Photocontrol of β-Amyloid Peptide - American Chemical Society

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J. Phys. Chem. B 2009, 113, 6164–6172

Photocontrol of β-Amyloid Peptide (1-40) Fibril Growth in the Presence of a Photosurfactant Andrea C. Hamill and C. Ted Lee, Jr.* Department of Chemical Engineering and Materials Science, UniVersity of Southern California, Los Angeles, California 90089-1211 ReceiVed: September 9, 2008; ReVised Manuscript ReceiVed: January 13, 2009

The effect of an azobenzene-based photoresponsive surfactant on fibril formation of β-amyloid (1-40) (Aβ40) has been studied using small-angle neutron scattering (SANS), atomic force microscopy (AFM), and light scattering (LS) measurements. Fibril formation is inhibited with a lag phase persisting for approximately 5 h in the presence of the trans isomer of the photosurfactant under visible light (i.e., the relatively hydrophobic, activated form). Conversely, only a 2-h lag phase is observed under UV light with the cis photosurfactant isomer (relatively hydrophilic, passive form), while large fibril networks are immediately observed for the pure protein. Furthermore, in situ UV illumination of a solution of trans surfactant and protein results in rapid fibril formation. Thus, the ability to photoreversibly inhibit and trigger the fibrilization process with light illumination is demonstrated. Shape-reconstruction analysis of the SANS data is used to obtain novel information on the conformation of the protein during the initial stages of protein aggregation. Small, cylindrical protein aggregates 5 nm in diameter and 7 nm long are initially observed during the lag phase independent of the sample conditions. AFM images confirm both the aggregate structure and the duration of the lag phase and further suggest that these early aggregates appear to be the nuclei for longer aggregates that develop over time. Introduction A variety of neurogenerative diseases, including Alzheimer’s, Huntington’s, and Parkinson’s diseases, are associated with amyloid fibrilogenesis where proteins self-associate into insoluble amyloid fibrils. Each disease is linked to protein aggregation: R-Synuclein to Parkinson’s disease, Huntingtin with poylQ expansion to Huntington’s disease, and Amyloid β peptide (Aβ) to Alzheimer’s disease,1 although amyloid fibrils have been observed in an array of proteins largely independent of the native secondary structure, including ribonuclease A,2 an SH3 domain,3 lysozyme,4 insulin,5 and R-chymotrypsin.6 While there are no clear similarities, in structure or sequence, between the wide range of proteins that have been shown to form amyloids, the resulting amyloid fibrils are found to exhibit similar features. As a result, the precise mechanism of fibril formation remains a challenging and as yet unsolved problem. It is generally believed that proteins need to unfold, at least partially, to aggregate into amyloid fibrils7-9 via a nucleated growth mechanism.1 Typically there is a lag phase prior to these partially folded states associating into nuclei consisting of small unstructured oligomers of 2-4 molecules in the case of Aβ40 and 5-6 for Aβ42.10 The small oligomers associate further into protofibrils, aggregates that are not yet fibrillar in their morphologies but have properties characteristic of amyloids (notably β sheet structure).1 Protofibrils then associate into protofilaments, elongated aggregates ∼2-5 nm in diameter. Eventually the species aggregate into long insoluble amyloid fibrils that consist of 2-6 intertwined protofibrils.11 These fibrils then accumulate, forming microscopic deposits (plaques) in the brain. Specifically in the case of the β-amyloid peptides (Aβ), implicated in Alzheimer’s disease, recent research has focused * To whom correspondence should be addressed. Phone: (213) 740-2066. Fax: (213) 740-8053. E-mail: [email protected].

