Controlling Amyloid beta -Peptide Fibril Formation with Protease-stable Ligands*

(Received for publication, November 1, 1996, and in revised form, February 10, 1997)

Lars O. Tjernberg Dagger , Christina Lilliehöök Dagger , David J. E. Callaway §, Jan Näslund Dagger , Solveig Hahne Dagger , Johan Thyberg par , Lars Terenius Dagger and Christer Nordstedt Dagger **

From the Dagger  Laboratory of Biochemistry and Molecular Pharmacology, Section of Drug Dependence Research, Department of Clinical Neuroscience, Karolinska Hospital, S-171 76 Stockholm, Sweden, the § Picower Institute for Medical Research, Manhasset, New York 11030, and the par  Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, S-171 77 Stockholm, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

We have previously shown that short peptides incorporating the sequence KLVFF can bind to the ~40amino acid residue Alzheimer amyloid beta -peptide (Abeta ) and disrupt amyloid fibril formation (Tjernberg, L. O., Näslund, J., Lindqvist, F., Johansson, J., Karlström, A. R., Thyberg, J., Terenius, L., and Nordstedt, C. (1996) J. Biol. Chem. 271, 8545-8548). Here, it is shown that KLVFF binds stereospecifically to the homologous sequence in Abeta (i.e. Abeta 16-20). Molecular modeling suggests that association of the two homologous sequences leads to the formation of an atypical anti-parallel beta -sheet structure stabilized primarily by interaction between the Lys, Leu, and COOH-terminal Phe. By screening combinatorial pentapeptide libraries exclusively composed of D-amino acids, several ligands with a general motif containing phenylalanine in the second position and leucine in the third position were identified. Ligands composed of D-amino acids were not only capable of binding Abeta but also prevented formation of amyloid-like fibrils. These ligands are protease-resistant and may thus be useful as experimental agents against amyloid fibril formation in vivo.


INTRODUCTION

Accumulating evidence ascribes the deposition of large fibrillar polymers of the Alzheimer amyloid beta -peptide (Abeta )1 in the brain parenchyma and vasculature (1-3) as the key step in the pathogenesis of Alzheimer's disease. In support of this contention, specific mutations in the gene encoding the beta -amyloid precursor protein (4) have been shown to co-segregate with familial forms of Alzheimer's disease (5, 6). These mutations have phenotypes with either increased production of the Abeta -peptide or formation of structural variants of the Abeta -peptide (e.g. terminating at amino acid Abeta 42) that more easily aggregate and form amyloid fibrils (7-9). Mutations in the genes encoding the presenilins that also co-segregate with early onset familial Alzheimer's disease (10-13) lead to an increased production of Abeta X-42 (14). Moreover, transgenic mice expressing mutant human Abeta precursor protein develop lesions similar to those found in Alzheimer's disease (15, 16).

A number of pharmacological strategies aimed at decreasing the production of the Abeta -peptide or precluding its tissue deposition have been suggested. These include the use of protease inhibitors to prevent amyloidogenic processing of Abeta precursor protein (17), altering Abeta precursor protein metabolism by pharmacological manipulation of signal transduction pathways (18), and inhibition of amyloid fibril formation with small molecules (19-23). Inhibition of fibril formation is essential since fibrils of Abeta , but not monomeric Abeta , are toxic to neurons (21, 24, 25) and protease-resistant (26), and therefore can accumulate in and probably also damage the tissue.

Previously, it was shown that residues 16-20 in the Abeta -peptide (KLVFF) are important in the Abeta -Abeta interaction (19, 27). The linear hexapeptide Ac-QKLVFF-NH2 was found to be capable of both binding to the Abeta -peptide and preventing its polymerization into fibrils (19). In the present work we have focused on (i) identifying the structure or structures in the Abeta -peptide that bind KLVFF-containing ligands, (ii) using molecular modeling to study the interaction between these ligands and model their binding site, (iii) developing novel peptide ligands based on amino acids in D configuration, and (iv) investigating the effects of these novel metabolically stable ligands on amyloid fibril formation in vitro.


