Special Issue Article Received: 9 June 2010, Revised: 15 September 2010, Accepted: 15 October 2010, Published online in Wiley Online Library: 2011 (wileyonlinelibrary.com) DOI:10.1002/jmr.1112 Structure and assembly–disassembly properties of wild-type transthyretin amyloid protofibrils observed with atomic force microscopyy Ricardo H. Pires a,b *, Maria J. Saraiva b,c, Ana M. Damas b,c ** and Miklós S. Z. Kellermayera *** Transthyretin (TTR) is an important human transport protein present in the serum and the cerebrospinal fluid. Aggregation of TTR in the form of amyloid fibrils is associated with neurodegeneration, but the mechanisms of cytotoxicity are likely to stem from the presence of intermediate assembly states. Characterization of these intermediate species is therefore essential to understand the etiology and pathogenesis of TTR-related amyloidoses. In the present work we used atomic force microscopy to investigate the morphological features of wild-type (WT) TTR amyloid protofibrils that appear in the early stages of aggregation. TTR protofibrils obtained by mild acidification appeared as flexible filaments with variable length and were able to bind amyloid markers (thioflavin T and Congo red). Surface topology and contour-length distribution displayed a periodic pattern of 15 nm, suggesting that the protofibrils assemble via an end-binding oligomer fusion mechanism. The average height and periodic substructure found in protofibrils is compatible with the double-helical model of the TTR amyloid protofilament. Over time protofibrils aggregated into bundles and did not form mature amyloid-like fibrils. Unlike amyloid fibrils that are typically stable under physiological conditions, the bundles dissociated into component protofibrils with axially compacted and radially dilated structure when exposed to phosphate-buffered saline solution. Thus, WT TTR can form metastable filamentous aggregates that may represent an important transient state along the pathway towards the formation of cytotoxic TTR species. Copyright ß 2011 John Wiley & Sons, Ltd. Keywords: transthyretin; atomic force microscopy; protofibril; periodicity; amyloid; fibrillogenesis; cytotoxic oligomers * Correspondence to: R. H. Pires, Department of Biophysics and Radiation Biology, Faculty of Medicine, Semmelweis University, Tűzoltó u. 37-47, Budapest IX, H1094, Hungary. E-mail: ricardo.pires@eok.sote.hu INTRODUCTION Amyloidoses encompass a wide spectrum of highly debilitating disorders involving systemic pathological lesions, neurodegeneration and several other tissue-specific dysfunctions (Chiti and Dobson, 2006). In these disorders, perturbation of the protein’s native fold results in aggregation, which ultimately leads to the formation of large deposits constituted mostly of amyloid fibrils. A related misfolding process is thought to result in the amyloidogenic conversion of transthyretin (TTR). TTR is a homotetrameric protein synthesized in the liver and the choroid plexus of the brain which is then exported to the blood serum and the cerebrospinal fluid, respectively (Fleming et al., 2009). Aggregation of TTR in the form of amyloid fibrils that deposit in extracellular space leads to a condition known as senile systemic amyloidosis in the case of wild type (WT) TTR (Westermark et al., 1990), while mutations in the TTR sequence are often associated with familial amyloidotic polyneuropathy (FAP) (Sousa and Saraiva, 2003). In the case of WT TTR and, similarly to other proteins, the in vitro amyloid fibrillogenesis is often initiated by acidifying the medium so as to mimic the lysosomal milieu (Colon and Kelly, 1992). The lysosome is a mildly acidic (pH 5) cytoplasmic vesicle whose housekeeping activity of degrading subcellular structures has been associated with amyloidogenic disorders (Nixon, 2007). ** Correspondence to: A. M. Damas, IBMC-Institute for Molecular and Cell Biology; Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. E-mail: amdamas@ibmc.up.pt *** Correspondence to: M. S. Z. Kellermayer, Department of Biophysics and Radiation Biology, Faculty of Medicine, Semmelweis University, Tűzoltó u. 37-47. Budapest IX, H1094, Hungary. E-mail: miklos.kellermayer@eok.sote.hu a R. H. Pires, M. S. Z. Kellermayer