Both the D-(+) and L-(-) enantiomers of nicotine inhibit Abeta aggregation and cytotoxicity

The underlying cause of Alzheimer's disease is thought to be the aggregation of monomeric beta-amyloid (Abeta), through a series of toxic oligomers, which forms the mature amyloid fibrils that accumulate at the center of senile plaques. It has been reported that L-(-)-nicotine prevents Abeta aggregation and toxicity, and inhibits senile plaque formation. Previous NMR studies have suggested that this could be due to the specific binding of L-(-)-nicotine to histidine residues (His6, His13, and His14) in the peptide. Here, we have looked at the effects of both of the L-(-) and D-(+) optical enantiomers of nicotine on the aggregation and cytotoxicity of Abeta(1-40). Surprisingly, both enantiomers inhibited aggregation of the peptide and reduced the toxic effects of the peptide on cells. In NMR studies with Abeta(1-40), both enantiomers of nicotine were seen to interact with the three histidine residues. Overall, our data indicate that nicotine can delay Abeta fibril formation and maintain a population of less toxic Abeta species. This effect cannot be due to a highly specific binding interaction between nicotine and Abeta, as previously thought, but could be due instead to weaker, relatively nonspecific binding, or to the antioxidant or metal chelating properties of nicotine. D-(+)-nicotine, being biologically much less active than L-(-)-nicotine, might be a useful therapeutic agent.     

Alzheimer's disease amyloid-beta binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits

Amyloid beta peptide (Abeta) is the major constituent of extracellular plaques and perivascular amyloid deposits, the pathognomonic neuropathological lesions of Alzheimer's disease. Cu(2+) and Zn(2+) bind Abeta, inducing aggregation and giving rise to reactive oxygen species. These reactions may play a deleterious role in the disease state, because high concentrations of iron, copper, and zinc have been located in amyloid in diseased brains. Here we show that coordination of metal ions to Abeta is the same in both aqueous solution and lipid environments, with His(6), His(13), and His(14) all involved. At Cu(2+)/peptide molar ratios >0.3, Abeta coordinated a second Cu(2+) atom in a highly cooperative manner. This effect was abolished if the histidine residues were methylated at N(epsilon)2, indicating the presence of bridging histidine residues, as found in the active site of superoxide dismutase. Addition of Cu(2+) or Zn(2+) to Abeta in a negatively charged lipid environment caused a conformational change from beta-sheet to alpha-helix, accompanied by peptide oligomerization and membrane penetration. These results suggest that metal binding to Abeta generated an allosterically ordered membrane-penetrating oligomer linked by superoxide dismutase-like bridging histidine residues.     

Zinc binding to Alzheimer's Abeta(1-16) peptide results in stable soluble complex

Aggregation of the human amyloid beta-peptide (Abeta) into insoluble plaques is a key event in Alzheimer's disease. Zinc sharply accelerates the Abeta aggregation in vitro, and the Abeta region 6-28 was suggested to be the obligatory zinc binding site. However, time-dependent aggregation of the zinc-bound Abeta species investigated so far prevented their structural analysis. By using CD spectroscopy, we have shown here for the first time that (i) the protected synthetic peptide spanning the fragment 1-16 of Abeta binds specifically zinc with 1:1 and 1:2 stoichiometry under physiologically relevant conditions; (ii) the peptide-zinc complex is soluble and stable for several months; (iii) zinc binding causes a conformational change of the peptide towards a more structured state. These findings suggest the region 1-16 to be the minimal autonomous zinc binding domain of Abeta.     

Histidine-13 is a crucial residue in the zinc ion-induced aggregation of the A beta peptide of Alzheimer's disease.

Metal ions such as Zn(2+) and Cu(2+) have been implicated in both the aggregation and neurotoxicity of the beta-amyloid (Abeta) peptide that is present in the brains of Alzheimer's sufferers. Zinc ions in particular have been shown to induce rapid aggregation of Abeta. Rat Abeta binds zinc ions much less avidly than human Abeta, and rats do not form cerebral Abeta amyloid. Rat Abeta differs from human Abeta by the substitution of Gly for Arg, Phe for Tyr, and Arg for His at positions 5, 10, and 13, respectively. Through the use of synthetic peptides corresponding to the first 28 residues of human Abeta, rat Abeta, and single-residue variations, we use circular dichroism spectroscopy, sedimentation assays, and immobilized metal ion affinity chromatography to show that the substitution of Arg for His-13 is responsible for the different Zn(2+)-induced aggregation behavior of rat and human Abeta. The coordination of Zn(2+) to histidine-13 is critical to the zinc ion induced aggregation of Abeta.     

