Soluble Amyloid Abeta -(1-40) Exists as a Stable Dimer at Low Concentrations*

(Received for publication, March 7, 1997, and in revised form, May 14, 1997)

William Garzon-Rodriguez Dagger §, Marisa Sepulveda-Becerra , Saskia Milton Dagger and Charles G. Glabe Dagger par

From the Departments of Dagger  Molecular Biology and Biochemistry and  Psychobiology, University of California, Irvine, California 92696 and the § Universidad Nacional Autonoma de Mexico, Facultad de Quimica, Mexico D.F., Mexico 04510

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Recent studies have implicated the amyloid Abeta peptide and its ability to self-assemble as key factors in the pathogenesis of Alzheimer's disease. Relatively little is known about the structure of soluble Abeta or its oligomeric state, and the existing data are often contradictory. In this study, we used intrinsic fluorescence of wild type Abeta -(1-40), fluorescence resonance energy transfer (FRET), and gel filtration chromatography to examine the structure of Abeta -(1-40) in solution. We synthesized a series of mono-substituted fluorescent Abeta -(1-40) derivatives to use as donors and acceptors in FRET experiments. We selected fluorescent peptides that exhibit aggregation properties comparable to wild type Abeta for analysis in donor-acceptor pairs; two labeled with 5-(2-((iodoacetyl)amino)ethyl)aminonaphthylene-1-sulfonic acid at Cys-25 or Cys-34 and fluorescein maleimide at Cys-4 or Cys-7. Another peptide containing a Trp substitution at position 10 was used as an acceptor for the intrinsic Tyr fluorescence of wild type Abeta -(1-40). Equilibrium studies of the denaturation of Abeta -(1-40) by increasing concentrations of dimethyl sulfoxide (Me2SO) were conducted by monitoring fluorescence, with a midpoint value for the unfolding transition of both the substituted and wild type peptides at among 40 and 50% Me2SO. Abeta -(1-40) is well solvated and largely monomeric in Me2SO as evidenced by a lack of FRET. When donor and acceptor Abeta derivatives are mixed together in Me2SO and then diluted 10-fold into aqueous Tris-HCl buffer at pH 7.4, efficient FRET is observed immediately for all pairs of fluorescent peptides, indicating that donor-acceptor dimers exist in solution. FRET is abolished by the addition of an excess of unlabeled Abeta -(1-40), demonstrating that the fluorescent peptides interact with wild type Abeta -(1-40) to form heterodimers that do not exhibit FRET. The Abeta -(1-40) dimers appear to be very stable, because no subunit exchange is observed after 24 h between fluorescent homodimers. Gel filtration confirms that nanomolar concentrations of 14C-labeled Abeta -(1-40) and fluorescein-labeled Abeta -(1-40) elute at the same dimeric position as wild type Abeta -(1-40), suggesting that soluble Abeta -(1-40) is also dimeric at more physiologically plausible concentrations.


INTRODUCTION

The extracellular deposition of beta -amyloid in senile plaques is one of neuropathological hallmarks of Alzheimer's disease. Biochemical analysis of the amyloid peptides isolated from Alzheimer's disease brain indicates that amyloid beta  (Abeta )1 1-42 is the principal species associated with senile plaque amyloid (1), while Abeta -(1-40) is more abundant in cerebrovascular amyloid deposit. Abeta is folded into the beta -sheet structure that is characteristic of amyloid fibrils. Amyloid plaque formation may involve two basic steps: the initial formation of a seeding aggregate that establishes the amyloid fibril lattice (2), followed by the elongation of the fibril by the sequential addition of subunits (3). Some of the key parameters that promote the assembly of amyloid fibril include high peptide concentration, long incubation time and low pH (pH 5-6) (4-6), solvent composition (7), and salt concentration (8). The length of the carboxyl terminus is also critical in determining the assembly dynamics. The Abeta -(1-42) isoform aggregates at a significantly greater rate and to a greater extent at pH 7.4 than Abeta -(1-40). Assembly of Abeta into the fibrils may also be promoted by molecules that interact with Abeta and increase its rate of aggregation in vitro including apolipoprotein E (9, 10), alpha 1-antichymotrypsin (11), complement C1q (1), heparin sulfate proteoglycan (12), and zinc ions (13, 14).

Although many of the parameters influencing fibril assembly have been elucidated, relatively little is known about the structure of soluble Abeta . Understanding the structure of Abeta may provide insight into how this peptide assembles into the amyloid fibrils characteristic of Alzheimer's disease and other amyloidoses. Gel filtration analyses of Abeta in solution have revealed the presence of multiple, discrete structures that have been variously interpreted as monomer, dimer, trimer, and higher order aggregates (8, 13, 15-17). While differences in the concentration of the peptide, time of incubation, and the structure of Abeta (16) can account for some of the discrepancies in the oligomeric species observed in the studies cited above, it is not clear that the identities assigned to the peaks are correct, since gel filtration only measures the effective hydrodynamic radius of a molecule and not its molecular mass.

