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. 2006 Jun;15(6):1334–1341. doi: 10.1110/ps.052013106

Dichotomous versus palm-type mechanisms of lateral assembly of amyloid fibrils

Natallia Makarava 1, Olga V Bocharova 1,1, Vadim V Salnikov 1,2, Leonid Breydo 1, Maighdlin Anderson 1, Ilia V Baskakov 1
PMCID: PMC2265092  PMID: 16731968

Abstract

Despite possessing a common cross-β core, amyloid fibrils are known to exhibit great variations in their morphologies. To date, the mechanism responsible for the polymorphism in amyloid fibrils is poorly understood. Here we report that two variants of mammalian full-length prion protein (PrP), hamster (Ha) and mouse (Mo) PrPs, produced morphologically distinguishable subsets of mature fibrils under identical solvent conditions. To gain insight into the origin of this morphological diversity we analyzed the early stages of polymerization. Unexpectedly, we found that despite a highly conserved amyloidogenic region (94% identity within the residues 90–230), Ha and Mo PrPs followed two distinct pathways for lateral assembly of protofibrils into mature, higher order fibrils. The protofibrils of Ha PrP first formed irregular bundles characterized by a peculiar palm-type shape, which ultimately condensed into mature fibrils. The protofibrils of Mo PrP, on the other hand, associated in pairs in a pattern resembling dichotomous coalescence. These pathways are referred to here as the palm-type and dichotomous mechanisms. Two distinct mechanisms for lateral assembly explain striking differences in morphology of mature fibrils produced from closely related Mo and Ha PrPs. Remarkable similarities between subtypes of amyloid fibrils generated from different proteins and peptides suggest that the two mechanisms of lateral assembly may not be limited to prion proteins but may be a common characteristic of polymerization of amyloidogenic proteins and peptides in general.

Keywords: conformational changes, prion, amyloid fibrils, electron microscopy, lateral assembly, protofibrils

The ability of proteins and peptides to polymerize into amyloid fibrils is considered to be a generic feature of the polypeptide backbone (Dobson 2002). It is linked to several neurodegenerative and systemic diseases, such as transmissible spongiform encephalopathies, Alzheimer's disease, Parkinson's disease, Huntington's disease, type II diabetes and others (Carrell and Lomas 1997; Prusiner 2001). Self-assembly of polypeptides into amyloid structures is also implicated in diverse biological functions such as the colonization of E. coli (Chapman et al. 2002), melanosome biogenesis (Berson et al. 2003), maintenance of long-term memory (Si et al. 2003), and protein-based inheritance in yeast and fungi (Wickner 1994). Mature fibrils produced from amyloidogenic proteins often consist of several filaments assembled in a hierarchical manner that results in a variety of fibril morphologies (Ionescu-Zanetti et al. 1999; Jimenez et al. 1999, 2002; Kad et al. 2001, 2003; Antzutkin et al. 2002; Baxa et al. 2003; Gosal et al. 2005). Remarkable similarities were found between subtypes of amyloid fibrils generated from different proteins and peptides suggesting the existence of common polymerization pathways.

In recent studies, the differences in fibrillar morphologies were linked to strain- and species-specific properties of amyloid fibrils produced from the yeast prion [PSI] or from the fragment of mammalian prion protein encompassing residues 23–144 (Diaz-Avalos et al. 2005; Jones and Surewicz 2005). While the link between prion strains and protein conformation has been well established in the past few years (Caughey et al. 1998; Safar et al. 1998; Chien et al. 2004; Krishnan and Lindquist 2005), the origin of conformational and morphological polymorphisms in amyloid fibrils is not well understood. Here we analyzed the early stages of nonseeded polymerization of full-length mouse (Mo) and Syrian hamster (Ha) prion proteins (PrP)1 and found striking differences in the mechanisms for their lateral assembly. The protofibrils of Ha PrP first formed irregular bundles characterized by a peculiar palm-type shape, which ultimately condensed into mature fibrils. The protofibrils of Mo PrP, on the other hand, associated in pairs in a pattern resembling dichotomous branching. These two distinct mechanisms for lateral assembly account for the differences in morphology of fibrils produced from Mo and Ha PrPs and may have important implications for providing insight into the molecular determinants of prion strains and species barriers.