EXPERIMENTAL PROCEDURES

Materials

Synthetic Abeta 1-40 was obtained from Dr. David B. Teplow, Biopolymer Laboratory at Harvard University. All other soluble peptides were obtained from Research Genetics, Huntsville, AL. Unless otherwise indicated, all reagents were from Sigma. The peptide KKLVFFA (this peptide will be referred to hereafter as LBMP1620) and Abeta 1-40 were iodinated using the Bolton-Hunter technique. Following the reaction, the iodinated peptide was purified on a Vydac C-4 reverse phase liquid chromatography column (0.21 × 15 cm) using a solvent system containing 0.1% trifluoroacetic acid in water (buffer A) and 0.1% trifluoroacetic acid in acetonitrile (buffer B).

Synthesis of Peptides on Cellulose Membranes

The technique used is essentially identical to the SPOT technique described by Frank (28). Cellulose membranes (Whatman 1Chr) were derivatized with N,N'-diisopropylcarbodiimide-activated Fmoc beta -alanine. A spacer, consisting of 2 molecules of beta -alanine, was coupled to derivatized filters. The indicated peptides were then synthesized using Fmoc-protected and pentafluorophenyl- or N,N'-diisopropylcarbodiimide-activated amino acids dissolved in N-methylpyrrolidone. Coupling efficiency was monitored by bromphenol blue staining.

Binding Experiments

Following the blocking of filters with 0.05% Tween 20 in Tris-buffered saline (TBS), they were incubated in the presence of 2-20 µM 125I-labeled Abeta 1-40 or 125I-labeled LBMP1620 at 20 °C for 12 h in TBS, pH 7.4, supplemented with 1% bovine serum albumin. Subsequently, the filters were washed repeatedly in the same buffer containing 0.5 M NaCl and dried. Radioactivity bound to the filters was visualized by autoradiography and quantitated using densitometry.

Molecular Modeling of KLVFF Docking to the Homologous Abeta Sequence

The docking of a KLVFF (Abeta 16-20) pentamer to an Abeta 13-23 segment was studied using the Insight/Discover 2.9.7 program suite (Biosym/Molecular Simulations, San Diego, CA). The two polypeptides were simulated in a periodic box (47 × 20 × 24 Å) with 607 explicit water molecules and an 8-Å cut-off. Default values were used for all other conventions. The Abeta 13-23 segment was initialized in a beta  sheet conformation.

Inhibition of Amyloid Fibril Formation

The ligands were dissolved in hexafluoroisopropyl alcohol and diluted with TBS to a final concentration of 100 µM. Abeta was dissolved in hexafluoroisopropyl alcohol before the addition of the freshly prepared ligand solution to a final Abeta concentration of 100 µM. The final concentration of hexafluoroisopropyl alcohol was 2%. In another experiment the samples were dissolved directly in TBS. The samples were thereafter incubated at 37 °C for 48 h. Subsequently, the peptides were examined by electron microscopy, and inhibition of fibril formation was estimated semiquantitatively.

Electron Microscopy

The peptides were dissolved in TBS (pH 7.4) and incubated at 37 °C for 48 h. Polymers were sedimented by centrifugation at 20,000 × g for 20 min, the supernatant was aspirated, and the pellet was resuspended in 100 µl of water by a short sonication. Aliquots (8 µl) of the resuspended material were placed on grids covered by a carbon-stabilized Formvar film. After 30 s, excess fluid was withdrawn, and the grids were negatively stained with 3% uranyl acetate in water. Finally, the specimens were examined and photographed in a Jeol electron microscope 100CX at 60 kV.


RESULTS

Identification of Sequences in the Abeta -peptide Serving as Binding Sites for KLVFF-containing Ligands

The previously used peptide sequence Ac-QKLVFF-NH2 has a limited solubility in aqueous buffers. We therefore synthesized a peptide sequence with improved water solubility, LBMP1620.

