Ultrasonication-dependent production and breakdownlead to minimum-sized amyloid fibrilsEri Chatania,1, Young-Ho Leea, Hisashi Yagia, Yuichi Yoshimuraa, Hironobu Naikib, and Yuji Gotoa,2

aInstitute for Protein Research, Osaka University and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Suita,Osaka 565-0871, Japan; and bDivision of Molecular Pathology, Department of Pathological Sciences, Faculty of Medical Sciences, University of Fukui andCore Research for Evolutional Science and Technology, Japan Science and Technology Agency, Eihei-ji, Fukui 910-1193, Japan

Edited by David S. Eisenberg, University of California, Los Angeles, CA, and approved May 21, 2009 (received for review February 13, 2009)

Because of the insolubility and polymeric properties of amyloidfibrils, techniques used conventionally to analyze protein structureand dynamics have often been hampered. Ultrasonication caninduce the monomeric solution of amyloidogenic proteins to formamyloid fibrils. However, ultrasonication can break down pre-formed fibrils into shorter fibrils. Here, combining these 2 opposingeffects on �2-microglobulin (�2-m), a protein responsible for dialysis-related amyloidosis, we present that ultrasonication pulses areuseful for preparing monodispersed amyloid fibrils of minimal sizewith an average molecular weight of �1,660,000 (140-mer). Theproduction of minimal and monodispersed fibrils is achieved by thefree energy minimum under competition between fibril productionand breakdown. The small homogeneous fibrils will be of use forcharacterizing the structure and dynamics of amyloid fibrils, ad-vancing molecular understanding of amyloidosis.

�2-microglobulin � dialysis-related amyloidosis � protein misfolding �analytical ultracentrifugation

Amyloid fibrils are supramolecular assemblies exhibiting along unbranched fibrillar morphology �10 nanometers in

diameter, the deposition of which is associated with �30 degen-erative diseases including Alzheimer’s disease, prion disease,and dialysis-related amyloidosis (1–3). The past decade has seenprogress in our biophysical understanding of amyloid fibrilsusing various approaches including solution and solid state NMR(4–6) and X-ray crystallography (7, 8). However, the polymericproperties of amyloid fibrils, where huge size and heterogeneousnature result in insoluble and noncrystalline assemblies, are anobstacle to techniques such as X-ray crystallography, solutionNMR spectroscopy and other conventional spectroscopic mea-surements. To overcome the analytical problems confrontingstudies of amyloid fibrils, it is worth establishing a strategy forproducing monodispersed samples of amyloid fibrils. If amyloidfibrils of a well-defined molecular weight and improved solubilityare formed reproducibly, a more general application of varioustypes of fibril samples to a series of preexisting spectroscopicmeasurements will be accomplished.

As a strategy to produce amyloid fibrils of uniform andminimal size, ultrasonication has several potential applications.Although ultrasonication was originally used to prepare seedsfrom preformed fibrils (9), which were further applied to theamplification of infectious prion proteins (10, 11), ultrasonica-tion-dependent fragmentation is becoming an important ap-proach to analyzing the properties of fibrils (12, 13). However,its strong effect of agitation has recently been found to acceleratethe fibril nucleation of several proteins and peptides (14–18).Interestingly, amyloid fibrils produced by ultrasonication-induced fibrillation were very short with apparently similarlengths as determined by AFM (15, 19), suggesting that shortfibrils of homogeneous molecular size are formed efficientlyunder ultrasonication in combination with the opposing effectsof fragmentation and of nucleation (Fig. 1).

In this study, by applying analytical ultracentrifugation, wehave investigated the size distribution of amyloid fibrils of

�2-microglobulin (�2-m) formed spontaneously under pulses ofultrasonication. �2-m, a light chain of the type I major histo-compatibility antigen, is the main component of the amyloidfibrils deposited in the synovia of the carpal tunnel of patientssuffering from dialysis-related amyloidosis (9, 20). We will firstdemonstrate the uniformity of amyloid fibrils generated byultrasonication based on the distribution of sedimentation co-efficients determined by a sedimentation velocity analysis. Wealso measured the size distribution of the amyloid fibrils ob-tained by the ultrasonication of long fibrils to evaluate the effectsof ultrasonication-induced fragmentation. Furthermore, weight-average molecular weight was determined by sedimentationequilibrium taking advantage of the small and uniform size ofultrasonicated fragments.