Copper selectively triggers beta-sheet assembly of an N-terminally truncated amyloid beta-peptide beginning with Glu3

Metal ions have been suggested to induce aggregation of amyloid beta-peptide (Abeta), which is a key event in Alzheimer's disease. However, direct evidence that specific metal-peptide interactions are responsible for the amyloid formation has not previously been provided. Here we present the first example of the metal-induced amyloid formation by an Abeta fragment, which exhibits a clear-cut dependence on the amino acid sequence. A heptapeptide, EFRHDSG, corresponding to the amino acid residues 3-9 of Abeta (Abeta(3-9)) undergoes a conformational transition from irregular to beta-sheet and self-associates into insoluble aggregates upon Cu(II) binding. A Raman spectrum analysis of the Cu(II)-Abeta(3-9) complex and aggregation assays of mutated Abeta(3-9) peptides demonstrated that a concerted Cu(II) coordination of the imidazole side chain of His6, the carboxyl groups of Glu3 and Asp7, and the amino group at the N-terminus is essential for the amyloid formation. Although Abeta(1-9) and Abeta(2-9) also contain the metal binding sites, neither of these peptides forms amyloid depositions in the presence of Cu(II). The results of this study may not only provide new insight into the mechanism of amyloid formation, but also be important as a step toward the construction of proteinaceous materials with a specific function under the control of Cu(II).     

Solution 1H NMR investigation of Zn2+ and Cd2+ binding to amyloid-beta peptide (Abeta) of Alzheimer's disease

Elevated levels of zinc2+ and copper2+ are found chelated to the amyloid-beta-peptide (Abeta) in isolated senile plaque cores of Alzheimer's disease (AD) patients. However, the precise residues involved in Zn2+ ligation are yet to be established. We have used 1H NMR and CD to probe the binding of Zn2+ to Abeta(1-28). Zinc binding to Abeta causes a number of 1H NMR resonances to exhibit intermediate exchange broadening upon Zn2+ addition, signals in slow and fast exchange are also observed. In addition, there is a general loss of signal for all resonances with Zn2+ addition, suggestive of the formation of high molecular weight polymeric species. Perturbations in specific 1H NMR resonances between residues 6 and 14, and analysis of various Abeta analogues in which each of the three His residues have been replaced by alanine, indicates that His6, His13 and His14 residues are implicated in Zn-Abeta binding. Complementary studies with Cd2+ ions cause perturbations to 1H NMR spectra that are strikingly similar to that observed for Zn2+. Binding monitored at Val12 indicates a 1:1 stoichiometry with Abeta for both Zn2+ and Cd2+ ions. Circular Dichroism (CD) studies in the far-UV indicate quite minimal ordering of the main-chain with Zn2+ or Cd2+ addition. Changes in the far-UV are quite different from that obtained with Cu2+ additions indicating that Zn2+ coordination is distinct from that of Cu2+ ions. Taken together, these observations seem to suggest that Zn2+ coordination is dominated by inter-molecular coordination and the formation of polymeric species.     

Binding of zinc(II) and copper(II) to the full-length Alzheimer's amyloid-beta peptide

There is evidence that binding of metal ions like Zn2+ and Cu2+ to amyloid beta-peptides (Abeta) may contribute to the pathogenesis of Alzheimer's disease. Cu2+ and Zn2+ form complexes with Abeta peptides in vitro; however, the published metal-binding affinities of Abeta vary in an enormously large range. We studied the interactions of Cu2+ and Zn2+ with monomeric Abeta(40) under different conditions using intrinsic Abeta fluorescence and metal-selective fluorescent dyes. We showed that Cu(2+) forms a stable and soluble 1 : 1 complex with Abeta(40), however, buffer compounds act as competitive copper-binding ligands and affect the apparent K(D). Buffer-independent conditional K(D) for Cu(II)-Abeta(40) complex at pH 7.4 is equal to 0.035 micromol/L. Interaction of Abeta(40) with Zn2+ is more complicated as partial aggregation of the peptide occurs during zinc titration experiment and in the same time period (within 30 min) the initial Zn-Abeta(40) complex (K(D) = 60 micromol/L) undergoes a transition to a more tight complex with K(D) approximately 2 micromol/L. Competition of Abeta(40) with ion-selective fluorescent dyes Phen Green and Zincon showed that the K(D) values determined from intrinsic fluorescence of Abeta correspond to the binding of the first Cu2+ and Zn2+ ions to the peptide with the highest affinity. Interaction of both Zn2+ and Cu2+ ions with Abeta peptides may occur in brain areas affected by Alzheimer's disease and Zn2+-induced transition in the peptide structure might contribute to amyloid plaque formation.     