Department of Biophysics and Radiation Biology, Faculty of Medicine, Semmelweis University, Tűzoltó u. 37-47, Budapest IX, H1094 Hungary b A. M. Damas, R. H. Pires, M. J. Saraiva IBMC-Institute for Molecular and Cell Biology; Rua do Campo Alegre, 823, 4150-180 Porto, Portugal c A. M. Damas, M. J. Saraiva ICBAS-Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal y This article is published in Journal of Molecular Recognition as a focus on AFM on Life Sciences and Medicine, edited by Jean-Luc Pellequer and Pierre Parot (CEA Marcoule, Life Science Division, Bagnols sur Cèze, France). Abbreviations: AFM, atomic force microscopy; CAC, circular autocorrelation; DFT, discrete Fourier transform; FAP, familial amyloidotic polyneuropathy; FFT, fast Fourier transform; nCAC, normalized circular autocorrelation; PBS, phosphate buffered saline; pI, isoelectric point; ThT, thioflavin T; TTR, transthyretin; WT, wild type. 467 J. Mol. Recognit. 2011; 24: 467–476 Copyright ß 2011 John Wiley & Sons, Ltd. R. H. PIRES ET AL. The acidic environment leads to the disassembly of TTR’s quaternary structure followed by partial unfolding of the monomer that becomes prone to aggregation (Lai et al., 1996; Foss et al., 2005). The acidification process has been proposed to be sufficient for initiating the assembly of TTR amyloid fibrils (Colon and Kelly, 1992). However, the process of fibrillogenesis depends on various additional conditions as well, such as temperature, protein concentration, ionic strength, agitation and pH. Manipulation of some of these parameters has been shown to lead to the appearance of various intermediate aggregation states prior to the appearance of mature amyloid fibrils (Kodali and Wetzel, 2007). These intermediate states have been receiving increased attention because they possess much greater cytotoxic potential than the fibrils (Caughey and Lansbury, 2003). One of the amyloid fibrillogenic intermediate states is the protofibril, which can be frequently found in diverse amyloidogenic protein preparations and displays high cytotoxic activity (Caughey and Lansbury, 2003). The term ‘protofibril’ is used in the literature rather loosely, often referring to a wide range of intermediates sometimes irrespective of their morphology. Here we adopt the terms ‘oligomer’, ‘protofibril’, ‘protofilament’, and ‘fibril’ as defined in a recent review (Kodali and Wetzel, 2007). Accordingly, protofibrils are flexible linear aggregates approximately 500 nm in length, and may (but usually do not) contain a periodic substructure. Protofibrils may be the direct precursors of protofilaments which, via a hierarchical assembly process, give rise to amyloid fibril structure (Khurana et al., 2003). In fact, in the case of the Aß1–40 peptide, the presence of protofibril-specific antibodies in the aggregation reaction effectively precludes the formation of mature amyloid fibrils, leading to the accumulation of protofibrils (Habicht et al., 2007). In contrast, in the case of ß2-microglobulin (ß2m), under certain conditions the in vitro fibrillation reaction leads to the accumulation of protofibrils without the formation of mature amyloid fibrils (Kad et al., 2001; Gosal et al., 2005). Furthermore, these protofibrils are unable to accelerate the assembly of amyloid fibrils under mature fibril-forming conditions, suggesting that they might be ‘off-pathway’ kinetic traps (Kad et al., 2001). In the case of TTR the structural features of intermediate states is relatively unexplored despite their high cytotoxic activity (Sousa et al., 2001; Sousa et al., 2002). Detailed structural studies have been carried out only down to the level of the TTR amyloid protofilament for which two main models have been proposed. One of them suggests that the protofilament is composed by a single array of monomers (Inouye et al., 1998), that laterally associate via a hierarchical assembly mechanism to form amyloid fibrils (Cardoso et al., 2002). Another model proposes a double helical structure containing an 11.55 nm repeat (Blake and Serpell, 1996; Blake et al., 1996). Since protofibrils have been proposed to direct precursors of amyloid fibrils displaying cytotoxic activity, studies addressing their structure and assembly dynamics are