ResultsUltrasonication-Induced Spontaneous Fibrillation. Previously (15),we found that repetitive ultrasonication pulses of 1 min with a

Author contributions: E.C., H.N., and Y.G. designed research; E.C., Y.-H.L., H.Y., and Y.Y.performed research; E.C., Y.-H.L., H.Y., and Y.Y. analyzed data; and E.C. and Y.G. wrote thepaper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1Present address: College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu, Shiga 525-8577, Japan.

2To whom correspondence should be addressed. E-mail: ygoto@protein.osaka-u.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0901422106/DCSupplemental.

Fig. 1. Schematic illustration representing the small-size and monodispersedsize distribution of amyloid fibrils under ultrasonication pulses. The schematicfree energy diagrams in the absence (black) and presence of ultrasonication(red) are shown, representing that the ultrasonication pulses modulate thefree energy landscape to form a minimum, producing minimum-sized andmonodispersed fibrils.

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quiescent incubation period of 9 min resulted in an overshoot offibrillation after a lag time of several hours, followed by thebreakdown of the preformed fibrils. The overshoot phenomenonsuggested that by reducing the period of quiescent incubation,one can obtain fibrils of minimal size by escaping the overshoot,the most efficient approach for forming them. Here, the ultra-sonication-induced fibrillation reaction was performed by ap-plying repetitive pulses of a fixed period of 1 min with variousquiescent incubation periods from 6 sec to 9 min. As for the NaClconcentration of the protein solutions, 38 mM, lower thanpreviously, was selected to prevent fibrils from associating duringthe sedimentation analysis (21) (see Figs. S1 and S2).

In all pulse cycles used, an abrupt increase in ThT fluores-cence was observed after a lag time (Fig. 2 A–D), consistent withultrasonication-induced spontaneous fibrillation (15). When thefibrils sampled after the 1-day incubation period (arrows 2 in Fig.2 A–D) were subjected to a sedimentation velocity analysis, allsamples exhibited considerably simple boundary profiles withstable plateaus, indicating a sharp distribution of s20W values at�36, 29, 27, and 17 S for quiescent periods of 9 min, 4 min, 2 min,and 6 sec, respectively (Fig. 2 E–H, closed circles in Fig. 2 I–L).

As expected, the overshoot became less pronounced as thepulse interval became shorter (Fig. 2 I–L), and with a 6-secquiescent period, the size of amyloid fibrils reached equilibriumalmost directly accompanied by only a slight decrease in average

size during the consecutive ultrasonication after the explosivephase, in accordance with negligible changes in ThT fluores-cence intensity (closed circles in Fig. 2 D and L). Furthermore,smaller fibrils were produced as the period became shorter,which suggests that the converged size of amyloid fibrils isregulated by the frequency of ultrasonication pulses appliedduring the fibrillation process. The fibril size obtained when thequiescent period was 6 sec long was the smallest possessing 17 Sand additionally, a significant unsedimenting fraction assigned tomonomeric �2-m with 1.6 S at 50,000 rpm (192,900 � g)coexisted (Fig. 2H). The results suggest that the decrease in thesize of fibrils accompanies the shift of equilibrium in thedirection of depolymerization, i.e., the critical monomer con-centration (CMC) increases with the decrease in fibril size. Thepresence of the overshoot phase was also suggested by the changein ThT fluorescence intensity, although the exact dependency ofThT fluorescence on fibril size is unknown (Fig. 2 A–D).