The 31 possible decamers corresponding to Abeta 1-10, Abeta 2-11, Abeta 3-12, ... , Abeta 31-40 were synthesized on a cellulose membrane with their COOH terminus covalently bound to the membrane as described (28). The membrane was incubated overnight at 20 °C in the presence of 2 µM 125I-LBMP1620. Following repeated washing in TBS, binding of 125I-LBMP1620 to the immobilized peptides was determined by autoradiography and subsequent densitometry. The radioligand bound to peptides Abeta 11-20, Abeta 12-21, Abeta 13-22, Abeta 14-23, Abeta 15-24, and Abeta 16-25. This binding pattern is similar to the one found when immobilized peptides were incubated with 125I-Abeta 1-40 (19). The decamer Abeta 13-22 (i.e. HHQKLVFFAE) was selected for further studies. The minimal sequence required for 125I-LBMP1620 binding was determined by systematic truncation at the NH2- and COOH terminus, respectively (Fig. 1). The results revealed that the LBMP1620-binding site had certain distinct features. (i) Only pentapeptides or larger peptides displayed significant binding, and (ii) binding peptides contained the sequence KLVFF or LVFFA (i.e. Abeta 16-20 or Abeta 17-21). It is concluded that the Abeta -ligand LBMP1620 binds to the Abeta -peptide by interacting with a domain with a homologous primary sequence.


Fig. 1. Screening of NH2- and COOH-terminal truncated versions of Abeta 13-22 with 125I-LBMP1620. The indicated peptides were synthesized on a cellulose filter as described under "Experimental Procedures." The filter was blocked with TBS-Tween and TBS-bovine serum albumin and incubated in the presence of 2 µM 125I-LBMP1620 for 5 h at 20 °C. Following incubation, the filter was washed extensively in TBS, and radioactivity was visualized by autoradiography and quantitated by densitometry.
[View Larger Version of this Image (29K GIF file)]

A peptide entirely composed of amino acids in D configuration with the sequence klvff (lowercase marks amino acids in D configuration) was synthesized using the SPOT technique and assayed for 125I-LBMP1620 binding. This peptide failed to bind 125I-LBMP1620 (data not shown) indicating that the KLVFF-KLVFF interaction is stereospecific.

Immobilized Abeta 16-20 Shows Decreased Binding to 125I-Abeta 1-40 after Incubation with LBMP1620

KLVFF was synthesized on cellulose membranes. They were then incubated overnight at 20 °C with incubation buffer alone, 20 µM Abeta 1-40, or LBMP1620. Following washing, the immobilized peptide was incubated with 20 µM 125I-Abeta 1-40 for 5 h. After the removal of non-bound radiolabeled peptide by repeated washing, binding of 125I-Abeta 1-40 was determined by autoradiography and densitometry. Preincubation of KLVFF with Abeta 1-40 did not affect 125I-Abeta 1-40 binding, whereas preincubation with LBMP1620 reduced 125I-Abeta 1-40 binding by 49%. This confirms that short KLVFF-containing peptides bind to Abeta 16-20 and thereby block further Abeta 1-40 binding. Therefore, it is reasonable to assume that a short peptide or a functionally similar molecule capable of binding to Abeta 16-20 may inhibit Abeta polymer growth.

Structural Modeling of KLVFF Docking to Abeta

Incubation of KLVFF in aqueous buffers leads to aggregation and precipitation (19), which complicates studies of the structural basis for binding using solution phase techniques (e.g. NMR). The small sizes of the present peptides made them particularly suitable for computer simulations using molecular modeling. Repeated runs of molecular dynamics were followed by conjugate gradient energy minimization. Peptides with the sequences HHQKLVFFAED (Abeta 13-23) and KLVFF (Abeta 16-20) were docked (Fig. 2). Two common docking motifs were observed roughly corresponding to parallel and anti-parallel beta -sheet conformations, respectively. The parallel conformation was considerably higher in energy, and hence the anti-parallel conformation was chosen for further studies. In dynamic simulations of up to several picoseconds in duration, a typical docking pattern involved the approach of the portion of the KLVFF pentamer comprised of the alkane-like basic side chain of the Lys and the Leu of the ligand to the Phe (Abeta 20) of Abeta 13-23. The COOH-terminal Phe of the pentamer typically projected outward. This motif would suggest a prominent role for Lys, Leu, and the COOH-terminal Phe residues in Abeta aggregation as observed in earlier experiments (19). Over the course of the run, the Abeta segment developed an elbow, with the docked pentamer in the interior. Presumably, during longer simulations this conformational shift could significantly enhance the binding of KLVFF pentamers to Abeta .