Effect of amino-acid substitutions on Alzheimer's amyloid-beta peptide-glycosaminoglycan interactions.

One of the major clinical features of Alzheimer's disease is the presence of extracellular amyloid plaques that are associated with glycosaminoglycan-containing proteoglycans. It has been proposed that proteoglycans and glycosaminoglycans facilitate amyloid fibril formation and/or stabilize these aggregates. Characterization of proteoglycan-protein interactions has suggested that basic amino acids in a specific conformation are necessary for glycosaminoglycan binding. Amyloid-beta peptide (Abeta) has a cluster of basic amino acids at the N-terminus (residues 13-16, His-His-Gln-Lys), which are considered critical for glycosaminoglycan interactions. To understand the molecular recognition of glycosaminoglycans by Abeta, we have examined a series of synthetic peptides with systematic alanine substitutions. These include: His13-->Ala, His14-->Ala, Lys16-->Ala, His13His14Lys16-->Ala and Arg5His6-->Ala. Alanine substitutions result in differences in both the secondary and fibrous structure of Abeta1-28 as determined by circular dichroism spectroscopy and electron microscopy. The results demonstrate that the His-His-Gln-Lys region of Abeta, and in particular His13, is an important structural domain, as Ala substitution produces a dysfunctional folding mutant. Interaction of the substituted peptides with heparin and chondroitin sulfate glycosaminoglycans demonstrate that although electrostatic interactions contribute to binding, nonionic interactions such as hydrogen bonding and van der Waals packing play a role in glycosaminoglycan-induced Abeta folding and aggregation.     

High-resolution NMR studies of the zinc-binding site of the Alzheimer's amyloid beta-peptide

Metal binding to the amyloid beta-peptide is suggested to be involved in the pathogenesis of Alzheimer's disease. We used high-resolution NMR to study zinc binding to amyloid beta-peptide 1-40 at physiologic pH. Metal binding induces a structural change in the peptide, which is in chemical exchange on an intermediate rate, between the apo-form and the holo-form, with respect to the NMR timescale. This causes loss of NMR signals in the resonances affected by the binding. Heteronuclear correlation experiments, (15)N-relaxation and amide proton exchange experiments on amyloid beta-peptide 1-40 revealed that zinc binding involves the three histidines (residues 6, 13 and 14) and the N-terminus, similar to a previously proposed copper-binding site [Syme CD, Nadal RC, Rigby SE, Viles JH (2004) J Biol Chem 279, 18169-18177]. Fluorescence experiments show that zinc shares a common binding site with copper and that the metals have similar affinities for amyloid beta-peptide. The dissociation constant K(d) of zinc for the fragment amyloid beta-peptide 1-28 was measured by fluorescence, using competitive binding studies, and that for amyloid beta-peptide 1-40 was measured by NMR. Both methods gave K(d) values in the micromolar range at pH 7.2 and 286 K. Zinc also has a second, weaker binding site involving residues between 23 and 28. At high metal ion concentrations, the metal-induced aggregation should mainly have an electrostatic origin from decreased repulsion between peptides. At low metal ion concentrations, on the other hand, the metal-induced structure of the peptide counteracts aggregation.     