Characterization of the Ultrasonication-Induced Amyloid Fibrils. Togain more information about the shape and molecular weight ofmonodispersed amyloid fibrils obtained by ultrasonication-induced spontaneous fibrillation, AFM, EM, and a sedimenta-tion equilibrium analysis were performed. Although the amyloidfibrils formed with 6-sec intervals were the smallest, it wasdifficult to reproduce them due to an occasional unexpected

Fig. 2. Size distribution of �2-m amyloid fibrils formed by ultrasonication-induced spontaneous fibrillation. Monomeric �2-m was subjected to the periodic1-min ultrasonication followed by a period of silence without sonication lasting 9 min (A, E, and I), 4 min (B, F, and J), 2 min (C, G, and K), or 6 sec (D, H, and L).(A–D) Kinetics of ultrasonication-induced fibrillation monitored using ThT fluorescence. The results of 3 independent experiments (filled circles, triangles, andgray squares) are shown. The products of the triangle and filled circle plots were subjected to a sedimentation velocity analysis at 2 different time points, labeled1 and 2, respectively. (E–H) Sedimentation boundary profiles of fibrils sampled after the continued ultrasonication (filled circles in A–D). The data were recordedat 20,000 rpm (30,900 � g) and 5 °C by monitoring the absorbance at 280 nm, in which several traces at intervals of 10 min (E–G) or 8 min (H) were presented.(I–L) Integral distribution plots (s20W) of ultrasonication-generated fibril products sampled immediately after the abrupt increase of ThT fluorescence (triangles)and after the continued ultrasonication (filled circles).

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overshoot (gray squares in Fig. 2D). Therefore, for the sake ofreproducibility, we selected the amyloid fibrils formed at 2-minintervals.

AFM images scanned immediately after the explosive fibril-lation phase and after the 1-day incubation period showed plentyof small fibrils without any large aggregates, consistent withobservations made in our study in ref. 15 (Fig. 3 A and B). Withregard to the uniformity of fibril size, the AFM images indicateduniform fibril lengths, which were further confirmed with EMimages (Fig. 3C). A time-dependent decrease in fibril length wasalso observed on comparison of the AFM images between the 2time points, supporting an overshoot of fibril size as observed inthe sedimentation velocity analysis (Fig. 3 A and B). No seriouschange in conformation or chemical properties was detected byfar-UV CD, HPLC, and MS analyses (SI Text and Fig. S3).

Furthermore, the small size of the ultrasonicated fibrils al-lowed applying the sedimentation equilibrium method in whichsedimentation and diffusion to be successfully balanced at 3,000rpm (700 � g) and 5 °C (Fig. 3D). By virtue of the uniformity ofthe sample fragments, the experimental data were fitted by anequation assuming a single size component and no interfibrillarinteractions (see SI Text). By using the solvent density at 5 °Ccalculated from the database in Ultrascan 8.0 and the partialspecific volume (v�) at 5 °C obtained in our previous experiment(12), we obtained a molecular weight (Mw) value of 1,660,000 �

20,000 (140 mer), where error is the fitting error. The A0 valuewas 0.2, slightly higher than that observed for the sedimentationvelocity (0.07), which would be a result of the difference in rotorspeed between sedimentation velocity and equilibrium: A smallnumber of fragments sedimented at 20,000 rpm (31,000 � g)remained as an offset at 3,000 rpm (700 � g) during theequilibrium analysis (see SI Text).

It is also important to investigate the distribution width ofmolecular weight and the Mw value of amyloid fibrils formed bythe ultrasonication pulses. As seen in small residual errors of thefitting even with the equation assuming a single size component(Fig. 3D), very sharp distribution of molecular weight wassuggested by sedimentation equilibrium measurements. In con-trast, when the s values in the abscissa of the sedimentationvelocity profile were converted into molecular weights by usingthe Mw value obtained from the sedimentation equilibrium, awide range of molecular weight distribution with the standarddeviation of 1,660,000 � 1,290,000 was estimated (refer to Fig.2K, plot 2). At the present stage, the difference in distributionwidth between these 2 types of ultracentrifugation methodsremains unclear, suggesting that more sophisticated processingof sedimentation profiles is required for more precise analysis ofmolecular weight distribution.