Results

Mature fibrils of Ha PrP and Mo PrP have similar secondary structures but show variation in fibrillar morphology

Amyloid fibrils of full-length Mo PrP and Ha PrP were produced using the same solvent conditions that we previously described for generating fibrils with limited infectivity from Mo PrP 89–230 (Baskakov et al. 2002; Legname et al. 2004). Fibrils produced from full-length Mo PrP and Ha PrP showed no significant differences in their secondary structure as judged from FTIR (Fig. 1). Within the amyloidogenic region of PrP (residues 90–230), Mo and Ha proteins differ by eight amino acid residues. While such differences in sequence appear to have no strong impact on the secondary structure, the electron micrographs showed significant differences in the morphologies of mature Mo and Ha fibrils (Fig. 2). Noteworthily, fibrils of both Ha and Mo PrPs appeared as heterogeneous mixtures with broad range of morphologies. The number of filaments, as well as the periodicity and the pattern of filament twisting varied from fibril to fibril and, occasionally, even within a single fibril. Such polymorphism is not surprising considering that a variety of cross-β ordered structures can be produced even from the seven-residue peptide derived from the yeast prion protein Sup 35 (Diaz-Avalos et al. 2003).

Figure 1.

Figure 1.

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Second derivatives of FTIR spectra of amyloid fibrils produced from full-length Ha (dashed lines) and Mo (solid lines) PrP under the same growth conditions. Briefly, to form amyloid fibrils, Mo and Ha PrPs (10 μM) were incubated in the presence of 1 M GndHCl, 3 M Urea, 150 mM NaCl, and 20 mM Na-acetate (pH 5.0) at 37°C. Duplicates correspond to two separate preparations of fibrils.

Figure 2.

Figure 2.

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Gallery of negatively stained mature fibrils produced from Ha PrP (panels 1–6) and from Mo PrP (panels 7–13). The left panel in each pair displays actual electron micrographs, while the right panel corresponds to the same image extended perpendicular to the fibril axis to visualize the details in the intertwining pattern of individual fibrils. Arrows mark sites where the twisting pattern changes or switches to untwisted flat morphology. Scale bars are 0.2 μm. Mo and Ha full-length recombinant PrPs encompassing residues 23–230 and 23–231, respectively, were expressed, purified, and folded into the amyloid form as described earlier (Bocharova et al. 2005a).

Detailed analysis of EM images revealed several morphological subtypes peculiar to Mo and Ha fibrils, respectively. Specifically, the major population of Mo fibrils resembled flat untwisted ribbons (Fig. 2, panel 7) or contained regions with untwisted ribbon-like flat morphologies (Fig. 2, panels 8–11). In contrast, the fibrils of Ha PrP were always twisted with regular periodicities in the twisting patterns (Fig. 2, panels 1–6); no ribbon-like untwisted morphologies were found in the preparations of Ha fibrils. A minor subpopulation of Mo fibrils also displayed twisted morphologies (Fig. 2, panels 12,13); however, this type of fibril was less common than the ribbon-like fibril. Surprisingly, Mo fibrils displayed more diversity in their twisting patterns than Ha fibrils. The periodicity of twisting in Mo fibrils was less regular. Another peculiar feature of Mo fibrils were changes in their twisting patterns or switches from a twisted to untwisted morphology occurred within a single fibril (Fig. 2, panels 9–13). Often, a single fibril switched or changed its twisting periodicity several times. Alteration of the twisting pattern within an individual fibril was rarely observed in Ha fibrils (Fig. 2, panels 2,5).

What is the origin of fibrillar polymorphism? Why do Mo and Ha fibrils, produced under identical solvent conditions, have different morphologies and twisting patterns? To get insight into the mechanism responsible for high fibrillar polymorphism we decided to analyze the early stages of fibrillization.

As measured by ThT assay, polymerization of full-length PrPs displayed typical nucleation–polymerization kinetics with a lag phase followed by a substantial increase in the ThT signal (Fig. 3A). In previous studies, we found that mature fibrils were already formed by the end of lag phase (Fig. 3B; Baskakov and Bocharova 2005). However, only a small fraction of PrP molecules (<5%) was converted into the fibrillar form by the end of lag phase, while >95% remained in nonfibrillar, monomeric, or oligomeric states as judged by size-exclusion chromatography (Baskakov et al. 2002; Bocharova et al. 2005a). Several lines of evidence indicated that two processes occurred in parallel during the so-called elongation stage: fragmentation of fibrils and actual elongation of fibrils (Baskakov and Bocharova 2005). Fragmentation resulted in the exponential multiplication of fibrillar ends allowing for rapid recruitment of PrP molecules and rapid growth of ThT fluorescence. In the current study, we have specifically focused on the lag phase of the polymerization reaction.