Fig. 2. Molecular modeling of the complex formed between Abeta 13-23 and KLVFF. The docking configuration between Abeta 13-23 (gray/yellow) and a KLVFF pentamer (violet/blue) as predicted by molecular simulations described in text is shown. Stick models are used for all residues except for three residues critical for binding, which are highlighted by ball-and-stick models. Residue Phe (Abeta 20) of Abeta is indicated in yellow, and Lys and Leu residues of KLVFF are indicated in blue.
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Identification of Abeta -ligands Exclusively Composed of Amino Acids in D Configuration Using Combinatorial Peptide Chemistry

LBMP1620 contains several proteolytic cleavage sites and is highly sensitive to proteolysis (26). We therefore decided to design a ligand with the same binding properties as LBMP1620 but that would be resistant to proteolysis. Since proteolytic enzymes are not capable of hydrolyzing peptide bonds between amino acids in D configuration, ligands entirely composed of D-amino acids should be able to withstand enzymatic proteolysis. Here it was shown that the shortest peptide binding efficiently to LBMP1620 was a pentamer (see Fig. 1). Therefore, pentapeptides exclusively composed of D-amino acids were synthesized. A combinatorial pentapeptide library, theoretically containing all 2,476,099 permutations of the 19 D-amino acids (cysteine was excluded to avoid unwanted disulfide bonds), was synthesized as described (29) and subsequently was assayed for 125I-LBMP1620 binding. By this design, an individual peptide spot on the membrane contained 130,321 different peptide sequences. As seen in Fig. 3, all amino acid residues mediating 125I-LBMP1620 binding had either basic side chains (h, k, r), aromatic side chains (f, y, w) or non-polar side chains (i, l, m). No amino acid residues with acidic side chains promoted binding, strongly suggesting that electrostatic interactions (i.e. with Lys in LBMP1620) were not critical for binding. Assaying an identical library for 125I-Abeta 1-40 binding yielded similar results (data not shown).


Fig. 3. Screening of a combinatorial D-pentapeptide library for 125I-LBMP1620 binding properties. D-pentapeptide libraries were synthesized as described using the SPOT technique and assayed for 125I-LBMP1620 binding as described under "Experimental Procedures." The NH2 termini of the synthesized peptides were acetylated. Amino acids in D configuration are indicated by lowercase 1-letter code. Positions indicated with x correspond to a mixture (equal molar ratios) of D-amino acids Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr (cysteine was excluded). In the positions indicated with o1-o5, the amino acid shown on the x axis has been coupled.
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The three amino acids in each position in the pentapeptide library that most prominently promoted 125I-LBMP1620 binding were selected for additional studies (Table I). The 243 possible pentapeptide combinations of these D-amino acids were synthesized individually and assayed for 125I-LBMP1620 binding. The 39 peptides binding most efficiently to 125I-LBMP1620 are listed in Fig. 4. As seen in the figure, f in the second and l in the third position were critical for binding. The f in position 2 could be replaced with y, and l in position 3 could be replaced with m, but not concurrently. In position 1, l and y showed higher binding than w, whereas l was favored in position 4. The fifth residue could be either one of the three amino acids tested. It is unlikely that binding observed in these experiments was the result of nonspecific protein adhesion since the reaction mixture contained relatively large quantities of bovine serum albumin.

Table I. The three amino acid residues that, in the indicated position, most efficiently promoted pentapeptide binding to 125I-Abeta 1-40


Position D-Amino acid

O-1 y l w
O-2 y f w
O-3 l m r
O-4 l m r
O-5 l m r


Fig. 4. D-Ligands capable of binding Abeta 16-20. The 243 permutations of the amino acids shown in Table I were synthesized and assayed for 125I-LBMP1620 binding. The indicated 39 peptides bound the radiolabeled peptide.
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Functional Properties of Pentapeptide Ligands Identified in Combinatorial Libraries of All D-Amino Acids