Effect of Ultrasonication on the Preformed Fibrils. �2-m amyloidfibrils with lengths of several micrometers prepared by a seed-dependent extension reaction were also subjected to repeated1-min pulses each of which was followed by a 9-min quiescentincubation at 7 °C and 700 watts (see SI Text). When cycles ofultrasonication pulses were applied, the sedimentation patternsshowed a marked decrease in fibril size and distribution width,demonstrating the production of isomeric fibrils (Fig. 4). AFMimages also showed the effective fragmentation of amyloid fibrilsby the pulses (Fig. 5 A–D). When 2 �2-m amyloid fragmentshaving sedimentation coefficients of 44 S and 32 S obtained after18 and 72 pulses of ultrasonications, respectively, the differenceconfirmed with EM images (Fig. 5 E and F), were subjected toa sedimentation equilibrium analysis assuming a single sizecomponent, they gave Mw of 3,310,000 � 70,000 (279 mer) and2,040,000 � 30,000 (172 mer), respectively (Fig. 5 G and H),where errors are the fitting errors. Far-UV CD, HPLC, and MSanalyses of conformation and chemical properties revealed noserious changes in either of these fibrils (Fig. S3).

By further applying repetitive ultrasonication, insoluble ag-gregates with white turbidity were formed as a nonideal by-product, and were prominent after 360 pulses (Fig. S4). Simul-taneously, the dissociation of a significant amount of monomerand/or small oligomers was also observed with a marked offsetfrom the baseline at 10,000 rpm (7,700 � g), which was assignedto monomeric and/or oligomeric �2-m upon slow sedimentationat 53,000 rpm (216,700 � g). This result indicates that thefragmenting effect of ultrasonication leads to a dissociation intomonomers without any convergence of fibril size at 7 °C, wherefibrillation does not occur.

To evaluate the effectiveness of the ultrasonication techniquein the measurements of NMR spectroscopy, the amyloid fibrilstreated by 18 pulses of ultrasonication were further subjected toone dimensional 1H-NMR measurements. The result clearlyrepresents that the ultrasonicated amyloid fibrils gave manysharp peaks of amyloid fibrils whereas the usual long amyloidfibrils without ultrasonication did not give any peaks, indicatingthat the ultrasonication-generated amyloid fibrils will be usefulfor the direct measurement of NMR spectra of amyloid fibrils(Fig. S5 and SI Text).

DiscussionThe mechanism underlying the ultrasonication-induced produc-tion of small monodispersed fibril fragments is thought to be a

Fig. 3. Characterization of the �2-m fibrils formed by ultrasonication-induced fibrillation. The amyloid fibrils formed by periodic 1-min ultrasoni-cation followed by a 2-min silence were subjected to AFM, EM, and sedimen-tation equilibrium analyses. (A and B) AFM images of fibrils sampledimmediately after the abrupt increase in ThT fluorescence (A) and thosesampled after applying 1-day ultrasonication (B). (C and D) EM image (C) andsedimentation equilibrium analysis (D) of fibrils sampled after the 1-dayultrasonication treatment. The sedimentation equilibrium measurement wasperformed by measuring absorbance at 280 nm at a rotor speed of 3,000 rpm(700 � g), and 5 °C. The solid line represents a theoretical fitted curve assum-ing a single species model with an offset. The corresponding residuals areshown in Upper. (Scale bars: B, 1 �m; C, average length of fibril fragmentsestimated from the result of sedimentation equilibrium and velocity experi-ments based on the rod-like model, details of which are given in SI Text.)