Structural changes of region 1-16 of the Alzheimer disease amyloid beta-peptide upon zinc binding and in vitro aging

Amyloid deposits within the cerebral tissue constitute a characteristic lesion associated with Alzheimer disease. They mainly consist of the amyloid peptide Abeta and display an abnormal content in Zn(2+) ions, together with many truncated, isomerized, and racemized forms of Abeta. The region 1-16 of Abeta can be considered the minimal zinc-binding domain and contains two aspartates subject to protein aging. The influence of zinc binding and protein aging related modifications on the conformation of this region of Abeta is of importance given the potentiality of this domain to constitute a therapeutic target, especially for immunization approaches. In this study, we determined from NMR data the solution structure of the Abeta-(1-16)-Zn(2+) complex in aqueous solution at pH 6.5. The residues His(6), His(13), and His(14) and the Glu(11) carboxylate were identified as ligands that tetrahedrally coordinate the Zn(II) cation. In vitro aging experiments on Abeta-(1-16) led to the formation of truncated and isomerized species. The major isomer generated, Abeta-(1-16)-l-iso-Asp(7), displayed a local conformational change in the His(6)-Ser(8) region but kept a zinc binding propensity via a coordination mode involving l-iso-Asp(7). These results are discussed here with regard to Abeta fibrillogenesis and the potentiality of the region 1-16 of Abeta to be used as a therapeutic target.     

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMRsolution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide Abeta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native Abeta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length Abeta peptides Abeta(1-40) and Abeta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which Abeta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of Abeta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation.     

Identifying the minimal copper- and zinc-binding site sequence in amyloid-beta peptides

With a combination of complementary experimental techniques, namely sedimentation assay, Fourier transform infrared spectroscopy, and x-ray absorption spectroscopy, we are able to determine the atomic structure around the metal-binding site in samples where amyloid-beta (Abeta) peptides are complexed with either Cu(II) or Zn(II). Exploiting information obtained on a selected set of fragments of the Abeta peptide, we identify along the sequence the histidine residues coordinated to the metal in the various peptides we have studied (Abeta(1-40), Abeta(1-16), Abeta(1-28), Abeta(5-23), and Abeta(17-40)). Our data can be consistently interpreted assuming that all of the peptides encompassing the minimal 1-16 amino acidic sequence display a copper coordination mode that involves three histidines (His(6), His(13), and His(14)). In zinc-Abeta complexes, despite the fact that the metal coordination appears to be more sensitive to solution condition and shows a less rigid geometry around the binding site, a four-histidine coordination mode is seen to be preferred. Lacking a fourth histidine along the Abeta peptide sequence, this geometrical arrangement hints at a Zn(II)-promoted interpeptide aggregation mode.     

Characterization of copper binding to the peptide amyloid-beta(1-16) associated with Alzheimer's disease

Amyloid-beta peptide (Abeta) is the principal constituent of plaques associated with Alzheimer's disease (AD) and is thought to be responsible for the neurotoxicity associated with the disease. Copper binding to Abeta has been hypothesized to play an important role in the neruotoxicity of Abeta and free radical damage, and Cu2+ chelators represent a possible therapy for AD. However, many properties of copper binding to Abeta have not been elucidated clearly, and the location of copper binding sites on Abeta is also in controversy. Here we have used a range of spectroscopic techniques to characterize the coordination of Cu2+ to Abeta(1-16) in solution. Electrospray ionization mass spectrometry shows that copper binds to Abeta(1-16) at pH 6.0 and 7.0. The mode of copper binding is highly pH dependent. Circular dichroism results indicate that copper chelation causes a structural transition of Abeta(1-16). UV-visible absorption spectra suggest that three nitrogen donor ligands and one oxygen donor ligand (3N1O) in Abeta(1-16) may form a type II square-planar coordination geometry with Cu2+. By means of fluorescence spectroscopy, competition studies with glycine and L-histidine show that copper binds to Abeta(1-16) with an affinity of Ka approximately 10(7) M(-1) at pH 7.8. Besides His6, His13, and His14, Tyr10 is also involved in the coordination of Abeta(1-16) with Cu2+, which is supported by 1H NMR and UV-visible absorption spectra. Evidence for the link between Cu2+ and AD is growing, and this work has made a significant contribution to understanding the mode of copper binding to Abeta(1-16) in solution.     

The effect of Abeta conformation on the metal affinity and aggregation mechanism studied by circular dichroism spectroscopy

The conformational change and associated aggregation of beta amyloid (Abeta) with or without metals is the main cause of Alzheimer's disease (AD). In order to further understand the effects of Abeta and its associated metals on the aggregation mechanism, the influence of Abeta conformation on the metal affinity and aggregation was investigated using circular dichroism (CD) spectroscopy. The Abeta conformation is dependent on pH and trifluoroethanol (TFE). The binding of metals to Abeta was found to be dependent on the Abeta conformation. The aggregation induced by Abeta itself or its associated metals is completely diminished for Abeta in 40% TFE. Only in 5% and 25% TFE can Abeta undergo an alpha-helix to beta-sheet aggregation, which involve a three-state mechanism for the metal-free state, and a two-state transition for the metal-bound state, respectively. The aggregation-inducing activity of metals is in the order, Cu2+ > Fe3+ > or = Al3+ > Zn2+.     