Figure 3.

Figure 3.

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(A) Schematic representation of the nucleation-polymerization kinetics that consists of a lag phase and elongation phase. The actual kinetics for Mo and Ha PrPs were measured using the ThT-binding assay; Ha PrP always displayed a longer lag time than mouse PrP. For this reason, the lag time was calculated for both proteins, and different time points, at which aliquots were taken, are expressed as a percentage of the total lag time as indicated by arrows (20%–40%, 40%–60%, or 60%–100% of lag time). The definition used for the lag time is described in Materials and Methods. (B) EM images of Mo PrP collected at the end of the lag phase that correspond to the time point marked by the dashed line in A. Scale bar is 0.2 μm.

Ha PrP follows a palm-like mechanism of lateral assembly

Aliquots were taken at different time points within the lag phase as shown in Figure 3A and analyzed using electron microscopy. The first structures visible by EM at the early stages of polymerization of Ha PrP (at the time points that correspond to 20%–40% of the lag time) were curved flexible filaments (Fig. 4A). The filaments elongated over time while remaining very flexible and sinuous (Fig. 4B). We considered these filaments to be protofibrils—minimal structural units of mature fibrils. At the later stages of polymerization (within 60%–100% of the lag time), the protofibrils developed weak lateral interactions with each other, producing irregular bundle-like structures (Fig. 4C, panels 1,2). The characteristic feature of this stage of assembly was the formation of thick but quite loosely packed bundles composed of numerous protofibrils. Occasionally, regular coiling motifs composed of several protofibrils could be seen.

Figure 4.

Figure 4.

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EM images of Ha PrP fibrils collected at early stages of assembly. Samples were taken at 20%–40% (A), 40%–60% (B), or 60%–100% (C) of the lag time. The lag time was calculated as described in Materials and Methods. Formation of the palm-like structures (marked by arrows) can be seen at the fibrillar edges (panels 2,3,5–7) or within the fibrils (panel 4). Panel 8 was extended perpendicular to the fibril axis to illustrate the formation of a complex interwoven pattern within a compact fibrillar region. Scale bars are 0.1 μm. At each time interval three aliquots were taken, and up to 10 EM images were collected from each sample and analyzed.

By the end of the lag phase, loosely packed bundles condensed into structures that had regular twisting morphologies and diameters similar to that found in mature fibrils (Fig. 4C, panels 3,5–7). Remarkably, the “condensation” of protofibrils into mature fibrils were often seen to begin somewhere in the middle regions of protofibrils and then appear to propagate toward one or both edges (Fig. 4C, panels 3,5). As a result, the fibrillar edges typically displayed palm-shaped structures composed of numerous unassembled protofibrils, while the middle regions were condensed and resembled mature fibrils (Fig. 4C, panels 5–8). Occasionally, regions with unassembled protofibrils were found between two condensed regions (Fig. 4C, panel 4). This suggests that condensation can be initiated from several points within a single fibril. Overall, we found that the formation of palm-like structures was the most distinguishing feature of the hierarchical assembly of Ha PrP fibrils.

Mo PrP follows a dichotomous mechanism for lateral assembly

In contrast to Ha PrP, protofibrils of Mo PrP were found to be assembled in pairs even at the earlier stages of polymerization. The protofibrils had flat, untwisted, ribbon-like morphologies (Fig. 5A, panels 2–4). Occasionally, single-filament structures were observed (Fig. 5A, panel 1). Over time, protofibrils elongated while maintaining ribbon-like morphologies (Fig. 5B). At this stage, fibrils had variable widths and exhibited some curvature—a characteristic of ribbon-like structures. Occasionally, early ribbons were observed splitting apart, either at their edges or at the middle, demonstrating that they were still composed of two individual protofibrils (Fig. 5B).

Figure 5.

Figure 5.