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combination of 2 opposing effects of ultrasonication, the for-mation and breaking down of fibrils (Fig. 1). Although theenergy barrier of the nucleation phase is generally high, pre-venting the spontaneous formation of amyloid fibrils underquiescent conditions, ultrasonication can promote nucleation bylowering the energy barrier resulting from the assistance ofagitating effects (14, 15). Once the nucleus is formed, the growthphase proceeds via the incorporation of the monomers into theends of seed fibrils in a template-dependent manner (9). Undernormal extension conditions without the fragmenting effect ofultrasonication, the energy landscape is broad, and might bemodulated by additional mechanisms including breaking downand rejoining reactions especially for long fibrils (22–25), result-ing in a wide range of fibril lengths (black line in Fig. 1). The

increase in the monomeric fraction with the decrease in fibrillength (Fig. 2 and Fig. 4) suggests that CMC decreases with anincrease in fibril length, implying a more complicated mecha-nism than that approximated by a unique constant of fibrilpolymerization. In contrast, the combination of fragmentation

Fig. 4. Dependency of the size distribution of fragmented amyloid fibrils onthe number of ultrasonication pulses applied. The frequency, power of out-put, and pulse length for the ultrasonicators are described in Materials andmethods. (A–D) Sedimentation boundary profiles of fragmented fibrils (0.3mg/mL) without sonication (A), or with 1 pulse (B), 18 pulses (C), and 72 pulses(D) of 1-min ultrasonication. Each sedimentation pattern was recorded at10,000 rpm (7,700 � g) (A) or 17,000 rpm (22,300 � g) (B–D) and 5 °C bymonitoring the absorbance at 280 nm, and several traces at intervals of 10 min(D), 11 min (A), or 12 min (B and C) are presented. (E) The integral distributionplots of �2-m amyloid fibrils treated by 1(black), 3 (red), 9 (blue), 18 (yellow),33 (green), 54 (magenta), and 72 pulses (cyan) of ultrasonication. The averagesedimentation coefficient reached 44 S after 3 pulses, and, although noremarkable change in fibril size was detected between 3 and 33 pulses, thesedimentation coefficient decreased again and eventually reached 32 S after72 pulses. (Inset) Result for amyloid fibrils before the ultrasonication treat-ment (white). A distorted integral distribution plot for the fibril sampleswithout ultrasonication represents too large a range of sedimentation coef-ficients with no stable plateau, failing to extrapolate correctly in the vanHolde–Weischet analysis. Fig. 5. Characterization of the �2-m fibrils fragmented by ultrasonication.

(A–D) AFM images of the fibrils before the ultrasonication treatment (A) andthose treated with 1 (B), 18 (C), and 72 pulses (D) of ultrasonication. (E and F)EM images of the fibrils treated by 18-pulse (E) and 72-pulse (F) ultrasonica-tion, with sedimentation coefficients of 44 S and 32 S, respectively (Fig. 4). (Gand H) Sedimentation equilibrium measurements of ultrasonicated fibrils of�2-m. The data for fibrils treated by 18-pulse and 72-pulse ultrasonication areplotted under the same condition as used in Fig. 3D. (Scale bars: D, 1 �m; E andF, average length of fibril fragments estimated from the result of sedimen-tation equilibrium and velocity experiments based on the rod-like model,details of which are given in SI Text.)

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with the dynamic nature of the equilibrium between the mono-meric dissociation and association occurring at the end of eachfibril, which has been demonstrated by H/D exchange of amyloidfibrils in the SH3 domain (13) (red line in Fig. 1), will serve toproduce small isometric amyloid fibrils. The position of theenergy minimum and the steepness of the landscape of frag-mentation would be determined by a balance between theelongation kinetics and fragmentation efficiency, the latter ofwhich is presumably determined by the frequency of ultrasoni-cation pulses. Additionally, the intrinsic physical properties suchas fragility of individual fibrils (23, 25–27) and intensity ofultrasonication would affect the equilibrated fibril size.

Another accomplishment of the current study is the directdetermination of molecular weight by applying the sedimenta-tion equilibrium method. The sedimentation velocity techniquehas recently been applied to the characterization of the sizedistribution of prefibrillar oligomers or larger aggregates involv-ing fibrillation (28). With regard to the application to amyloidfibrils themselves, worm-like amyloid fibrils of apolipoproteinC-II with an advantageous soluble nature represent the firstinstance of successful sedimentation velocity measurements, bywhich interfibrillar tangling (29) and fibril breaking and rejoin-ing (24) during the process of fibril formation were successfullyobserved. However, there has been no report for the sedimen-tation equilibrium of amyloid fibrils.