From the 39 125I-LBMP1620 binding peptides showed in Fig. 4, yfllr and lflrr were selected for further studies. The reason for selecting these peptides was that they contain one or two D-arginine residues, thus making them more hydrophilic and water-soluble than other binding peptides. We also selected a non-binding peptide with the sequence yyrrl as control. The peptides were incubated with synthetic Abeta 1-40 at equimolar concentrations (100 µM) as described (19). When Abeta 1-40 was incubated alone, a dense network of amyloid fibrils with a diameter of about 6-8 nm and undefined lengths was formed (Fig. 5A). The addition of the non-binding control peptide yyrrl had no effect on the polymerization of Abeta 1-40 (Fig. 5B). On the other hand, lflrr markedly reduced the amount of polymers produced and instead of elongated fibrils short rodlike structures were formed (Fig. 5C). The putative toxicity of these structures has not been studied. However, it has been shown that it is the fibrillar form, not the amorphous form, of aggregated Abeta that mediates neurotoxicity (21). A less distinct effect was obtained with yfllr under these conditions, and a mixture of elongated fibrils and diffuse aggregates appeared (Fig. 5D). When the D-pentapeptides were incubated alone no detectable polymers or aggregates were formed (not shown), in partial contrast to the previously studied KLVFF-containing peptide ligand made up of L-amino acids (19).


Fig. 5. Effects of D-pentapeptides on polymerization of Abeta 1-40 at equimolar concentration. Abeta 1-40 (100 µM) was incubated in TBS (pH 7.4) for 48 h at 37 °C either alone (A) or together with equimolar concentrations of the D-pentapeptides yyrrl (B), lflrr (C), or yfllr (D). Electron micrographs were made after negative staining with uranyl acetate. Scale bars, 100 nm.
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In another set of experiments, a lower concentration of Abeta 1-40 (5 µM) was incubated with a 10-fold excess of the D-pentapeptides (50 µM). In this case, Abeta 1-40 alone gave rise to fibrils with a similar width but shorter length than described above. Both lflrr and yflrr almost completely blocked the formation of fibrils, whereas yyrrl again had no effect (not shown). No differences between samples predissolved in hexafluoroisopropyl alcohol and samples dissolved directly in TBS were detected. The present method is designed to assay the inhibition of amyloid fibril formation, and hence we cannot exclude the possibility that small, potentially toxic, aggregates are being formed in the presence of ligands. However, preliminary results from fluorescence correlation spectroscopy studies indicate that LBMP1620 dissolves preformed soluble Abeta aggregates completely.2


DISCUSSION

Our previous data indicated that the KLVFF (Abeta 16-20) sequence was critical for Abeta fibril formation. However, it was not clear whether fibril formation involved interaction between homologous sequences or not. Here, we have used LBMP1620 as the labeled probe for screening defined sequences in Abeta 1-40, ranging from tripeptides to decapeptides, and thereby identified the sequence that KLVFF-containing ligands bind to within the Abeta 1-40 molecule. The identified binding site docking to KLVFF was then subjected to molecular modeling.

Our next aim was to use the acquired data to search for a protease-stable inhibitor of Abeta fibril formation. Random combinatorial pentapeptide libraries made up from D-amino acids, and consequently protease-stable, were probed with labeled LBMP1620. The three D-amino acids most efficiently promoting binding in each position were selected, and out of these all 243 possible pentapeptides were synthesized and assayed for LBMP1620 binding. Two of the binding peptides and one non-binding peptide were synthesized on a larger scale, and their capacity of inhibiting Abeta fibril formation in solution was tested.

The first experiments conclusively showed that the KLVFF-containing ligand binds to the homologous sequence in Abeta (Fig. 1). The data obtained in binding studies were used in computer simulations of docking between KLVFF and Abeta 13-23. An anti-parallel arrangement was shown in the minimum energy conformation. Residues Lys and Leu in KLVFF interact with the Phe (Abeta 20) in the homologous sequence in Abeta 13-23, thereby stabilizing the formation of a beta -sheet structure (Fig. 2). This is in agreement with previous experimental data showing that the substitution of Lys, Leu, and the COOH-terminal Phe in KLVFF with Ala leads to loss of binding capacity (19).