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EM images of Mo PrP fibrils collected at early stages of assembly. Samples were taken at 20%–40% (A), 40%–60% (B), or 60%–100% (C) of the lag phase. Scale bars are 50 nm in A, and 0.1 μm in B and C. Mo PrP fibrils display a dichotomous pattern of coalescence, where the “coalescence forks” are marked by arrows. Several events of hierarchical assembly observed within an individual fibril are shown on panels 1 and 2 in c. Inserts in panel 1 correspond to the areas marked with dashed lines and contain enlarged regions showing morphological details. At each time interval three aliquots were taken, and up to 10 EM images were collected from each sample and analyzed.

Several early ribbons associated with each other and formed higher-order fibrils with a dichotomous pattern of coalescence (Fig. 5C). Dichotomous coalescence was a peculiar feature of the lateral assembly of Mo PrP; we never observed palm-like coalescence in polymerization of Mo PrP. Multiple consecutive “coalescence forks” were often seen even within an individual growing fibril, illustrating that the process of lateral assembly is highly hierarchical (Fig. 5C, panels 1,2). Occasionally, the branching forks were found at both edges of a growing fibril, suggesting that the lateral association may proceed in opposite directions. Furthermore, individual coalescence forks were sometimes oriented toward each other, illustrating that more than one initiation point can be formed within a single fibril on the same hierarchical level (Fig. 5C, panels 3,4). Fibrils with dichotomous coalescence were seen predominantly during the lag phase of the polymerization process and were rare during the subsequent “elongation” stage. This argues that dichotomous morphology represents a stage of lateral assembly rather than dissociation of preassembled fibrils into protofilaments.

The striking differences between the mechanisms for lateral assembly of Ha and Mo fibrils were also seen using atomic force microscopy conducted in a liquid cell (Fig. 6A, B). The polymerization of Ha PrP was captured at a loosely packed bundle stage, where several single protofibrils had developed lateral interactions but had not yet condensed into mature fibrils (Fig. 6A). The polymerization of Mo PrP was captured at the stage of a “coalescence fork,” when two laterally assembling ribbons were splayed apart (Fig. 6B).

Figure 6.

Figure 6.

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The intermediate stages of the lateral assembly of Ha PrP (A) and Mo PrP (B), as revealed by Atomic Force Microscopy. Three-dimensional images capture the palm-type structure formed during assembly of Ha PrP fibrils and a “coalescence fork” typically observed in polymerization of Mo PrP. Arrows indicate single protofibrils. The images were taken at time points corresponding to 80% of lag time. Scale bars are 0.2 μm. Fibrils were imaged with a PicoSPM LE AFM (Molecular Imaging), operating in MAC (alternating magnetic field) mode and using a MAC II silicon cantilever (tip radius <7 nm, spring constant 2.8 N/m) and a liquid cell (Molecular Imaging).

Discussion

Analysis of the early stages of fibrillar polymerization proved to be extremely illuminating. Our studies revealed two types of lateral assembly for amyloid fibrils: palm-like and dichotomous. The palm-like mechanism is characterized by simultaneous association of several protofilaments, while the dichotomous mechanism appears limited to the interaction of two filaments or ribbons, each composed of two protofilaments, at a time in each “coalescence fork” (Fig. 7). Because several consecutive coalescence forks can be formed along an individual growing fibril, the mature fibrils produced via the dichotomous mechanism appears to include as many protofilaments as those produced via palm-like mechanism. Several general observations can be made regarding the morphology of fibrils: (1) An array of morphological variants can be generated through each mechanism; (2) the palm-like mechanism, however, produces only fibrils with twisted morphologies, whereas the dichotomous mechanism produces fibrils with both flat and twisted morphologies; (3) in comparison to the palm-like mechanism, the dichotomous mechanism results in (a) a broader diversity of twisting patterns and (b) frequent alteration of a twisting patterns within individual fibrils. Surprisingly, as judged from EM images, certain types of mature fibrils of Mo and Ha PrP appear to have similar twisting morphologies, although they are generated through different types of lateral assembly (Fig. 2, cf. panels 3 and 13).

Figure 7.

Figure 7.

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Schematic illustration of the palm-type and dichotomous mechanisms of lateral assembly.

The two types of lateral assembly are different in several other key aspects. The palm-like mechanism seems to have apparent analogies with current concept of protein folding mechanisms. On the way to a native fold, a disordered polypeptide chain is believed to collapse forming relatively compact, nonnative, molten-globule type structures. Such compact states are characterized by reduced conformational entropy and, therefore, provide an efficient mechanism in the search for the native conformation. In a similar manner, protofibrils of Ha PrP first form relatively compact irregular bundles, in which protofibrils still maintain large motional freedom. Physical proximity of protofibrils in a bundle reduces the conformational space and could speed up a search for the thermodynamically most favorable packing. Eventually, bundles with irregular morphologies condensed into fibrils with the regular twisting morphologies (Fig. 7). Coexistence of morphologically distinct fibrils in one preparation suggests that several alternative types of packing are possible.

