and 0.3 ns with relative amplitudes that agree with the chi(1) rotamer populations determined by NMR. The major 3.8-ns lifetime component is assigned to the chi(1) = -60 degrees rotamer. The multiple fluorescence lifetimes are attributed to differences among rotamers in the rate of excited-state electron transfer to peptide bonds. Electron-transfer rates were calculated for the six preferred side chain rotamers using Marcus theory. A simple model with reasonable assumptions gives excellent agreement between observed and calculated lifetimes for the 3.8- and 1.8-ns lifetimes and assigns the 1.8-ns lifetime component to the chi(1) = 180 degrees rotamer. Substitution of phenylalanine by lysine on either side of tryptophan has no effect on fluorescence quantum yield or lifetime, indicating that intramolecular excited-state proton transfer catalyzed by the epsilon-ammonium does not occur in these peptides.

The major protein constituents of amyloid deposits in Alzheimer's disease (AD) are the 40-residue beta-amyloid (Abeta) (1-40) peptide and the 42-residue Abeta(1-42) peptide. The Abeta(1-42) is more pathogenic and produced in greater quantities in familial forms of AD. A major goal of research is to uncover a suitable inhibitor that either slows down or inhibits Abeta formation (beta-amyloidosis). During beta-amyloidosis, structural changes associated with the conversion of monomeric Abeta peptide building blocks into the aggregated fibrillar beta-sheet structures occur (alpha-helix-->beta-sheet or random, extended chain-->beta-sheet). In previous work, we and others established that nicotine, a major component of cigarette smoke, inhibits beta-amyloidosis of the Abeta(1-42), which may result from nicotine binding to the alpha-helical structure. These conclusions were based on solution nuclear magnetic resonance (NMR) spectroscopic studies with the nonnative 28-residue Abeta(1-28). This information suggests that, when administered therapeutically to AD patients, nicotine may not only affect cholinergic activation, but could also conceivably alter amyloid deposition. In this report, NMR studies were augmented with the naturally occurring Abeta(1-42), under conditions where the peptide folds into a predominantly alpha-helical or random, extended chain structure. The major result is that nicotine shows only modest binding to these conformations, indicating that the nicotine inhibition to beta-amyloidosis probably results from binding to a small, soluble beta-sheet aggregate that is NMR invisible.

Publisher Summary The immense difficulties and unexpected questions presented by hundreds of amyloid toxicity studies beg for a careful study of A β assembly processes, evaluating the kinetics and structures of different aggregation pathways that convert the soluble monomer into the higher order A β assemblies that harbor neurotoxic properties. A large array of sophisticated instrumental techniques, including analytical ultracentrifugation, atomic force microscopy, solid-state and solution NMR, and fluorescence tracer techniques, will help elucidate the bioactive A β structure(s), whereas specific cellular and biochemical techniques will elucidate the specific steps involved in the aftermath of A β cellular interactions. Nonetheless, all these potentially useful high technology methods are ineffective unless proper care regarding A β sample preparation is taken into account. This chapter briefly summarizes potential problems involved with A β sample preparation and has also described methods to deal with these issues. The most critical complications are related to the starting structures and aggregation states of the A β .