Copper binding to the amyloid-beta (Abeta) peptide associated with Alzheimer's disease: folding, coordination geometry, pH dependence, stoichiometry, and affinity of Abeta-(1-28): insights from a range of complementary spectroscopic techniques

There is now direct evidence that copper is bound to amyloid-beta peptide (Abeta) in senile plaque of Alzheimer's disease. Copper is also linked with the neurotoxicity of Abeta and free radical damage, and Cu(2+) chelators represent a possible therapy for Alzheimer's disease. We have therefore used a range of complementary spectroscopies to characterize the coordination of Cu(2+) to Abeta in solution. The mode of copper binding is highly pH-dependent. EPR spectroscopy indicates that both coppers have axial, Type II coordination geometry, square-planar or square-pyramidal, with nitrogen and oxygen ligands. Circular dichroism studies indicate that copper chelation causes a structural transition of Abeta. Competition studies with glycine and l-histidine indicate that copper binds to Abeta-(1-28) at pH 7.4 with an affinity of K(a) approximately 10(7) m(-1). (1)H NMR indicates that histidine residues are involved in Cu(2+) coordination but that Tyr(10) is not. Studies using analogues of Abeta-(1-28) in which each of the histidine residues have been replaced by alanine or in which the N terminus is acetylated suggest that the N terminus and His(13) are crucial for Cu(2+) binding and that His(6) and His(14) are also implicated. Evidence for the link between Alzheimer's disease and Cu(2+) is growing, and our studies have made a significant contribution to understanding the mode of Cu(2+) binding to Abeta in solution.     

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

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

Humoral immune response to fibrillar beta-amyloid peptide

The beta-amyloid peptide (Abeta) is a normal product of the proteolytic processing of its precursor (beta-APP). Normally, it elicits a very low humoral immune response; however, the aggregation of monomeric Abeta to form fibrillar Abeta amyloid creates a neo-epitope, to which antibodies are generated. Rabbits were injected with fibrillar human Abeta(1-42), and the resultant antibodies were purified and their binding properties characterized. The antibodies bound to an epitope in the first eight residues of Abeta and required a free amino terminus. Additional residues did not affect the affinity of the epitope as long as the peptide was unaggregated; the antibody bound Abeta residues 1-8, 1-11, 1-16, 1-28, 1-40, and 1-42 with similar affinities. In contrast, the antibodies bound approximately 1000-fold more tightly to fibrillar Abeta(1-42). Their enhanced affinity did not result from their bivalent nature: monovalent Fab fragments exhibited a similar affinity for the fibrils. Nor did it result from the particulate nature of the epitope: monomeric Abeta(1-16) immobilized on agarose and soluble Abeta(1-16) exhibited similar affinities for the antifibrillar antibodies. In addition, antibodies raised to four nonfibrillar peptides corresponding to internal Abeta sequences did not exhibit enhanced affinity for fibrillar Abeta(1-42). Antibodies directed to the C-terminus of Abeta bound poorly to fibrillar Abeta(1-42), which is consistent with models where the carboxyl terminus is buried in the interior of the fibril and the amino terminus is on the surface. When used as an immunohistochemical probe, the antifibrillar Abeta(1-42) IgG exhibited enhanced affinity for amyloid deposits in the cerebrovasculature. We hypothesize either that the antibodies recognize a specific conformation of the eight amino-terminal residues of Abeta, which is at least 1000-fold more favored in the fibril than in monomeric peptides, or that affinity maturation of the antibodies produces an additional binding site for the amino-terminal residues of an adjacent Abeta monomer. In vivo this specificity would direct the antibody primarily to fibrillar vascular amyloid deposits even in the presence of a large excess of monomeric Abeta or its precursor. This observation may explain the vascular meningeal inflammation that developed in Alzheimer's disease patients immunized with fibrillar Abeta. Passive immunization with an antibody directed to an epitope hidden in fibrillar Abeta and in the transmembrane region of APP might be a better choice in the search for an intervention to remove Abeta monomers without provoking an inflammatory response.     