on the small, early oligomers as they have been detected in the brains of Alzheimer’s disease patients.12 These small oligomers have become increasingly viewed as the primary pathogenic species as the severity of cognitive impairment in Alzheimer’s disease has been correlated with the amount of prefibrillar species of Aβ.13-15 Furthermore, the premature development of Alzheimer’s disease has been linked to enhanced protofibril formation.16 Thus, unique insight into the disease pathway could be obtained if the mechanisms of the early stages of the Aβ protein assembly process were better understood. Furthermore, if techniques could be developed to prevent and possibly reverse these early stages of protein association, potential novel disease treatment strategies could be envisioned. As a result, the development of novel structural characterization methods capable of examining partially folded proteins in non-native conformations and supramolecular complexes undergoing self- or heteroassociation is highly desired. Small-angle neutron scattering (SANS) represents a technique capable of studying the various stages of fibril formation over length scales ranging from nanometer-sized early oligomers through protofibrils and protofilaments and eventually micrometer-sized fibrils.17 From the range of momentum vectors Q ) 4πλ-1 sin(θ/2), where λ is the neutron wavelength (∼5 Å) and θ is the scattering angle, in a typical SANS experiment (Q ) 0.005-0.5 Å-1), it can be seen that the data span length scales (l ) 2π/Q) ranging from 12.5 to 1250 Å, ideal for protein association studies. For example, through the use of SANS the low-resolution in vitro structure of early Aβ40 oligomers was found to be cylindrical with radii and length of 24 and 110 Å, respectively.18 Furthermore, it was recently shown that light illumination could be used to induce photoreversible changes in the degree of R-chymotrypsin association during the initial stages of amyloid fibril formation.19 Through the use of an azobenzenebased photoresponsive surfactant having an active (visible light)

10.1021/jp8080113 CCC: $40.75  2009 American Chemical Society Published on Web 04/03/2009

β-Amyloid Peptide (1-40) Fibril Growth and inactive (UV light) isomeric conformation, light could be used to control protein association by photoreversibly replacing protein-protein interactions with protein-surfactant interactions. In addition, a novel shape-reconstruction technique20 was applied to the SANS data to determine the structure of the preamyloid R-chymotrypsin complexes at relatively high resolution. Under visible light the activated trans form of the photosurfactant caused partial unfolding of the protein, which led to the formation of protein hexamers, while shapereconstruction showed these hexamers to have a corkscrew like structure. Under UV light the passivated cis form of the surfactant caused the hexamers to laterally associate and intertwine two at a time to form dodecamers. The dodecamers were found to have an elongated twisted conformation with cross-sectional radii similar to amyloid protofilaments. FT-IR was also used to show that this photoinduced hexamer-tododecamer transition occurred through intermolecular β sheets stabilized with hydrogen bonds. The present study sets out to investigate the effect of such a photosurfactant on the Aβ40 peptide. SANS, AFM, and light scattering are used to fully investigate the Aβ40 fibril growth process over time as a function of surfactant concentration and light conditions. The ability to inhibit, and then trigger, the growth process of amyloid fibrils with light illumination is examined. As a result, shape reconstruction can be applied to the SANS data to allow structural determination of early Aβ40 oligomers. Furthermore, the effect of light illumination on the fibril growth process in the presence of the photosurfactant is examined as a means to inhibit protein-protein interactions and lead to delayed fibril formation under visible light, while subsequent UV illumination triggers the association process. Experimental Methods Materials. An azobenzene trimethlyammonium bromide (azoTAB) surfactant of the form below was synthesized and

used as in previous studies.19,21-25 The surfactant undergoes a photoisomerization from a trans form to a predominately cis form (90%) when exposed to 365 nm UV light. This isomerization can then be reversed upon exposure to 436 nm visible light (75% trans) or under dark conditions for ∼24 h (100% trans). Photoconversion was achieved with the 365 nm line from a 200 W mercury arc lamp (Oriel model no. 6283), isolated with the combination of a 320 nm band-pass filter (Oriel model no. 59800) for UV conversion or a 400 nm long-pass filter (Oriel model no. 59472) for visible conversion, and an IR filter (Oriel model no. 59060). Exposure of the peptide to UV light was avoided by exposure of surfactant solutions to UV light prior to addition of the peptide in the solid form. β-Amyloid peptides, proteolytic fragments of the amyloid-β precursor protein, are small 39-43 amino acid peptides26 with most abundant forms of Aβ40 and Aβ42. β-Amyloid peptide (1-40), Ultra Pure, HCl,27 expressed in E. coli (rPeptide cat. no. A-1156) was used as received. All other chemicals were obtained from Aldrich in the highest purity. The Aβ40 was resuspended in a 1% NH4OH solution as suggested by the supplier. Attempts to resuspend in a pH 7.2 phosphate buffer gave rapid protein aggregation. While Aβ42 is secreted at only ∼10% of the rate of Aβ40, Aβ42 is generally more predisposed