Previous studies from this laboratory indicate that the smallest species in aqueous solution elutes from a gel filtration column with an apparent molecular mass appropriate for a dimer for both Abeta -(1-40) and Abeta -(1-42) and at higher concentrations, larger micelle-like oligomers also exist in solution (16). The presence of these micelle-like oligomers has been confirmed by dynamic light scattering measurements (18, 19) and analytical ultracentrifugation (19). In this work, we have used the intrinsic fluorescence of wild type Abeta -(1-40) and fluorescence resonance energy transfer in conjunction with gel filtration chromatography to define the oligomeric state of soluble Abeta -(1-40). Here we report that soluble Abeta -(1-40) forms a stable dimer at concentrations from the low nanomolar range up to the critical concentration of approximately 25 µM, as evidenced by the observation of efficient FRET between several different combinations of donor and acceptor peptides. This suggests that soluble Abeta -(1-40) adopts an ordered conformation in solution as a prelude to fibril assembly and that dimerization is an initial event in amyloid self-assembly.


EXPERIMENTAL PROCEDURES

Materials

All Abeta peptide analogs were synthesized by fluoren-9-ylmethoxy carbonyl chemistry using a continuous flow semiautomatic instrument as described previously (5). The peptides were purified by reverse phase high performance liquid chromatography, and the purity and expected structure was verified by electrospray mass spectrometry. Only peptides exhibiting 90.0% or greater purity with less than 5.0% of a single contaminant were used. Cys substitution mutants were synthesized simultaneously by the same method, except that at locations where Cys was substituted, a portion of the resin was coupled separately with Cys. [3H]Abeta -(1-40) and [14C]Abeta -(1-40) were synthesized by incorporation of Fmoc-[3H]Phe or Fmoc-[14C]Ala at positions 4 and 2 respectively, yielding specific activities of 200 mCi/mmol for [3H]Abeta -(1-40) and 36 mCi/mmol for [14C]Abeta -(1-40). 1,5-IAEDANS and FM were obtained from Molecular Probes (Eugene, OR). All other reagents were of the highest analytical grade commercially available. We use a shorthand notation to refer to the Abeta -(1-40) analogs that indicates the position of the Cys substitution with the understanding that all peptides are 40 residues long and the rest of the sequence is that of wild type Abeta as described in the abbreviations list.

Fluorescence Labeling of Mutant Abeta -(1-40) with 1,5-IAEDANS and FM

Since Abeta -(1-40) was modified with a single Cys at different positions, the sulfydryl-specific reagents FM and 1,5-IAEDANS were used to prepare fluorescent derivatives. The Abeta -(1-40) analog peptide was dissolved in 10 mM MOPS, pH 8.5, at a concentration of 25 µM (pH 7.4 in the case of fluorescein labeling). 1,5-IAEDANS or FM was added to this solution from a stock solution of 10 mM at a 20-fold (for 1,5-IAEDANS) or 5-fold (for FM) molar excess over Abeta -(1-40). The reaction was allowed to proceed at room temperature in the dark for 6 h. Free fluorophore was then removed by filtration on a Sephadex G-25 column equilibrated with 10 mM MOPS at pH 7.4. Labeled Abeta -(1-40) was aliquoted, lyophilized, and stored at -20 °C. Protein was determined by Coomassie R protein assay reagent (Pierce). The concentrations of 1,5-IAEDANS or FM were spectrophotometrically determined by using their molar extinction coefficients (5.7 mM-1 at 336 nm or 83 mM-1 at 490 nm, respectively). The labeling stoichiometry of the final products was 1.0. The stoichiometry was confirmed by laser desorption mass spectrometry that demonstrated all of the precursor has been converted to a mass appropriate for fluorescent peptide.

Aggregation Measurements

Aggregation was determined using a sedimentation assay as described previously (5). 75 µM [3H]Abeta -(1-40) (specific activity 36 mCi/mmol) was mixed with 5 µM fluorescent Abeta -(1-40) in either 0.1 M NaAc, pH 5.0, 0.1 M NaCl, 20 mM Tris-HCl, pH 7.4 or 0.1 M NaCl, 20 mM Tris-HCl, 70 µM ZnCl2, and incubated for 48 h at room temperature. The radiolabeled and fluorescent peptides were diluted 10-fold from water stock solutions upon mixing. The samples were centrifugated at 15,000 × g for 10 min in a Beckman Microfuge 11. Afterward, the amount of fluorescent and tritiated wild type Abeta in both the supernatant and pellet was determined by measuring the fluorescence intensity and the radioactivity by scintillation counting.

Gel Filtration Chromatography

Gel filtration analysis was performed with a Pharmacia Superdex 75 HR 10/30 column using a Waters 490 multiple wavelength UV absorbance detector and Hewlett Packard 3250 fluorescence detector. Data were collected with a Waters Maxima chromatography data system, with a flow rate of 0.4 ml/min. Because the refolding experiments of Abeta -(1-40) were carried out by diluting the Me2SO to subdenaturing concentrations, the running buffer used was 50 mM Tris-HCl, 0.1 M NaCl, pH 7.4 (buffer A) in the presence of 10% or 2% Me2SO. The column was calibrated several times using the buffer A in the presence of 10% or 2% Me2SO, the standards were chromatographed both separately and in mixtures, obtaining the same calibration curve in all conditions. The standards used to calibrate the column and their masses (Da) are: thyroglobulin (670,000), bovine serum albumin (68,000), ovalbumin (43,000), soybean trypsin inhibitor (23,000), ubiquitin (8500), aprotinin (6500), and acetone (58). The peptides were detected by UV absorbance at 280 nm and by fluorescence emission at 482 nm for 1,5-IAEDANS (or at 520 nm for FM) upon excitation at 336 nm (or 490 nm for FM).