relevant in understanding their cytotoxic behavior as well as their role within the amyloid aggregation pathway. However, so far no study of amyloid protofibrils has been undertaken with high enough resolution to allow the direct comparison of their morphology with structural models of the amyloid protofilament. In the current study we analyzed the morphological properties of TTR amyloid protofibrils with atomic force microscopy (AFM) under liquid buffer conditions. We show that protofibrils formed by mild acidification display characteristics that are more closely related to the double-helical model of the amyloid protofilament. These structures are able to bind thioflavin T and Congo red and appear to grow via an oligomer fusion mechanism. However, maturation of the protofibrils into amyloid-like fibrils was not seen on a time scale of several months. Rather, they showed a tendency to form unorganized bundles that undergo dissociation at neutral pH. Thus, the protofibrils described in our work possibly represent a distinct structural entity whose potential cytotoxic activity—a hallmark of oligomeric amyloid species—may be modulated by environmental conditions that either preserve their protofibrillar state, or induce their dissociation. MATERIALS AND METHODS Protein purification Recombinant wild type (WT) TTR expressed in BL21 E. coli cells was isolated and purified as described previously (Almeida et al., 1997) followed by high-affinity anion exchange chromatography (MonoQ column, GE Healthcare) equilibrated with 100 mM BisTris pH 6.8. The protein eluting with approximately 150 mM NaCl was subsequently concentrated and loaded onto a calibrated analytical size exclusion chromatography superdex S75 column (GE Healthcare) equilibrated with 50 mM HEPES pH 7.0 and 150 mM NaCl. Protein eluting in a peak corresponding to tetrameric TTR (55 kDa) was collected and dialyzed overnight against a weakly buffered solution (10 mM HEPES pH 7.0). The protein was then concentrated to 10–15 mg/ml. Protein concentration was determined with the Bradford method (BioRad). Samples were approximately 98% pure as judged by Coomassie-blue staining of SDS-PAGE gels. According to dynamic light scattering measurements (Zetasizer Nano ZS, Malvern) the samples were considered monodisperse. The hydrodynamic radius of the particles ranged between 6.4 and 6.8 nm, consistent with previous observations (Hou et al., 2007). Sample preparation TTR protofibrils were prepared by incubating WT TTR in 50 mM sodium acetate buffer at pH 3.6, 378C and at a concentration of 1 mg/ml for a period of 1 year. At several time points following acidification, samples were taken and diluted before surface deposition to reduce molecular overcrowding. Thus, samples were diluted 500-fold in acetate buffer at pH 3.6 and imaged as described below. To test the stability of protofibrils at more physiological conditions, samples in an advanced state of aggregation were diluted 25-fold with phosphate-buffered saline solution (PBS), followed by incubation at room temperature for 1 min. Subsequently, the samples were diluted 20-fold in ultrapure water (pH 6.3) prior to further investigation. Atomic force microscopy AFM imaging was carried out by using procedures reported in previous papers on Aß fibrils (Karsai et al., 2005; Kellermayer et al., 2005; Karsai et al., 2006; Karsai et al., 2007; Karsai et al., 2008; Kellermayer et al., 2008) with modifications. Hundred microliters of the diluted WT TTR sample was deposited on freshly cleaved mica, incubated for 5 min at room temperature; the surface was then rinsed with buffer. Imaging was carried out on a daily basis over a period of 2 months and then periodically for up to 1 year. AFM images of the samples were acquired with an MFP-3D AFM instrument (Asylum Research, Santa Barbara, CA) in non-contact 468 wileyonlinelibrary.com/journal/jmr Copyright ß 2011 John Wiley & Sons, Ltd. J. Mol. Recognit. 