J. Phys. Chem. B, Vol. 113, No. 17, 2009 6165 to fibril formation. Thus, Aβ40 was selected because it is less fibrillogenic than Aβ42, allowing focus on the early oligomer species as opposed to the full fibrils. Small-Angle Neutron Scattering. SANS experiments were performed on the 30m NG3 and NG7 SANS instruments at NIST.28 Measurements were performed at 4 °C in order to avoid any association/growth during data collection.18,29,30 A neutron wavelength of λ ) 6 Å and a detector offset of 25 cm with two sample to detector distances of 1.33 and 7 m were utilized to obtain a Q range of 0.0048-0.47 Å-1. The net intensities were corrected for the background and empty cell (pure D2O), accounting for the detector efficiency using the scattering from an isotropic scatterer (Plexiglas) and converted to an absolute differential cross section per unit sample volume (in units of cm-1) using an attenuated empty beam. A constant background was subtracted (an average of the scattering from the 15 highest Q values) in order to correct for incoherent scattering. Shape reconstruction was done similar to previous studies19,21,23 using the GA_STRUCT algorithm.20 The program fits the experimental data starting with an initial guess of randomly oriented scattering centers and rearranges them until the best fit is achieved. Each displayed structure represents the consensus envelope of a family of 10 fits. A peptide concentration of 0.75 mg/mL was used to obtain significant scattering and when employed a surfactant concentration of 8 mM was used. Reduction and analysis of the SANS data were done using Igor Pro.31 A Lorentzian model, I(Q) ) I0/[1 +(QL)2] + B, where L is the screening length, I0 is the scattering intensity extrapolated to zero angle, and B is the background scattering intensity, was used to fit the scattering curves from the pure protein solutions. Each model was smeared to account for instrumental resolution. A cylinder model with circular cross section and uniform scattering length density was used to fit the data from the 8 mM azoTAB solutions at time t ) 0, 2, and 3 h under visible and t ) 0 under UV light. The Guinier approximation32 was used to calculate the radii of gyration through the equation I(Q) ) I(0)exp (-Q2Rg2/3), where I(Q) is the scattering intensity, I(0) is the extrapolated intensity at Q ) 0, and Rg is the radius of gyration, valid for globular species. When the protein was in a rod-like formation a modified Guinier analysis33 for rod-like forms was used Q · I(Q) ) IC(0)exp(-Q2R2C/2), where RC is the cross-sectional radius of gyration, related to the geometric radius by RC2 ) R2/2. A model-independent approach was taken to obtain information about the protein conformation through the calculation of a pair distance distribution function (PDDF), a measure of the probability P(r) of finding two scattering centers at a distance r apart34,35

I(Q) ) 4π

dr ∫0∞ P(r) sin(Qr) Qr

(1)

The GNOM program36 was used to calculate the PDDFs over a Q range of 0.013-0.29 Å-1. The maximum particle diameter, Dmax, was selected to be the lowest value that gave a smooth return of the PDDF to zero. Atomic Force Microscopy. Tapping mode AFM in air at 25 °C was performed using an Autoprobe CP atomic force microscope (Park Scientific Instruments). The microscope was equipped with a 100 µM scanner with a maximum xy scan range of 100 µM and a maximum z range of 6 µM. The images were acquired by using rectangular silicon cantilevers 135 µM long with resonant frequencies in the 200-400 kHz range and nominal spring constants in the 20-80 N/m range. The

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Figure 1. SANS data of Aβ40 in the presence and absence of 8 mM azoTAB as a function of time and light illumination. Pure Aβ40 incubated for 0 (O), 5 (O), 10 (O), or 15 h (O). Incubated with 8 mM trans azoTAB under visible light for 0 (9), 2 (b), 3 (2), or 5 h ([). Incubated with 8 mM cis azoTAB under UV light for 0 (0), 2 (O), or 3 h (4). Incubated with 8 mM trans azoTAB under visible light for 5 h, followed by 1 h of UV illumination (1). [Aβ] ) 0.75 mg/mL.