Denaturation of Abeta -(1-40) in Me2SO and Its Refolding

11 µM Abeta -(1-40) or Abeta C25AEDANS was incubated in increasing concentrations of Me2SO, at 24 °C. Emission spectra were recorded after 30 min incubation from 290 to 400 nm upon excitation at 280 nm (or from 346-600 upon excitation at 336 nm for Abeta C25AEDANS). Emission spectra were not recorded at longer incubation times as Abeta aggregates (19). For refolding experiments, samples were incubated in 100% Me2SO for 30 min at room temperature and refolding was initiated by 10-or 50-fold dilution of the Me2SO solution in Tris-HCl buffered solution. The concentration of peptide ranged between 3 and 10 µM.

Absorption and Fluorescence Measurements

Absorption measurements were measured with a Perkin Elmer Lambda 3B UV-Vis spectrophotometer. Fluorescence spectra (excitation band pass 4 nm; emission band pass 8 nm) were measured either on an Aminco SLM 48000 or a SPEX Fluorolog F112A spectrofluorometer. Intrinsic Tyr fluorescence was measured from 285 to 400 upon excitation at 275 nm. For Abeta C25AEDANS or in energy transfer experiments, excitation was at 330, and the spectra were obtained from 340 to 620 nm. In all cases, emission fluorescence spectra of identical samples (without protein) were recorded. These were subtracted from the experimental samples. The lifetime measurements for FM were acquired using the 488 nm line of argon ion laser for excitation using a multiharmonic frequency-domain spectrofluorometer (Aminco 48000S).

Fluorescence Resonance Energy Transfer

The efficiency (E) of fluorescence resonance energy transfer (FRET) between probes was determined by measuring the fluorescence intensity of the donor (Abeta C-AEDANS or Abeta -(1-40)) both in the absence (Fd) and presence (Fda) of the acceptor (Abeta C-FM or Abeta Y10W), as given by Equation 1.
E=1−(F<SUB><UP>da</UP></SUB>/F<SUB><UP>d</UP></SUB>) (Eq. 1)
The efficiency of FRET depends on the inverse sixth power of the distance between donor and acceptor (20). This allows FRET measurements to be used with high sensitivity to follow the association of fluorescent-labeled Abeta -(1-40) monomers during refolding of the peptide in aqueous solution. Stock solutions of peptide in Me2SO were mixed at an equal molar ratio and diluted 10-fold or 50-fold (to dilute Me2SO to subdenaturing concentrations) into 50 mM Tris-HCl, 0.1 M NaCl, pH 7.4, and the fluorescence spectra recorded at various times after dilution. Controls included the donor and acceptor peptides diluted separately and either donor or acceptor in Me2SO, mixed with an equal amount of nonlabeled Abeta -(1-40), and then allowed to refold by dilution of Me2SO.


RESULTS

Aggregation Properties of Fluorescent Abeta Probes

To demonstrate the feasibility of the experiments employing fluorescent Abeta analogs, we synthesized a series of Abeta -(1-40) variants containing a single Cys substitution. We chose Abeta -(1-40) because it is stable over the time interval required for these experiments, while Abeta -(1-42) rapidly forms higher order oligomers that would complicate the interpretation of the fluorescence data (16, 21). Cys was chosen because of its unique chemical reactivity and its absence in the wild type Abeta sequence. We initially synthesized a series of Cys substitutions by replacing every third residue, because the Cys side chain would be expected to alternate on opposite sides of the strand in a beta -sheet structure. The Cys-containing probe peptides were covalently labeled with a variety of extrinsic fluorescent probes. For this work we used Abeta -(1-40) Cys mutants labeled with 1,5-IAEDANS at positions 25 and 34 and FM-labeled Abeta -(1-40) Cys mutants at positions 4 and 7. Mass spectrometry confirmed the expected mass of the final product, and the absence of the precursor peptide indicated that the labeling reaction was complete (data not shown). We also synthesized a probe containing Trp instead of Tyr at position 10 to use as an acceptor for the wild type peptide intrinsic Tyr fluorescence. Some of the other peptides synthesized were not analyzed because they either labeled inefficiently or displayed significantly altered aggregation properties (data not shown).

An obvious concern in modifying the structure of Abeta to make fluorescent derivatives is whether the modification significantly alters the structure and properties of Abeta . We compared the aggregation properties of the fluorescent derivatives to wild type Abeta under physiological conditions where it is largely soluble (e.g. Tris-HCl buffered saline at pH 7.4) and under conditions that are known to promote fibril assembly (pH 5.0 and at pH 7.4 in the presence of ZnCl2) (Fig. 1). At pH 7.4 and at pH 5.0, the sedimentation behavior of all of the fluorescent peptides used is indistinguishable from wild type Abeta -(1-40). In the presence of 70 µM Zn2+, the fluorescein- and AEDANS-labeled Abeta peptides aggregate to approximately 50-75% of the extent of wild type Abeta . However, Trp substitution at residue 10 did not alter the aggregation behavior in response to Zn2+. Several of the other Cys substitution mutant peptides we synthesized were not suitable for further analysis because either they failed to label efficiently or the labeled peptides displayed significantly altered aggregation properties (data not shown).