The Abeta peptide is the major protein component of amyloid deposits in Alzheimer's disease (AD). Age-related microenvironmental changes in the AD brain promote amyloid formation that leads to cell injury and death. Altered levels of metals (such as Cu and Zn) exist in the AD brain, and because Cu and Zn can be bound to the Abeta in the amyloid plaques, it is thought that these binding events in vivo may trigger or prevent Abeta amyloid formation in the AD brain. Although several structural models have been proposed, all of these are undefined due to the lack of definitive structural data. The present NMR studies utilized uniformly 15N-labeled Abeta(1-40) peptide and 1H-15N HSQC experiments and demonstrate for the first time that the Abeta binds Cu and Zn in a distinct manner. The binding promotes NH signal disappearance of E3-V18, which was not due to the paramagnetic effect of Cu2+, as identical NMR studies were seen with Zn2+, which is diamagnetic. NMR titration experiments showed that the amide NH peak intensities of R5-L17 showed the most pronounced intensity reduction, and that the 1H signals for the side chain aromatic signals of the three histidines shift upfield (H6, H13, and H14). We propose that initially Cu2+ is anchored to the Abeta monomer (fast exchange rate) and is followed by deprotonation and/or severe line broadening of the backbone amide NH for E3-V18 (intermediate exchange rate). By contrast, Cu2+ binding to soluble Abeta aggregates leads to rapid aggregation and nonfibrillar amorphous structures, and without metal, the Abeta can undergo the normal time-dependent aggregation, eventually producing more ordered, late-stage parallel beta-sheet structures. These anomalous (rare) binding events may account for some of the unique properties associated with the Abeta, such as its proposed "dual role", where sequestration of metal ions by the monomer is neuroprotective, while that by beta-aggregates generates oxygen radicals and causes neuronal death.

The 42-residues beta-(1-42) peptide is the major protein component of amyloid plaque cores in Alzheimer's disease. In aqueous solution at physiological pH, the synthetic beta-(1-42) peptide readily aggregates and precipitates as oligomeric beta-sheet structures, a process that occurs during amyloid formation in Alzheimer's disease. Using circular dichroism (CD) and ultraviolet spectroscopic techniques, we show that nicotine, a major component in cigarette smoke, inhibits amyloid formation by the beta-(1-42) peptide. The related compound cotinine, the major metabolite of nicotine in humans, also slows down amyloid formation, but to a lesser extent than nicotine. In contrast, control substances pyridine and N-methylpyrrolidine accelerate the aggregation process. Nuclear magnetic resonance (NMR) studies demonstrate that nicotine binds to the 1-28 peptide region when folded in an alpha-helical conformation. On the basis of chemical shift data, the binding primarily involves the N-CH3 and 5'CH2 pyrrolidine moieties of nicotine and the histidine residues of the peptide. The binding is in fast exchange, as shown by single averaged NMR peaks and the lack of nuclear Overhauser enhancement data between nicotine and the peptide in two-dimensional NOESY spectra. A mechanism is proposed, whereby nicotine retards amyloidosis by preventing an alpha-helix-->beta-sheet conformational transformation that is important in the pathogenesis of Alzheimer's disease.

The three-dimensional solution structure of residues 1-28 of the amyloid beta-peptide was determined using nuclear magnetic resonance spectroscopy, distance geometry, and molecular dynamic techniques. The nuclear magnetic resonance data used to derive the structure consisted of nuclear Overhauser enhancements, vicinal coupling constants, and temperature coefficients of the amide-NH chemical shifts. The beta-peptide is the major proteinaceous component of amyloid deposits in Alzheimer's disease. In membrane-like media, the peptide folds to form a predominately alpha-helical structure with a bend centered at residue 12. The side chains of histidine-13 and lysine-16 are close, residing on the same face of the helix. Their proximity may constitute a binding motif with the heparan sulfate proteoglycans. The molecular details of the structure shown here could facilitate the design of rational treatments to curtail the binding of heparan sulfate proteoglycans or to prevent an alpha-helix-->beta-sheet conversion that may occur during the early stages of amyloid formation in Alzheimer's disease.

beta-peptide is a normal component of amyloid deposits in Alzheimer's disease. In aqueous solution, beta-peptide is extremely insoluble and rapidly aggregates forming oligomeric beta-sheet structures that eventually precipitate from solution. Presumably, this process is related to the production of amyloid deposits in Alzheimer's disease. Formic acid is commonly used to dissolve the beta-peptides and prevent aggregation in biological and biophysical studies. However, a side-reaction which covalently modifies beta-peptide is encountered with formic acid. In this report, fast atom bombardment mass spectrometry and tandem mass spectrometry demonstrate that both Ser8 and Ser26 become O-formylated in 70% aqueous formic acid solutions. The implications of this O-formylation upon the aggregational properties of beta-peptide are discussed.