cantilevers have integrated tips with a radius of curvature below 10 nm (Veeco Probes, model no. RTESPA). Samples were imaged at scan sizes between 0.2 and 6 µM using line scan rates below 2 Hz. All of the images were processed by plane fitting and then three-dimensional shade rendering. All peptide solutions were diluted to 0.01 mg/mL using a 1% NH4OH solution. A 10 µL aliquot of solution was then deposited onto freshly cleaved mica. The samples were then wicked dry using filter paper with imaging occurring within 2 h of sample preparation. Light Scattering. Light scattering measurements were performed at 25 °C on a Brookhaven model BI-200SM instrument (Brookhaven Instrument Corp.) with a 35 mW (Melles Griot, model number 05-LHP-928) helium neon (λ ) 632.8 nm) laser at a scattering angle of 90°. The wavelength of the laser is far enough away from the 436 nm absorbance peak of azoTAB to avoid any conversion back to the trans state while in the cis state. A BI-9000AT digital correlator (Brookhaven Instrument Corp.) was used. Prior to measurements solutions were filter through a 200 nm Anatop filter. After the initial filtration, if possible, the solution was further filtered through a 20 nm Anatop filter (only done for the measurements under visible light). A peptide concentration of 0.25 mg/mL was used along with a surfactant concentration of 8 mM. Results and Discussion Aβ40 fibril growth was studied over time in the presence of 8 mM azoTAB photosurfactant under both visible and UV light using SANS, as shown in Figure 1. Both time and azoTAB significantly affect the observed scattering. Scattering at different Q values indicates structures of different length scales (Q ) 2π/l), where l is the approximate length scale of the scattering species. Pure peptide solutions are seen to form large structures as the scattering is shifted to low Q values (20 mM).23 Furthermore, the critical micelle concentration of cis azoTAB is ∼11 mM,58 well below the conditions used in this study. Thus, the fact that the scattering observed for the cis azoTAB sample at 0 h under UV light, which could not possibly contain micelles, is nearly identical to the other oligomer conditions (i.e., trans azoTAB samples after 0, 2, and 3 h) further shows that the plateau-like scattering observed in the SANS data in Figure 1 is indeed a result of small Aβ40 oligomers. The AFM images taken on samples aged for 5 h under visible light (Figure 5a) or 15 h under UV light (Figure 5d) exhibit intermediates structures anywhere from tens to hundreds of nanometers long and typically around 11 nm in diameter (as measured from the height in the z direction). The images in the center column of Figure 5b and 5e were taken on the 8 mM azoTAB SANS samples incubated at room temperature for 15 h and then stored on dry ice for transport from NIST. These fibrillike structures have a z height of ∼15 nm, and they range in length from several hundred nanometers to several micrometers. The z height agrees with the diameter of the cylinder determined from the radii calculated from modified Guinier analysis, R ≈ 56 Å. The fact that the AFM heights are slightly larger is most likely due to the fact that although amyloid fibrils share the same characteristics no two fibrils are exactly identical (fibril thicknesses typically vary). Figure 5c illustrates the axial periodicity observed in some fibril samples, very clearly seen in the 3D rendering. Periodicity of this type has been observed in protein fibrils in the β-amyloid peptide (Alzheimer’s disease),59-61 human amylin (type II diabetes),62 the peptide hormone calcitonin (medullary carcinoma of the thyroid),63 and Sup35 (prion disease).64 The image in Figure 5f illustrates the twisting of fibrils as observed in some samples, a well-known characteristic of amyloid fibrils that has been reported previously with AFM60,62,65,66 and TEM.67-71 On the basis of the SANS data along with the AFM images, the following aggregation mechanism involving two different pathways is proposed. Both pathways start with the same initial

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Figure 6. (a) Light scattering intensity of pure Aβ40 (b) and Aβ40 in the presence of 8 mM trans azoTAB under both visible (9) and UV light (2) as a function of time. (b) Light scattering intensity as a function of surfactant light illumination: 5 h visible light followed by 1 h of UV light exposure (]) and 5 h UV light followed by 1 h visible light exposure ([). [Aβ] ) 0.25 mg/mL.