2011; 24: 467–476 WILD-TYPE TTR PROTOFIBRIL STRUCTURE (AC) mode. Imaging was done by scanning in liquid, either in 50 mM acetate buffer, pH 3.6 or in ultrapure water for samples which were incubated in PBS. Imaging was performed by using low-noise cantilevers (Biolever, Lever A, Olympus) with a spring constant of 30 pN/nm and a resonance frequency of 9.2 kHz. The free amplitude was set to 0.3 V and the amplitude set point to 0.2 V, and the images were recorded at a typical scanning frequency of 0.8 Hz. Image processing and analysis Raw AFM images were flattened by using a linear function followed by masking out particles with heights above 50 pm and applying another first-order flattening to the (unmasked) background to correct for artifacts arising from the first flattening step. For any given image, a distribution of pixel heights was used to find the background ‘height’ relative to which all height measurements were carried out. Images were smoothed with two passes of a Gaussian convolution filter with one pixel neighborhood. Protofibril length was measured by taking the tip convolution effect into account (Figure 1). The cantilever tip was approximated by a hemisphere with a radius (R) of 30 nm (manufacturer’s specifications) and the protofibrils as cylinders with diameter (h) corresponding to the average protofibril height. Protofibril length (L) was obtained from the measured fibril length (M) as: pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi L ¼ M2 2hRh2 : (1) For small oligomers that appeared nearly spherical under the AFM, the molecular volume (V) was calculated by measuring the height (h) and taking the full width at half height as the diameter (d) which significantly compensates for the tip convolution effect and these were used as follows (Carnally et al., 2008): ph 2 3d 2 V¼ h þ (2) 6 4 The number of TTR monomers present in a given oligomer was estimated by considering the dimensions of the native WT TTR monomer: 2 3 4.5 nm (Blake et al., 1978) or 27 nm3. Statistical analysis of periodicity was carried out on several protofibrillar axial sections which exhibited a clearly corrugated structure. The data were analyzed in two independent ways. In the first, the distance between consecutive local height maxima or shoulders was measured, based on which a histogram of periodicities was constructed. In the second, analytical approaches (autocorrelation and Fourier transformation) were applied to axial height sections of 20 protofibrils taken from high-resolution (1024 1024 pixel) images. The height values were treated as a discrete homogeneous periodic signal by ignoring the slant height profile at the protofibril termini and considering only the harmonic component of the axial topology. Thus, for any given harmonic segment of an axial section the height variations (z) can be approximated as zðdÞ ¼ h þ A sinð2p v d þ uÞ; (3) where h is the average height of the protofibril, A is the height amplitude, v is the frequency, d is the distance along the protofibril axis, and u is the phase. To obtain a statistical representation of the major frequency components in protofibril periodicity, an amplified waveform signal f(d) was obtained by summing each i axial height section decremented by their individual average height as f ðdÞ ¼ n n X X zi ðd Þhi ¼ ðAi sinð2p vi d þ uÞÞ; i¼1 (4) i¼1 where d 2 N0, 0 d 99. To allow amplification of the periodic signal by limiting destructive phase interference, zi(0) was set at u ¼ 908 which gives zi ð0Þ ¼ hi þ Ai . Since the periodic pattern is expected to repeat itself over the analyzed protofibril length (99 nm), a circular autocorrelation (CAC) was then obtained from f(d) of protofibrils grown in pH 3.6 and from f(d) of protofibrils obtained upon disassembly of bundles after PBS incubation. To allow direct comparison between the two data sets a power spectral density (PSD) was obtained from a normalized CAC (nCAC). Thus, a discrete Fourier transform (DFT) of nCAC was computed through a fast Fourier transform (FFT) algorithm with a hanning window function and the data analyzed between periods of 2–50 nm. All analyses were performed by using IGOR Pro v6.06 (Wavemetrics, Oregon, USA). Measurement of spectral properties Thioflavin T Assay: 10 mM thioflavin-T (ThT) (e441 ¼ 2.2 104 M1 cm1) stock solution was prepared fresh in ThT buffer (50 mM glycine/NaOH, pH 9.0). The solution was passed through a 0.22 mm PVDF filter and stored in the dark and on ice. Native WT TTR protein solution and protofibril suspension (1 mg/ml of protein) were diluted 10-fold