In dichotomous assembly, on the other hand, only two ribbons were seen to bind to each other at any one time. Therefore, only a few alternative geometries would be available under these conditions for the most favorable type of packing. As judged from EM, after forming an initial complex, the lateral contact between a pair of interacting protofibrils propagates along the fibrillar axis toward one or both directions (Fig. 7). One can assume that the number of alternative possibilities for packing increases as the lateral assembly progresses to the upper level and higher order fibrils are formed. If it is true, the mechanism of dichotomous assembly predicts that an incremental change in the range of fibril morphologies at the higher level of the hierarchical assembly. Frequent switches in morphologies within individual fibrils suggest that alternative types of packing are interconvertible, thermodynamically equivalent, and separated by a very modest energetic barrier. Alterations in morphologies were previously observed in the paired helical filaments found in patients with Alzheimer disease (Crowther 1991). The factors that regulate the twisting pattern and its alterations remain to be determined. In our previous studies, Cu2+ was found to be one of the factors that favored twisted over flat morphologies (Bocharova et al. 2005b). Low levels of methionine oxidation could also influence the interactions between filaments and affect the modes of their lateral association (Breydo et al. 2005).

It is totally unexpected and not intuitive that that Ha and Mo PrPs follow distinct pathways of lateral assembly under identical solvent conditions and produce two morphologically distinguishable subsets of mature fibrils. Since the amyloidogenic regions of Mo and Ha PrPs (residues 90–230) are 94% identical, minor variations in the primary sequence must determine the type of lateral assembly and result in the dramatic differences observed in fibril morphology. For example, using a series of Ha PrP-derived peptides, Petty et al. (2005) showed that the side chain of single residue could modulate the morphology of protofibrils. The question of great interest that needs to be addressed in future studies is whether the different pathways of lateral assembly arise due to formation of structurally different nuclei or to minor variations in the protofibrillar interface.

In the current studies, the morphological differences between Ha and Mo protofibrils appeared at very early stages of polymerization. Flexible and curvy protofibrils were commonly observed for Ha PrP, while straight flat ribbons composed of two filaments appeared as the earliest structures of Mo PrP grown under the same solvent conditions. Whether the earliest paired ribbons of Mo PrP emerged via an association of two preformed filaments, undetected by EM, or whether they appeared by an extension of a single nucleus with paired substructures remains to be clarified. Nevertheless, the difference between polymerization of Mo and Ha seems to arise already at the very early stages of polymerization, possibly as early as nucleation. These differences then promulgated through the higher levels of lateral assembly. Taken together, our studies suggest that morphological differences in early protofilaments determine two different types of the lateral assembly, each of which generates morphologically polymorphous subsets of higher order fibrils. Therefore, the polymorphism is regulated on both levels: First, it appears at the stage of early polymerization, and then, it is reinforced further throughout distinct mechanisms of lateral assembly.

Our current and former studies demonstrate that the formation of mature fibrils and fibril elongation already take place during the so-called “lag phase” (Baskakov and Bocharova 2005). However, only a small fraction of PrP molecules (∼5%) converts into mature fibrils during the lag phase. The subsequent “elongation” phase is accompanied by a strong increase in the ThT signal reflecting the polymerization of the remaining PrP molecules. The elongation phase consists of two parallel processes: fibril fragmentation and the actual elongation of fibrillar fragments. Fragmentation results in a rapid multiplication of the active centers of polymerization permitting the fast recruitment of PrP molecules and a rapid increase in ThT fluorescence (Baskakov and Bocharova 2005). Noteworthily, fibril elongation by itself cannot be referred to as an autocatalytic reaction, unless fragmentation and multiplication of the active centers take place.