step, the formation of very short cylindrical oligomers (Figure 3a and 3b). In the first pathway after the nuclei have formed they initially associate end-to-end (Figure 3c, green highlight) and once they have established some length they twist around one another to form thicker and longer cylinders (Figure 5d). These elongated structures then continue to grow (Figure 5e) to form full fibrils (Figure 5f). In the second pathway after the nuclei have formed they initially associate side-to-side (Figure 3d) with the fatter bead-like cylinders then beginning to “string” together (Figure 5a); the beads then grow in length (Figure 5b) and tightly pack to form long fibrils (Figure 5c). It is anticipated that both of these proposed mechanistic pathways are occurring simultaneously, as no two full fibrils are identical. A similar mechanism for the formation of insulin fibrils has been proposed as “multipathway” fibrillization, where both intertwining of protofilaments and the lateral association of early prefibrillar forms followed by the lateral association of protofilaments into fibrils occur.72 In addition, a recently proposed73 colloidal aggregation pathway for amyloid formation could be in operation, where the formation of colloids (nuclei) through surface energy minimization leads to eventual amyloid formation. Light scattering intensity was used to further investigate the kinetics of fibril growth of Aβ40 in the presence of azoTAB. In Figure 6a the scattering intensity versus time for Aβ40 in the absence and presence of 8 mM azoTAB is shown. Both the pure Aβ40 and the 8 mM cis azoTAB solution under UV light initially have high scattering rates. From the SANS data we would expect to observe relatively low light scattering intensity consistent with the small oligomers found for the 8 mM cis azoTAB SANS sample at time t ) 0. However, due to the extremely high sensitivity of light scattering to any large structures, a very small amount of fibrils or other large aggregates would mask the scattering from these small particles. Thus, although these small oligomers are likely present, they are not detectable in the light scattering intensity. Also note the decrease over time in light scattering intensity from the 8 mM cis azoTAB sample, which also supports the idea of very large aggregates that may be settling over time. In contrast, under visible light it was possible to observe an increase in light scattering over time representing the growth process from the early oligomers to full fibrils. The lag phase observed with light scattering is ∼2.5 h, based on the time elapsed before a steep upturn in scattering intensity is observed. This time for the lag phase agrees well with the lag phase seen with both SANS and AFM. However, it should be noted that the kinetics of amyloid aggregation is strongly dependent on protein concentrations, and different protein concentrations were used in the SANS (0.75

Hamill and Lee mg/mL), AFM (0.01 mg/mL), and DLS (0.25 mg/mL) measurements, requirements of the techniques. Thus, a direct comparison of the results obtained from these three methods is not possible. Furthermore, these concentrations are well beyond those found under physiological conditions, where an alternate aggregation mechanism may apply. Figure 6b illustrates the ability to trigger the growth of amyloid fibrils with UV light. The light scattering intensity is displayed versus time from 8 mM azoTAB under both visible and UV light. After 5 h, both samples are then exposed to the opposite light source for 1 h (visible to UV and UV to visible). The sample converted from visible to UV light (trans to cis azoTAB) exhibits a jump in scattering intensity following the 1 h of UV illumination, agreeing well with the scattering intensity of the UV sample, which indicates an increase in the aggregation state to larger structures. In contrast, the sample converted from UV to visible light (cis to trans azoTAB) exhibits no change in scattering intensity. This implies that the larger aggregates, once formed, are not reversible, which agrees with the literature, which reports that even powerful denaturants cannot dissociate amyloid aggregates.43 Comparing this to the SANS data of 8 mM visible t ) 5 h and 8 mM visible-to-UV t ) 5 h samples in Figure 1, it can be stated that that the fibril growth process can be triggered with UV light. This agrees with the literature, which has shown that certain variables (both temperature18,29,30 and pH66,74) can have an effect on the speed of the fibrilization process of β-amyloid peptide (1-40). The time scale at which these parameters affect fibril growth ranges anywhere from a few hours to a few days. Low pH (∼5) results in an accelerated fibril growth process; however, it was shown that these fibrils are actually less neurotoxic than those formed at neutral pH.18,66,74 An increase in temperature has been shown to accelerate fibril growth, while a decrease slows growth.29,30 Thus, it is hypothesized that in the presence of the visible light trans form of the surfactant, surfactant-protein interactions are inhibiting protein-protein interactions. When the system is then illuminated with UV light, the cis form of the surfactant no longer strongly interacts with the protein, allowing for protein-protein interactions to occur and “triggering” the growth process. Under the current experimental conditions any potential reversibility of fibril formation with visible light exposure could not be investigated, due mostly to the high absorbance of azoTAB and, thus, the long times required for photoisomerization. For example, immediately after the UV light has triggered growth (surfactant has switched from the trans to the cis state) it is likely that if the surfactant was switched quickly back to the visible (trans) form the triggered growth could be stopped or even reversed. This is likely because at some point during nucleation a critical size is reached where monomer addition becomes favorable. This critical size defines the nucleus, and eventually the nucleus will be more stable than the monomer.75,76 The point at which this happens is called the supercritical concentration; after this point nucleation becomes an irreversible polymerization.77 Thus, it is possible that there is a point early on during UV exposure at which the growth process is reversible or that the accelerated fibril growth could result in less neurotoxic species. Further studies examining these two possibilities are underway. Conclusions The ability to photoreversibly inhibit and then trigger amyloid fibril formation of Aβ40 in the presence of a light-responsive, azobenzene-based surfactant has been demonstrated with small-