Fig. 1. Aggregation properties of fluorescent derivatives of Abeta -(1-40). The aggregation properties of the fluorescent derivatives and wild type Abeta -(1-40) are compared under physiological conditions where it is largely soluble (Tris-HCl-buffered saline at pH 7.4) and under two other different conditions that are known to promote fibrillization: at pH 5.0 and at pH 7.4 in the presence of 70 µM ZnCl2. 75 µM [3H]Abeta -(1-40) was mixed with 5 µM fluorescent Abeta -(1-40) as described under "Experimental Procedures" and incubated for 48 h at room temperature. The samples were centrifugated at 15,000 × g for 10 min in a Beckman Microfuge 11. Afterward the amounts of [3H]Abeta -(1-40) and fluorescent Abeta -(1-40) in the pellet and the supernatant were determined.
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The oligomeric structure of the fluorescent peptides was characterized by gel filtration chromatography, and we found that the fluorescent peptides Abeta C25AEDANS and Abeta C7FM elute at the same position as wild type Abeta -(1-40) (Fig. 2). The elution position corresponds to an apparent molecular mass of 9000 Da established by the elution behavior of a series of calibration standards as reported previously (16) (Fig. 2A, inset). The other fluorescent peptides used in this study also elute as a dimer (data not shown). The calibration curve also indicates that the expected elution position for a peptide of the mass of monomeric Abeta -(1-40) is well separated from the observed elution position of dimeric Abeta -(1-40). Nanomolar concentrations of 14C-labeled Abeta -(1-40) also elute at the position expected for a dimer (Fig. 2B). Since gel filtration only measures the effective Stokes' radius, the elution behavior is not definitive evidence for the interpretation that the peak represents a dimer.


Fig. 2. Gel filtration analysis of fluorescent Abeta -(1-40). In panel A, chromatogram a shows the elution profile of the wild type Abeta -(1-40) as detected by absorbance at 280 nm, and chromatograms b and c show the elution of Abeta C25AEDANS and Abeta C7FM, respectively, as detected by fluorescence at 482 nm and 520 nm, respectively. The peptides were dissolved in Me2SO for 30 min and then 10-fold diluted in buffer A at a final protein concentration of 5-10 µM. A 200-µl aliquot was loaded onto a Superdex 75HR 10/30 column and eluted at a rate of 0.4 ml/min. The inset in the upper right corner shows the calibration curve for the column using a series of peptide and protein standards eluted in buffer A in the presence of 10% Me2SO, as described under "Experimental Procedures." Panel B shows the elution profile of 2.0 nM [14C]Abeta -(1-40) run under the same conditions above and detected by scintillation counting. The letters in the figure correspond to: D, dimer; M, monomer; Vo, void volume, Vt, total volume.
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Denaturation and Renaturation of Abeta -(1-40) in Me2SO

To more definitively determine the structure of soluble Abeta -(1-40) by FRET, we had to first establish conditions for the denaturation and renaturation of Abeta -(1-40). Previous studies using a combination of Fourier transform infrared spectroscopy, and dynamic light scattering (7, 19) suggested that Abeta is a random coil monomer in Me2SO. The intrinsic fluorescence of proteins provides a signal commonly used to monitor conformational changes and unfolding (22). In the present work we used the intrinsic Tyr fluorescence of wild type Abeta -(1-40) to study the denaturation of Abeta in Me2SO and its renaturation. The Tyr emission of most native proteins and peptides is frequently small or undetectable due to the presence of more highly fluorescent Trp residues (20, 22), but Trp is absent in Abeta . The spectral properties of extrinsic fluorescent probes can also be exploited to obtain information about the environment surrounding the probe and to examine whether the environment changes upon aggregation and assembly (20). The denaturation curve for wild type Abeta -(1-40) showed a single, smooth cooperative transition (Fig. 3). Increasing concentrations of Me2SO increased the intrinsic fluorescence intensity of Abeta -(1-40), indicating that a significant increase in the exposure of the Tyr residue occurs in the unfolded state. The midpoint of intrinsic fluorescence changes occurred at approximately 40% Me2SO. The emission maximum of Tyr is not affected by Me2SO, remaining the same at all concentrations (e.g. 308 nm) because the Tyr fluorescence emission maximum is not sensitive to the polarity of the solvent (20). The data have been corrected for the relatively small solvent effect of Me2SO on free Tyr (less that 5% of the intensity change observed for Abeta -(1-40)) to ensure that the curve accurately reflects the unfolding of Abeta -(1-40).