The accomplishment of sedimentation equilibrium for amy-loid fibrils made possible the estimation of their hydrodynamicshapes. By using Mw obtained from the sedimentation equilib-rium measurement with the sedimentation coefficient obtainedfrom the sedimentation velocity measurement, we estimated thevalues of diameter and length for each fragment as 5.0 nm � 99nm for the 27-S amyloid fibrils formed at 2-min intervals and 6.9nm � 104 nm and 5.7 nm � 93 nm for the 44-S and 32-S amyloidfibrils formed by the fragmentation, respectively, assuming thatthe shape of ultrasonicated �2-m fibril fragments is approxi-mately cylindrical (SI Text) (30). All of the estimated sizes ofamyloid fibrils agreed with the EM images (Figs. 3C and 5 E andF; the scale bar in each panel represents the length calculatedfrom the frictional coefficient).

From a series of analytical ultracentrifugation investigationsperformed for fibrils of �2-m, we conclude that ultrasonicationis a powerful technique for producing monodispersed fibrils witha well-defined molecular size. The use of ultrasonication-generated amyloid fibrils combined with various conventionaltechniques is expected to bring about clarification of the widespectrum of physicochemical properties of amyloid fibrils. Inparticular, the faster rotational motion with the decrease in sizeof ultrasonicated amyloid fibrils, which has brought about prop-erties closer to those of a soluble and homogeneous proteinsolution, might extend to direct applications in solution NMRstudies, or the formation of amyloid crystals using isometricfragments as a building block for X-ray crystallography (7, 8).Indeed, the ultrasonicated amyloid fibrils gave many sharp NMRpeaks specific to the amyloid fibrils by the faster rotationalmotion accompanied by the decrease in size by ultrasonication,whereas the usual long amyloid fibrils obtained by seeding didnot give any peaks because of an extensive broadening of signals(Fig. S5). This result demonstrates that the ultrasonicationtechnique will become a promising procedure to achieve thedirect measurements of NMR spectra of amyloid fibrils to revealmore detailed structural and dynamic properties. Additionally, itmight also be expected that the ultrasonication diminishes thepolymorphic feature of amyloid fibrils at the molecular level (i.e.,various patterns of twisting between protofilaments or variousmicroscopic structures inside the fibrils), resulting in an improve-ment of the quality of solid-state NMR spectra. Other beneficialapplications include the detailed evaluation of kinetic andthermodynamic feature of the fibril formation and the quanti-

tative analysis of the cytophysiological effects such as toxicityand infectiveness of amyloid fibrils, both of which had beendifficult to be accomplished because of poor dispersibility andunclarified concentrations of seed ends in the conventionalmethod for fibril preparation. Overall, the development ofanalytical techniques in combination with the ultrasonication-induced fragmentation method should help to clarify the prop-erties of fibril formation and structure.