β-Amyloid Peptide (1-40) Fibril Growth angle neutron scattering, atomic force microscopy, and light scattering intensity measurements. Pure Aβ40 solutions are observed to immediately form large, 3D fibril networks with fibril radii ≈ 56 Å and mesh sizes decreasing from 1000 to 600 Å over time as the degree of aggregation increases. In the presence of the photosurfactant, however, cylindrical protein oligomers with average diameters and lengths of 46 and 70 Å, respectively, were observed during early incubation times, while large fibrils with radii ≈ 55 Å were eventually formed at long incubation times. This is an indication of a lag phase prior to fibril formation, which was found to last ∼5 h under visible light and ∼2 h under UV exposure. The presence of cylindrical oligomers during the lag phase was further confirmed with AFM images exhibiting z-direction heights on the order of 50 Å as well as novel shape reconstruction analysis of the SANS data detailing cylindrically shaped oligomers. The shapes and dimensions of the oligomers were found to be largely independent of the incubation time and light/sample conditions. Furthermore, following the lag phase, images from both shape reconstruction of the neutron scattering data and AFM measurements indicated that the samples contained a combination of these cylindrically shaped oligomers and fibrils. This suggests that the observed oligomers represent an important early intermediate in fibril formation. As early intermediates have become increasingly viewed as the primary pathogenic species, these combined SANS and AFM images provide unique insight into the early stages of the disease. Furthermore, since the lag phase prior to fibril formation was seen to last longer under visible than UV light, exposure to UV light could be utilized to trigger the growth of oligomers into fibrils, providing a novel method of exploring the disease pathway. Acknowledgment. This material is based upon work supported by the National Science Foundation under Grant No. 0554115. We thank W. T. Heller for graciously supplying the GA_STRUCT program. We acknowledge the support of the National Institute of Standards and Technology, U.S. Department of Commerce, in providing the neutron research facilities used in this work. References and Notes (1) Chiti, F.; Dobson, C. M. Annu. ReV. Biochem. 2006, 75, 333. (2) Sambashivan, S.; Liu, Y.; Sawaya, M. R.; Gingery, M.; Eisenberg, D. Nature 2005, 437, 266. (3) Zurdo, J.; Guijarro, J. I.; Jimenez, J. L.; Saibil, H. R.; Dobson, C. M. J. Mol. Biol. 2001, 311, 325. (4) Booth, D. R.; Sunde, M.; Bellotti, V.; Robinson, C. V.; Hutchinson, W. L.; Fraser, P. E.; Hawkins, P. N.; Dobson, C. M.; Radford, S. E.; Blake, C. C. F.; Pepys, M. B. Nature 1997, 385, 787. (5) Brange, J.; Andersen, L.; Laursen, E. D.; Meyn, G.; Rasmussen, E. J. Pharm. Sci. 1997, 86, 517. (6) Pallares, I.; Vendrell, J.; Aviles, F. X.; Ventura, S. J. Mol. Biol. 2004, 342, 321. (7) Dobson, C. M. Trends Biochem. Sci. 1999, 24, 329. (8) Kelly, J. W. Curr. Opin. Struct. Biol. 1998, 8, 101. (9) Uversky, V. N.; Fink, A. L. Biochim. Biophys. Acta 2004, 1698, 131. (10) Bitan, G.; Kirkitadze, M. D.; Lomakin, A.; Vollers, S. S.; Benedek, G. B.; Teplow, D. B. Proc. Natl. Acad. Sci. 2003, 100, 330. (11) Serpell, L. C.; Sunde, M.; Benson, M. D.; Tennent, G. A.; Pepys, M. B.; Fraser, P. E. J. Mol. Biol. 2000, 300, 1033. (12) Roher, A. E.; Chaney, M. O.; Kuo, Y.-M.; Webster, S. D.; Stine, W. B.; Haverkamp, L. J.; Woods, A. S.; Cotter, R. J.; Tuohy, J. M.; et al. J. Biol. Chem. 1996, 271, 20631. (13) Lue, L. F.; Kuo, Y. M.; Roher, A. E.; Brachova, L.; Shen, Y.; Sue, L.; Beach, T.; Kurth, J. H.; Rydel, R. E.; Rogers, J. Am. J. Pathol. 1999, 155, 853. (14) McLean, C. A.; Cherny, R. A.; Fraser, F. W.; Fuller, S. J.; Smith, M. J.; Beyreuther, K.; Bush, A. I.; Masters, C. L. Ann. Neurol. 1999, 46, 860.

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