Fig. 3. Unfolding of Abeta -(1-40) measured by Tyr fluorescence and 1,5-IAEDANS fluorescence. Equilibrium unfolding curves were monitored by measuring the intrinsic Tyr fluorescence at 308 nm upon excitation at 280 nm of wild type Abeta -(1-40) (black-square) and Abeta C25AEDANS- fluorescence with excitation at 336 nm (bullet ). The samples were incubated in buffer A containing the indicated Me2SO (DMSO) concentrations; the protein concentration was 11 µM Abeta .
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We also examined the denaturation of the extrinsically-labeled fluorescent Abeta -(1-40) probes. For environment-sensitive fluorophores (like 1,5-IAEDANS) the emission maximum shifts to a shorter wavelength (blue shift) as the polarity of the surrounding environment decreases (20). Conversely the emission maximum shifts to the longer wavelengths (red shift) in a more polar environment. For Abeta C25AEDANS, a marked blue shift (42 nm) of the emission was observed upon unfolding by Me2SO (Fig. 3), indicating that the fluorophore is increasingly exposed to the surrounding media at increasing Me2SO concentrations. As with the intrinsic Tyr fluorescence, the midpoint of the blue shift of AEDANS occurred at approximately 50% Me2SO. The unfolding transition was recorded after 30 min of incubation in Me2SO, as it has been reported that longer incubation times lead to an aggregated comformation of Abeta (19). At concentrations of Me2SO below 10%, there is little further change in the intrinsic fluorescence emission of Abeta -(1-40) or the extrinsic fluorescence of Abeta C25AEDANS (Fig. 3). These results indicate that AEDANS-labeled and wild type Abeta -(1-40) peptides have similar stabilities and suggest that there is relatively little change in the overall structure of Abeta -(1-40) over the range of Me2SO from 0 to 10%.

The renaturation of Abeta -(1-40) from Me2SO solution was also examined. Upon 10-fold dilution of Me2SO into aqueous buffer solution, the emission spectrum of wild type Abeta -(1-40) and Abeta C25AEDANS showed the same maximum at 308 and 494 nm, respectively, as observed for the peptides dissolved directly in buffer A, indicating that the denatured peptide returns to the same overall structure (Fig. 4, A and B). Time course studies indicated that the refolding of the peptide was immediate (data not shown). Similar denaturation and renaturation results were obtained with guanidine HCl (data not shown). Me2SO stock solutions of peptide were employed for all of the subsequent experiments.


Fig. 4. Renaturation of Abeta -(1-40) measured by Tyr fluorescence and AEDANS fluorescence. The emission spectra of peptide freshly dissolved in buffer A (- · - · -), peptide dissolved in 100% Me2SO (------), and peptide dissolved in 100% Me2SO and then diluted 10-fold in buffer A ···) are shown. The wild type peptide is presented in panel A, and Abeta C25AEDANS is presented in panel B. Emission spectra were recorded from 346 to 600 nm with an excitation at 280 nm for Tyr fluorescence and 346-600 nm with an excitation at 336 nm for Abeta C25AEDANS.
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Association of Fluorescent Abeta -(1-40) Peptides

Fluorescence resonance energy transfer between AEDANS and fluorescein was used initially to monitor association of Abeta -(1-40) monomers following dilution of Me2SO into aqueous buffer solution. For these experiments, Abeta C25AEDANS and Abeta C7FM Me2SO stock solutions were mixed 1:1 (donor:acceptor), and then subsequently diluted 10-fold in buffer A. The final concentration of the peptide was 3 µM. The resulting fluorescence spectra are shown in Fig. 5A. Efficient FRET was observed, as evidenced by a quenching of the donor emission at 474 nm and an increase in the acceptor fluorescence at 520 nm, compared with the control spectra, indicating that hybrid Abeta -(1-40) dimers had formed in the mixture containing both donor and acceptor (Fig. 5A). The efficiency of FRET did not change significantly upon subsequent incubation for 24 h (data not shown). To control for possible effects of peptide structure on the fluorescence intensity of labeled peptides, we carried out control measurements in which either Abeta C25AEDANS or Abeta C7FM were individually mixed with an equal amount of non-labeled peptide in Me2SO, and then diluted 10-fold in buffer A. The emission spectra obtained for Abeta C25AEDANS or Abeta C7FM are shown as arithmetic sum of the individual spectra (i.e. the expected emission in the absence of energy transfer). We also observed efficient FRET with several other pairs of Abeta -(1-40) peptides (Table I). Fig. 5B shows the spectra of the energy transfer experiment where another donor-acceptor pair, Abeta C34AEDANS-Abeta C4FM, was used. The efficiency of FRET for this combination is higher than that observed for Abeta C25AEDANS and Abeta C7FM, suggesting that the peptide structure is ordered and that the fluorophores at positions 34 and 4 may be in closer proximity in the structure than those at positions 25 and 7. Once formed, the Abeta -(1-40) dimers appear to be relatively stable in solution. If fluorescent homodimers are formed first by individually diluting the stock solutions 10-fold into buffer A, and then the homodimers are subsequently mixed, no resonance energy transfer is observed over an incubation of 24 h (data not shown), indicating that subunit exchange between homodimers is not detectable over this period. This suggests that the dimer is very stable.