The secondary structures in solution of the synthetic, naturally occurring, amyloid beta peptides, residues 1 to 42 [beta (1-42)] and beta (1-39), and related fragments, beta (1-28) and beta (29-42), have been studied by circular dichroism and two-dimensional nuclear magnetic resonance spectroscopy. In patients with Alzheimer's disease, extracellular amyloid plaque core is primarily composed of beta (1-42), whereas cerebrovascular amyloid contains the more soluble beta (1-39). In aqueous trifluoroethanol solution, the beta (1-28), beta (1-39), and beta (1-42) peptides adopt monomeric alpha-helical structures at both low and high pH, whereas at intermediate pH (4 to 7) an oligomeric beta structure (the probable structure in plaques) predominates. Thus, beta peptide is not by itself an insoluble protein (as originally thought), and localized or normal age-related alterations of pH may be necessary for the self-assembly and deposition of beta peptide. The hydrophobic carboxyl-terminal segment, beta(29-42), exists exclusively as an oligomeric beta sheet in solution, regardless of differences in solvent, pH, or temperature, suggesting that this segment directs the folding of the complete beta (1-42) peptide to produce the beta-pleated sheet found in amyloid plaques.

A structural study of trehalostatin 1, a specific inhibitor of blowfly trehalase, revealed that 1 contains an unusual five-membered pseudocyclitol.

Im Gegensatz zum Endoperoxid (I) wird das Dioxabicycloheptan (IIa) in H2O mit einer Geschwindigkeit zersetzt, die um 2 Grosenordnungen hoher ist als die von Reaktionen in Losungsmitteln vergleichbarer Polaritat. Fur die Aldehydbildung aus (IIa) und (IIb) ergibt sich ein kinetischer Isotopieeffekt (KIE) von l.5 aber kein D-Verlust und keine pH-Abhangigkeit der Reaktionsgeschwindigkeit.

The neem tree Azadirachta indica A. Juss and the related Chinaberry tree Melia azedarach L. are attracting considerable Interest, particularly because of their insect-repelling properties.1,2 The most potent constituent is azadirachtin, a limonoid 3 which exerts strong physiological4 and phagorepellent activities.5 Incorporating the earlier chemical and spectroscopic studies carried out by Morgan and coworkers 6 and applying the technique of continuous wave proton decoupling in partially relaxed Fourier transform 13C NMR, we proposed structure 1 for azadirachtin in 1975.7 Since evidence for this complex structure was not convincing, we have been using this molecule as a test sample for the application of modern NMR methods, however, without arriving at a conclusive structure.8 After proposals of revised structures by Kraus and coworkers. Ley and coworkers, and ourselves starting In mid-1985,9 its structure was finally established as 2 by Kraus, et al.10 and by Ley, et al.11 In the following we summarize the results of our NMR studies and some of the unique difficulties encountered during the investigation of this compound and its 7,11,20-trimethyl ether.

Arc repressor is a 53-residue sequence-specific DNA binding protein. We report the assignment of the proton NMR spectrum and the secondary structure for the thermostable PL8 variant of Arc. This mutant, which differs from wild type by a Pro-8----Leu substitution, was chosen for study because its enhanced stability allows spectra to be acquired at elevated temperatures where spectral resolution is higher. The first five residues of the protein play important roles in DNA binding but appear to be disordered in solution. Residues 6-14 form the remaining part of the N-terminal DNA binding region of the protein and assume an antiparallel beta-conformation. This indicates that Arc is a member of a new class of DNA binding proteins. The observed interresidue nuclear Overhauser effects are consistent with a beta-strand, gamma-turn, beta-strand structure for the residue 6-14 region, although other structures are also consistent with the data. The remaining portion of the protein is predominantly alpha-helical. Residues 16-26 and 35-50 form amphipathic alpha-helices which may pack together in a four-helix bundle in the protein dimer.