Secondary structure conversions of Alzheimer's Abeta(1-40) peptide induced by membrane-mimicking detergents

The amyloid beta peptide (Abeta) with 39-42 residues is the major component of amyloid plaques found in brains of Alzheimer's disease patients, and soluble oligomeric peptide aggregates mediate toxic effects on neurons. The Abeta aggregation involves a conformational change of the peptide structure to beta-sheet. In the present study, we report on the effect of detergents on the structure transitions of Abeta, to mimic the effects that biomembranes may have. In vitro, monomeric Abeta(1-40) in a dilute aqueous solution is weakly structured. By gradually adding small amounts of sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate to a dilute aqueous solution, Abeta(1-40) is converted to beta-sheet, as observed by CD at 3 degrees C and 20 degrees C. The transition is mainly a two-state process, as revealed by approximately isodichroic points in the titrations. Abeta(1-40) loses almost all NMR signals at dodecyl sulfate concentrations giving rise to the optimal beta-sheet content (approximate detergent/peptide ratio = 20). Under these conditions, thioflavin T fluorescence measurements indicate a maximum of aggregated amyloid-like structures. The loss of NMR signals suggests that these are also involved in intermediate chemical exchange. Transverse relaxation optimized spectroscopy NMR spectra indicate that the C-terminal residues are more dynamic than the others. By further addition of SDS or lithium dodecyl sulfate reaching concentrations close to the critical micellar concentration, CD, NMR and FTIR spectra show that the peptide rearranges to form a micelle-bound structure with alpha-helical segments, similar to the secondary structures formed when a high concentration of detergent is added directly to the peptide solution.     

Manipulating the amyloid-beta aggregation pathway with chemical chaperones.

Amyloid-beta (Abeta) assembly into fibrillar structures is a defining characteristic of Alzheimer's disease that is initiated by a conformational transition from random coil to beta-sheet and a nucleation-dependent aggregation process. We have investigated the role of organic osmolytes as chemical chaperones in the amyloid pathway using glycerol to mimic the effects of naturally occurring molecules. Osmolytes such as the naturally occurring trimethylamine N-oxide and glycerol correct folding defects by preferentially hydrating partially denatured proteins and entropically stabilize native conformations and polymeric states. Trimethylamine N-oxide and glycerol were found to rapidly accelerate the Abeta random coil-to-beta-sheet conformational change necessary for fiber formation. This was accompanied by an immediate conversion of amorphous unstructured aggregates into uniform globular and possibly nucleating structures. Osmolyte-facilitated changes in Abeta hydration also affected the final stages of amyloid formation and mediated transition from the protofibrils to mature fibers that are observed in vivo. These findings suggest that hydration forces can be used to control fibril assembly and may have implications for the accumulation of Abeta within intracellular compartments such as the endoplasmic reticulum and in vitro modeling of the amyloid pathway.     

Zinc coordination and substrate catalysis within the neuropeptide processing enzyme endopeptidase EC 3.4.24.15. Identification of active site histidine and glutamate residues.

Endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is broadly distributed within the brain, pituitary, and gonads. Its substrate specificity includes a number of physiologically important neuropeptides such as neurotensin, bradykinin, and gonadotropin-releasing hormone, the principal regulatory peptide for reproduction. In studying the structure and function of EP24.15, we have employed in vitro mutagenesis and subsequent protein expression to genetically dissect the enzyme and allow us to glean insight into the mechanism of substrate binding and catalysis. Comparison of the sequence of EP24.15 with bacterial homologues previously solved by x-ray crystallography and used as models for mammalian metalloendopeptidases, indicates conserved residues. The active site of EP24.15 exhibits an HEXXH motif, a common feature of zinc metalloenzymes. Mutations have confirmed the importance, for binding and catalysis, of the residues (His473, Glu474, and His477) within this motif. A third putative metal ligand, presumed to coordinate directly to the active site zinc ion in concert with His473 and His477, has been identified as Glu502. Conservative alterations to these residues drastically reduces enzymatic activity against both a putative physiological substrate and a synthetic quenched fluorescent substrate as well as binding of the specific active site-directed inhibitor, N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, the binding of which we have shown to be dependent upon the presence, and possibly coordination, of the active site zinc ion. These studies contribute to a more complete understanding of the catalytic mechanism of EP24.15 and will aid in rational design of inhibitors and pharmacological agents for this class of enzymes.