Fig. 5. Association of AEDANS-labeled and FM-labeled Abeta -(1-40) in diluted aqueous solution as determined by FRET. Panel A shows emission spectrum of an equal molar amount of Abeta C25AEDANS (donor) and Abeta C7FM (acceptor) mixed in Me2SO, and then diluted 10-fold in buffer A (···). The final protein concentration was 3 µM. The control spectrum (------) corresponds to the mathematical sum of the following control samples: an equal molar mixture of Abeta C25AEDANS and Abeta -(1-40) and an equal molar mixture of Abeta C7FM and Abeta -(1-40). Efficient FRET is evident by the quenching of the donor and an increased emission of the acceptor. Panel B shows the emission spectrum for the other donor-acceptor pair, Abeta C34AEDANS and Abeta C4FM. Panel C shows the emission spectrum of the mixture of Abeta C25AEDANS, Abeta C7FM plus a 10-fold molar excess of wild type Abeta -(1-40) (···). The control spectrum (------) corresponds to the arithmetical sum of the controls for donor alone and acceptor alone in the presence of 10-fold excess of wild type peptide. The addition of an excess of unlabeled Abeta abolishes the FRET between Abeta C25AEDANS and Abeta C7FM.
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Table I. Efficiencies of energy transfer for different donor-acceptor pairs


Donor-acceptor pair E (efficiency of energy transfer) Me2SO

%
Abeta C25AEDANS-Abeta C7FM 0.14 10.0
Abeta C34AEDANS-Abeta C4FM 0.20 10.0
Abeta -(1-40)-Abeta Y10W 0.21 10.0
Abeta -(1-40)-Abeta Y10W 0.46 2.0

Several controls were conducted to ensure that the FRET observed is due to interactions between peptides that occurs in solution with wild type Abeta -(1-40). After each FRET experiment, we confirmed that the fluorescent peptide mixture eluted at the same apparent dimer position as the wild type peptide by gel filtration chromatography (data not shown). We also determined that FRET is nearly abolished when a 10-fold molar excess of wild type peptide is added to the fluorescent peptide mixture in Me2SO and then subsequently diluted (Fig. 5C), indicating that the wild type peptide can compete for the fluorescent peptides and form fluorescent and wild type Abeta -(1-40) heterodimers that do not exhibit FRET. Finally, we exploited the endogenous Tyr fluorescence of wild type Abeta -(1-40) as a donor for Abeta -(1-40) in which Trp replaces the wild type Tyr at position 10 (Abeta Y10W). Efficient FRET was also observed for the mixture of Abeta -(1-40) and Abeta Y10W (Fig. 6). This experiment was conducted in two different conditions: where the mixture was diluted to 10% Me2SO (Fig. 6, A and B) and to 2% Me2SO to dilute the solvent to a more subdenaturing concentration (Fig. 6, C and D). A deconvolution analysis of FRET between Abeta -(1-40) and Abeta Y10W was conducted as reported (23) (Fig. 6, B and D). The efficiency of FRET is significantly higher in 2% Me2SO than in 10% Me2SO, suggesting that the structure in 10% Me2SO may be partially unfolded (Table I).


Fig. 6. Association of Abeta -(1-40) and Abeta Y10W in dilute aqueous solution as determined by FRET. The association of Abeta -(1-40) and Abeta Y10W in 10% Me2SO in buffer A is shown in panels A and B and in 2% Me2SO in buffer A is shown in panels C and D. Panels A and C represent the steady-state emission data, and panels B and D represent the corresponding deconvoluted spectra. The traces correspond to the emission spectrum of the mixture of Abeta -(1-40) and Abeta Y10W (spectrum 1), and the following controls: donor Abeta -(1-40) alone (spectrum 2), and acceptor Abeta Y10W alone (spectrum 3). Spectrum 4 represents the mathematical sum of spectra 2 and 3. Spectrum 5 represents the deconvoluted emission spectrum of the acceptor in the mixture and is obtained by multiplying spectrum 3 by the factor (FDA/FA), where FDA is the emission of the acceptor in the mixture and FA is the emission of the acceptor alone. Spectrum 6 is the deconvoluted emission spectrum of the donor in the mixture and is obtained by subtraction of spectrum 5 from 1. The Abeta -(1-40) concentration was 10 µM. The association of Abeta -(1-40) and Abeta Y10W is more readily evident in the deconvoluted spectra in panels B and D, as indicated by the quenching of the donor Tyr and an increase in the emission of the acceptor Trp.
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Our experiments with three different donor-acceptor pairs demonstrate that efficient FRET is observed when the peptides are mixed in Me2SO prior to dilution in aqueous buffer, suggesting that they form dimers in solution. It is conceivable that the aggregates may actually represent higher order structures (e.g. trimers or tetramers). We measured the lifetime of the FM-labeled Abeta -(1-40) by phase modulation frequency domain methods in the range 3 µM to 100 nM. The fluorescence lifetime data fit to a single decay exponential that remained constant over the concentration range examined (data not shown). This suggests that there is a single distance between the fluorophores and that it does not change over the concentration range examined. A single distance would be expected in a population of structurally homogeneous dimers, while higher order aggregates would be expected to contain more than one distance between fluorophores that would be observable as different lifetimes. Taken together with the gel filtration data, the simplest conclusion is that Abeta -(1-40) exists as a stable dimer in aqueous saline solution at pH 7.4 over the concentration range from nanomolar to micromolar.