Other amyloidogenic proteins and peptides have been shown to produce fibrils morphologically similar to either the curvy twisted fibrils of Ha PrP or to the rigid ribbon-like fibrils of Mo PrP (Harper et al. 1999; Ionescu-Zanetti et al. 1999; Lashuel et al. 2000; Aggeli et al. 2001; Jimenez et al. 2001, 2002; Kad et al. 2001, 2003; Antzutkin et al. 2002; Gosal et al. 2005). Depending on the pH and ionic strength of solvent, β2-microglobulin was shown to polymerize via two competing pathways producing either semiflexible worm-like fibrils or straight and rigid ribbon-like fibrils (Kad et al. 2001, 2003; Gosal et al. 2005). Considering that two competing pathways may also coexist in polymerization of PrP, it is reasonable to speculate that the mechanism for lateral assembly may switch between the palm-like and dichotomous type depending on solvent conditions.

Uncovering two alternative pathways of lateral assembly provides new insight into the complex mechanism of prion polymerization. Morphological similarities observed between subtypes of mature fibrils produced from a variety of proteins and peptides suggest that the two mechanisms of lateral assembly may not be limited to prion proteins but may be a common characteristic of polymerization of amyloidogenic proteins and peptides in general.

Materials and methods

Protein expression, purification, and conversion into amyloid fibrils

Mo and Ha full-length recombinant PrPs encompassing residues 23–230 and 23–231, respectively, were expressed and purified as described earlier (Bocharova et al. 2005a; Breydo et al. 2005). To form amyloid fibrils, a stock solution of PrP in 6 M GdnHCl was diluted to a final concentration of 10 μM in the presence of 1 M GdnHCl, 3 M Urea, 150 mM NaCl and 20 mM Na-acetate (pH 5.0), and incubated in 1.5 mL conical plastic tubes (Fisher) in a reaction volume of 0.45–0.6 mL at 37°C with continuous shaking at 600 rpm on a Delfia plate shaker (Wallac) (Bocharova et al. 2005a).

Monitoring the kinetics of amyloid formation

The kinetics of the amyloid formation was monitored using ThT-binding assay as described earlier (Bocharova et al. 2005b). The data were analyzed by fitting time-dependent changes in fluorescence of ThT (F) versus time of the reaction (t) to the following equation (Cohlberg et al. 2002):

graphic file with name 1334equ1.jpg

where A is the initial level of ThT fluorescence during the lag phase, B is the difference between final level of ThT fluorescence and the initial level during the lag phase, k is rate constant of amyloid formation (h−1), tm is the midpoint of transition, and c is an empirical parameter describing changes in ThT fluorescence after transition. The length of the lag time (tl) of amyloid formation was calculated as tl = tm − 2/k.

Negative stain electron microscopy (EM) and Fourier transform infrared (FTIR) spectroscopy

EM and FTIR were performed as described earlier (Breydo et al. 2005). To prepare samples for EM, aliquots were taken during the early stages of fibril formation, and diluted 1:1 in 10 mM Na-acetate (pH 5.0). To prepare samples for FTIR, mature amyloid fibrils were dialyzed overnight against 10 mM sodium acetate (pH 5.0). The FTIR bands were resolved by Fourier self-deconvolution in the Opus 4.2 software package using a Lorentzian line shape and parameters equivalent to 20 cm−1 bandwidth at half height and a noise suppression factor of 0.3.

Atomic force microscopy (AFM)

Fibrils were imaged with a PicoSPM LE AFM (Molecular Imaging), operating in MAC (alternating magnetic field) mode and using a MAC II silicon cantilever (tip radius <7 nm, spring constant 2.8 N/m) and a liquid cell (Molecular Imaging). Samples were deposited onto a glass coverslip (22 mm circumference; Fisherbrand), left to adhere for 30–40 min and then washed three times with filtered ultrapure H2O. The slide was then placed in the fluid cell and immersed in 200 μL 10 mM Tris-HCl buffer (pH 7.5). All imaging was performed at a scan rate of 0.5 lines/sec at a drive frequency of 25–30 kHz.

Acknowledgments

We thank Pamela Wright for editing the manuscript. This work was supported by NIH grant NS045585 to I.V.B.

Footnotes

Reprint requests to: Ilia V. Baskakov, Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 W. Lombard Street, Baltimore, MD 21201, USA; e-mail: baskakov@umbi.umd.edu; fax: (410) 706-8184.

Abbreviations:PrP, full-length recombinant prion protein; Mo, mouse; Ha, Syrian hamster; EM, electron microscopy; FTIR, Fourier Transform Infrared Spectroscopy; ThT, Thioflavin T; GdnHCl, guanidine hydrochloride.

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