Materials and MethodsUltrasonication Treatments. Recombinant human �2-m expressed as reportedin ref. 31 was dissolved at a concentration of 0.3 mg/mL in a 3.2 mM HClsolution (pH 2.5) containing 38 mM NaCl and was placed on a water bath-typeultrasonicator (ELESTEIN SP070-PG-M, Elekon, Tokyo). Ultrasonication pulseswere applied to the samples for 1 min followed by a quiescent period varyingfrom 6 second to 9 min, a process that was repeated during the incubation. Thesamples were ultrasonicated from 3 directions, i.e., bottom and 2 side walls ofthe incubating bath, the ultrasonication pulses from which cross one anotherat the sample position (15). The frequency and output of the sonication wereset to 17–20 kHz and 350 watts, respectively, and temperature was maintainedat 37 °C. To monitor the formation of amyloid fibrils, a 5-�L aliquot of samplewas mixed with 1 mL of 5 �M ThT in a 50 mM glycine-NaOH buffer (pH 8.5) at25 °C, and the fluorescence intensity at 485 nm of this solution was measuredwith an excitation wavelength of 445 nm at different points of time (9). Thefragmentation of long amyloid fibrils several micrometers in length was alsocarried out by using the ELESTEIN SP070-PG-M. Long �2-m amyloid fibrilsseveral micrometers in length were formed by the seed-dependent fibrilextension method established by Naiki et al. (9). �2-m was dissolved at aconcentration of 0.3 mg/mL in a 3.2 mM HCl solution (pH 2.5) containing 38mM NaCl and the extension reaction was carried out by adding 5 �g/mL ofultrasonicated fibrils as seeds at 37 °C under quiescent conditions. Then,500-�L aliquots of the formed fibrils were sealed into tubes and ultrasonicatedfor 1 min, before being incubated for 9 min without sonication, a process thatwas repeated. The frequency and the power of output were set to 17–20 kHzand 700 watts, respectively, and the temperature was maintained at 7 °Cthroughout the treatment.

Analytical Ultracentrifugation. The size distribution of the amyloid fibrils wasmonitored by sedimentation velocity and sedimentation equilibrium mea-surements that were performed using a Beckman-Coulter Optima XL-1 ana-lytical ultracentrifuge. For the sedimentation velocity experiments, the sam-ples were first centrifuged at 3,000 rpm (700 � g) for 5 min to stabilize thetemperature, and after precentrifugation, the rotor speed was increased to10,000–20,000 rpm (7,700–30,900 � g) and absorbance data at 280 nm werecollected at intervals of 10–20 min. All measurements were carried out at aconstant temperature of 5 °C with a radial increment of 0.003 cm in thecontinuous scanning mode. The sedimentation coefficients that were cor-rected to s20W (standard solvent conditions: The density and velocity of purewater at 20 °C) were obtained from the data by the van Holde–Weischetmethod with the software UltraScan 8.0 (www.ultrascan.uthscsa.edu) (32),using the partial specific volume of amyloid fibrils determined in our study inref. 12.

For the sedimentation equilibrium experiments, samples were centrifugedat 3,000 rpm (700 � g) at 5 °C and equilibrium concentration profiles wererecorded by monitoring absorbance at 280 nm across the centrifugation cellwith a radial increment of 0.001 cm in the continuous scanning mode. Detailedmethods for the data analysis of sedimentation equilibrium results are de-scribed in SI Text.

AFM Measurements. Fibril samples were diluted 5-fold with water and 50 �Lwas spotted onto a freshly cleaved mica plate. After 1 min, the residualsolution was blown off with compressed air. AFM images were obtained usinga Nano Scope IIIa (Digital Instruments). The scanning tip used was a phospho-rus (n)-doped Si (Veeco, spring constant � 20–80 N/m, resonance frequency �245–289 kHz), and the scan rate was 0.5 Hz.

Transmission Electron Microscopy. Amyloid fibrils (0.3 mg/mL) were diluted10-fold with water and immediately placed on a 400-mesh carbon-coatedcopper grid (Nissin EM). The excess solution was removed with filter paperafter the sample had stood for 1 min and the fibrils adsorbed on the grid werenegatively stained with a 2% (wt/vol) uranyl acetate solution. Electron micro-graphs were acquired using a transmission microscope (JEM100CX andJEM1200EX; JEOL) at 80 kV with magnification at �29,000 or �30,000.

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ACKNOWLEDGMENTS. We thank Rumi Adachi for help with the ultrasonica-tion experiments, Miyo Sakai for the ultracentrifugation experiments, andYuki Kobayashi for the expression and purification of recombinant �2-mprotein. Electron micrographs were taken at the Research Center for Ultra-

high Voltage Electron Microscopy, Osaka University, Japan. This work wassupported by the Japanese Ministry of Education, Culture, Sports, Science andTechnology (Y.G.) and a Japan Society for Promotion of Science PostdoctoralFellowship (to E.C. and Y.-H.L.).

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