DISCUSSION

The purpose of the present study was to characterize the oligomeric structure of Abeta -(1-40) in aqueous solution. The published literature on this subject is confusing because different distributions of monomer, dimer, trimer, tetramer, and higher order oligomers have been reported (8, 13, 15, 16, 21, 24-30). Some of the discrepancies can be attributed to structural differences in amyloid isolated from brain tissue (21, 26, 27, 29) in comparison to synthetic Abeta (8, 13, 15, 16, 21, 25). The aggregates observed in amyloid isolated from brain tissue may be covalent because they are stable under denaturing conditions and may reflect peptide-peptide associations occurring in vivo (21). Even though many of the differences in the oligomeric state of synthetic Abeta can be explained by differences in the concentrations of peptide examined and the conditions and methodology employed, a controversy remains about whether a monomer or dimer is the smallest structure that exists under physiological conditions. Hilbich et al. (8) reported that a dimer is the predominant species at pH 7.0 and at physiological salt concentrations for Abeta -(10-43), in good agreement with our own observations on Abeta -(1-40) and Abeta -(1-42) (16). Barrow et al. (15) reported a mixture of monomer, dimer, trimer, and tetramer for Abeta -(1-39) and Abeta -(1-42), and Roher and colleagues reported that a monomer is the predominant species for both Abeta -(1-40) and Abeta -(1-42) at pH 7.4 (21). While this distinction may not be important for screening for compounds that inhibit fibril assembly, it is important for understanding the mechanism of amyloid assembly and critical for correctly assigning the identity of NMR cross peak resonances in 2D structural analysis of Abeta , where it has been assumed that the peptide is monomeric (17, 31).

In this study, we used fluorescence spectroscopy and FRET to clarify the oligomeric structure of soluble Abeta -(1-40). Fluorescence spectroscopy has been used to probe the structure and dynamics of a wide variety of biological self-assembly or association reactions, including triose-phosphate isomerase (23), actin (32, 33), tubulin (34), and neurofilaments (35). FRET between two fluorophores can be used as a spectroscopic ruler for measurements of distances in the range of 10-100 Å, distances that begin where NMR methods leave off and that end at the diameter of a typical amyloid fibril (36). We found that Abeta -(1-40) is well solvated and monomeric in Me2SO solution, in agreement with previously published evidence (7, 19), and we found that it renatures upon dilution in aqueous buffers. Upon dilution from Me2SO solution, Abeta -(1-40) elutes as a single peak at a position corresponding to the expected molecular weight of a dimer and fluorescent Abeta -(1-40) derivatives exhibit efficient FRET, indicating that a stable complex is formed between donor and acceptor probes. Together, these observations suggest that Abeta -(1-40) exists as dimer in solution. The fact that a single peak is observed on gel filtration suggests that Abeta -(1-40) monomer is not detectable under these conditions. It is conceivable that the monomer and dimer might co-migrate on gel filtration, but this seems unlikely because the Abeta -(1-40) peak elutes in the mid range of the fractionation volume and the column is capable of resolving monomeric and dimeric Abeta under denaturing conditions (21). The fact that a single fluorescent lifetime was observed for Abeta C7FM is also consistent with the interpretation that the peak does not contain a mixture of monomer and dimer.

The utilization of fluorescent-labeled Abeta -(1-40) peptides in these studies requires altering the structure of Abeta -(1-40), so it is conceivable that the properties of the probes may not be precisely the same as Abeta -(1-40). Several lines of evidence argue against the interpretation that dimer formation is an artifact of fluorescent labeling. The elution behavior of the fluorescent peptides on gel filtration is identical to wild type Abeta -(1-40), indicating that they have the same hydrodynamic radius in solution. Denaturation-renaturation experiments also demonstrate that the stability of the fluorescent peptides is indistinguishable from the wild type peptide. When we assayed the aggregation properties of the fluorescent probe peptides, we found that they were nearly identical to the wild type peptides. Finally, we employed several different combinations of donor and acceptor peptides labeled at different positions and all of the peptides used behaved as dimers. One of the donor-acceptor pairs used Tyr fluorescence from wild type Abeta -(1-40) as the donor and Trp fluorescence as the acceptor from the Abeta peptide substituting Trp for Tyr at position 10. Tryptophan substitution has been often used to study proteins where is not desirable to drastically modify its structure (37). This donor-acceptor pair may be expected to be efficient since the Förster distance for Tyr-Trp transfer is about 10-18 Å, a size comparable to the diameter of many proteins (20). The observation of efficient energy transfer between Tyr and Trp confirms that wild type Abeta -(1-40) forms a dimer with the Trp substitution probe.

The finding that Abeta -(1-40) forms a stable dimer in solution suggests that dimerization is the initial event in amyloid aggregation and that it represents the fundamental building block for further fibril assembly as has been proposed previously (7, 16). This model of amyloid assembly is very similar to the model recently proposed for immunoglobulin light chain amyloid fibrils on the basis of molecular modeling studies (38). In this model, the two light chains align in a parallel fashion creating a dimer with a 2-fold axis of symmetry. It seems likely that the Abeta -(1-40) dimer may also have the same arrangement because it behaves as if it is axially amphipathic with one end polar and the other end hydrophobic (16). This implies that at least some of the dimer may be arranged in a parallel fashion, because if the dimer were arranged in a simple head-to-tail fashion, as has been proposed previously (20), the hydrophobic moment of the resulting dimer might be expected to be symmetric with respect to the ends of the dimer. Previous CD and Fourier transform infrared spectroscopic studies indicate that soluble Abeta has substantial beta -sheet content, suggesting that the dimer adopts a beta  structure (4, 7, 15, 17).