Since the first experiment revealed that LBMP1620 binding requires a sequence of at least five amino acids, we chose to use a combinatorial library consisting of pentamers of D-amino acids. By using a short peptide capable of binding to a region critical for polymerization of Abeta (i.e. LBMP1620), the risk of identifying D-pentapeptides binding to non-relevant regions of Abeta 1-40 (i.e. NH2- and COOH-terminal to Abeta 16-20) was minimized. Of the 243 pentapeptides synthesized from the most efficiently binding amino acids (Table I), 39 were found to bind 125I-LBMP1620. The composition of these binding peptides indicates that hydrophobic interactions are necessary for binding (Fig. 4). Although r can be found in position 4 or 5, l is more frequent in these positions. Positions 2 and 3 were most critical for binding, being l and f, respectively, in most of the binding peptides. In position 1, l and f were preferred over w. Some of the binding peptides showed similarities with a reversed KLVFF, e.g. yfllr (Fig. 4), indicating the formation of a parallel beta -sheet (KLVFF forming an anti-parallel beta -sheet according to molecular modeling). However, the positions critical for binding to LBMP1620, 2 (f) and 3 (l), would then correspond to non-critical residues in KLVFF. Molecular modeling of the D-amino acid ligands docking to KLVFF might reveal the nature of this interaction.

An interesting question is how Abeta -ligands, such as those described here, affect the physiological and pathological properties of aggregated and non-aggregated Abeta -peptide. It is well known that aggregated full-length and truncated variants of the Abeta -peptide are toxic to several neuronal and non-neuronal cell types (30). Moreover, substances capable of inhibiting the polymerization have been shown to prevent Abeta -associated toxicity (21-23), which points to the fact that the higher order structure is important for the toxicity of these peptides. An Abeta -ligand has to meet several important criteria to be a useful inhibitor of fibril formation in vivo. These include acceptable bioavailability, pharmacokinetics allowing passage of the blood-brain barrier, access to the desired site of action, and low toxicity. The present D-ligands are resistant to proteolysis and should therefore have acceptable bioavailability. Those that were studied functionally were chosen because of their relatively high hydrophilicity. Ligands to be used in vivo should probably be more hydrophobic because this increases the probability that they will pass the blood-brain barrier (31). Several of the 39 D-ligands presented in Fig. 4 have a high content of hydrophobic residues and hence should have properties allowing them to pass the blood-brain barrier. At present, there is no information regarding the toxicity of these compounds. On balance, several of the D-ligands are potentially useful for testing the hypothesis that Abeta -amyloidogenesis in vivo may be inhibited by ligands binding to the KLVFF motif.

A fragment of the Abeta -peptide corresponding to amino acids 25-35 undergoes polymerization and has neurotoxic properties (30, 32, 33). In these and our previous studies (19), we have not been able to identify ligands capable of binding efficiently to this region of the peptide. There are several possibilities for this failure. (i) A ligand efficiently binding to this region has to be larger than the penta- to decapeptides tested here and previously (19); (ii) the covalent linkage of ligands to a matrix via their COOH termini may prevent the adoption of the conformation necessary for interaction with (i.e. binding to) amino acids 25-35; and (iii) the polymerization of the hydrophobic 25-35 segment is not directly related to the polymerization of full-length Abeta -peptide.

The overall conclusion from these studies is that not only the KLVFF peptide (Abeta 16-20) but also structurally different peptides consisting of non-natural amino acids have the capability of binding Abeta and preventing its assembly into amyloid fibrils. Therefore, it is reasonable to assume that pharmacologically useful organic non-peptide molecules with similar functional properties as the present ligands can be synthesized.


FOOTNOTES

*   This work was supported in part by The Swedish Medical Research Council (to J. T. and L. T.), The Swedish Heart Lung Foundation (to J. T.), and The King Gustaf V 80th Birthday Fund (to J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Supported in part by a fellowship from The Swedish Society for Medical Research.
**   Recipient of fellowships from The Swedish Medical Research Council, The Berth von Kantzow Foundation, The Swedish Society for Medical Research, The Axel and Margret Ax:son Johnson Foundation, and The Nicholson Foundation. To whom correspondence should be addressed. Tel.: 46-8-517 73 919; Fax: 46-8-517 76 80; E-mail: Christer.Nordstedt{at}cmm.ki.se.
1   The abbreviations used are: Abeta , Alzheimer amyloid beta -peptide; LBMP1620, Lys-Lys-Leu-Val-Phe-Phe-Ala; Fmoc, N-(9-fluorenyl)methoxycarbonyl; TBS, Tris-buffered saline.
2   L. O. Tjernberg, A. Pramanik, J. Thyberg, R. Rigler, L. Terenius, and C. Nordstedt, manuscript in preparation.

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