In the amyloid light chain model, the next step in polymerization is head to tail association of dimers related by a 90° rotation around the 2-fold axis to form a tetramer that establishes a "proamyloid" filament lattice that is capable of propagating filaments of indefinite length. Elongation of the fibril is accomplished by the stepwise addition of dimers onto the filament. This "proamyloid" filament may correspond to the "beta crystallite" proposed for Abeta from fibril diffraction measurements (39) and observed in atomic force microscopy images (40). In this step, the free energy contribution of individual amino acid side chains is effectively doubled because of the 2-fold symmetry of the interacting surfaces (38). It seems likely that Abeta is also capable of forming a similar stable tetramer. Discrete aggregate species migrating at the position expected for a tetramer have been observed by SDS-PAGE in samples of Abeta -(1-42) (5, 16). In Abeta , the formation of this SDS-resistant higher order aggregate depends on the length of the carboxyl terminus of Abeta (e.g. only Abeta -(1-42) and Abeta -(1-43)) and it is also concentration-dependent, occurring at approximately the critical micelle concentration defined by surface tension measurements (16). These results suggest that the formation of higher order aggregates in Abeta may be mediated predominantly by hydrophobic contacts.

The formation of amyloid fibrils in the light chain model is proposed to proceed by the lateral association of the "proamyloid" filaments or subfibrils (38). This step corresponds mechanistically to the formation of the nucleating center proposed for Abeta (41). Evidence for the existence of subfibrils has been obtained for Abeta by rapid-freeze, deep-etch electron microscopy (42) and more recently by atomic force microscopy (40). In the light chain model, four strands are proposed to associate in an antiparallel fashion, but it is not clear how many subfibrils are contained within Abeta fibrils. The number of subfibrils for amyloid Abeta fibrils is not clear, but electron micrographs show images that appear to contain five subfibrils (42), and this number gives the best fit in modeling the observed reflections in fiber diffraction studies (39). It is also conceivable that this number could vary within a population of Abeta fibrils, and this could account for differences in the diameter and morphology of fibrils that has been reported (40) and the fact that sheet and ribbon morphologies are also known to occur in synthetic Abeta aggregates (5, 43). Other than the initial dimerization event, the details of this model of amyloid fibril formation remain to be verified experimentally for Abeta .

Fluorescent derivatives of Abeta -(1-40) may also prove useful for exploring other aspects of amyloid structure. Quantitative measurements of distances between fluorescent dipoles by FRET are also possible (20). The fact that Abeta -(1-40) forms a dimer in solution simplifies the interpretation of FRET measurements, because there is only one distance between fluorophores in a dimer. If a sufficient number of distance measurements are available, it may be possible to discern the structural organization of the polypeptide within the dimer, albeit at a lower resolution than might be achievable by x-ray crystallography or NMR. Until now, this is the first report in which Abeta amyloid intrinsic fluorescence and FRET between Abeta fluorescent derivatives have been used to study amyloid structure. Different fluorescent Abeta -(1-40) analogs may also be useful for mapping the solvent-accessible surface of the amyloid fibril by quenching studies (20). Since the aggregation state of Abeta has been shown to be important for in vitro toxicity, there is a growing interest in molecules that inhibit Abeta aggregation as a candidates for potential therapeutic strategies based on blocking amyloid deposition. Potentially interesting classes of inhibitors for therapeutic evaluation would be molecules that bind tightly with Abeta monomer and prevent dimerization and molecules that prevent oligomerization of dimers or the extension of fibrils.


FOOTNOTES

*   This work was supported by Grant NS31230 from the National Institutes of Health.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.
par    To whom all correspondence should be addressed. Tel.: 714-824-6081; Fax: 714-824-8551; E-mail: cglabe{at}uci.edu.
1   The abbreviations used are: Abeta , amyloid beta ; FRET, fluorescence resonance energy transfer; 1,5-IAEDANS, 5-(2-((iodoacetyl)amino)ethyl)aminonaphthylene-1-sulfonic acid; FM, fluorescein maleimide; Me2SO, dimethyl sulfoxide; Abeta C25AEDANS, Abeta with Cys in position 25 and labeled with 1,5-IAEDANS; Abeta C7FM, Abeta with Cys in position 7 and labeled with FM; Abeta C34AEDANS, Abeta with Cys in position 34 and labeled with 1,5-IAEDANS; Abeta C4FM, Abeta with Cys in position 4 and labeled with FM; Abeta -(1-40), Abeta wild type; Abeta Y10W, Abeta with Trp in position 10; [3H]Abeta -(1-40), Abeta -(1-40) radioactively labeled with tritium ([3H]Phe) at position 4; [14C]Abeta -(1-40), Abeta -(1-40) radioactively labeled with 14C ([14C]Ala) at position 2; MOPS, 4-morpholinepropanesulfonic acid.

ACKNOWLEDGEMENTS

This work was made possible, in part, through access to the Laser Microbeam and Medical Program (LAMMP) and the Clinical Cancer Center Optical Biology Shared Resource at the University of California, Irvine (facilities are supported by National Institutes of Health Grants RR-01192 and CA-62203). W. G.-R. and M. S.-B. gratefully acknowledge the Center for Fluorescence Spectroscopy in the Department of Biological Chemistry at the University of Maryland School of Medicine for training in fluorescence spectroscopy. We thank Drs. Nancy Allbritton and Bruce Tromberg for many helpful discussions and suggestions.


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