in ThT buffer. Emission was detected at 482 nm with the excitation and emission slits set to 5 nm and the spectrum recorded at room temperature on a Perkin-Elmer LS50B fluorescence spectrometer. Congo Red Redshift Assay: Measurements were carried out as reported earlier (Bonifacio et al., 1996). Briefly, 5 ml of TTR aggregates (1 mg/ml of protein) were mixed with 55 ml of a 10 mM Congo red solution prepared in 100 mM Tris/HCl, pH 7.0. Spectra were recorded at room temperature in a Shimadzu UV-2401 PC spectrophotometer. RESULTS Figure 1. Schematics of correction for AFM tip convolution in calculating the real length of TTR protofibrils. High-resolution AFM investigation of WT TTR protofibrils was carried out in the present work. Amyloidogenic transformation of WT TTR was induced by acidification of the buffer solution. Within the first week of incubation it was possible to observe monomers and different size oligomeric structures (Figure 2A, inset) as well as short flexible fibrillar structures of worm-like shape and nodular morphology (Figure 2A). While monomers/dimers were present throughout the entire incubation period (note the background in Figures 2C and 3A), the number of oligomers 469 J. Mol. Recognit. 2011; 24: 467–476 Copyright ß 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jmr R. H. PIRES ET AL. Figure 2. Morphology and spectroscopic properties of WT TTR aggregates formed by incubation at pH 3.6. (A) WT TTR imaged by AFM on the 5th day of incubation showing the presence of small protofibrils and oligomers (image 1024 512 pixels). Inset: magnified view of oligomeric structures (arrows) with the indication of the estimated number of TTR monomers. (B) On the 6th day of incubation protofibrils appear longer, seem more frequent than small oligomers, and already display some tendency to associate (image 1024 1024 pixels). Inset: magnified view of a protofibril displaying periodic axial topographical substructure; the green line indicates trajectory along which the profile plot was obtained (see Figure 3D). (C) After 2 weeks of incubation protofibrils show increased tendency to associate via their termini, forming a network containing nodules from which protofibrils extend in quasi-radial symmetry as highlighted by circles (image 1024 2014 pixels). (D) 10 weeks of incubation lead to a formation of protofibril networks of increased density forming protofibril bundles (image 512 512 pixels). (E) Fluorescence excitation spectra of Thioflavin T only (green), in the presence of native WT TTR (blue), and in the presence of WT TTR protofibrils (red). A red shift is well observable in the latter case as expected for an amyloid aggregate. (F) UV/VIS spectra of Congo red in the absence of protein (green), in the presence of native WT TTR (blue), and upon addition of protofibrils (red). The presence of protofibrils induces a red shift in the absorbance spectrum that is characteristic of amyloid aggregates with a shoulder at 540 nm as seen from the difference spectrum (black). Comparatively, the presence of native WT TTR does not induce any spectral changes as seen from difference spectrum (gray). 470 wileyonlinelibrary.com/journal/jmr Copyright ß 2011 John Wiley & Sons, Ltd. J. Mol. Recognit. 2011; 24: 467–476 WILD-TYPE TTR PROTOFIBRIL STRUCTURE Figure 3. Morphological properties of protofibrils before and after transient (1 min) incubation in PBS. (A) Protofibril bundle imaged 8 weeks after sample incubation in 50 mM sodium acetate at pH 3.6. (B) Incubation for 1 min in PBS of the sample shown in (A) leads to the disassembly of bundles into its constituting protofibrils. Inset: enlarged view of protofibrils displaying pronounced axial periodicity; the red line indicates trajectory along which profile plot was obtained (see Figure 3D). (C) Contour length histogram of protofibrils in assembling conditions (pH 3.6, blue histogram) and following the disassembly of bundles (PBS, red histogram). The arrowheads indicate local maxima spaced 15 nm apart for assembling protofibrils: 35, 50, 65, 80, 95 nm, and spaced by 12 nm for protofibrils in disassembly conditions: 14, 26, 38 and 50 nm. (D) Topographical profile plots along the long axis of protofibrils in assembling (blue, from Figure 2B) and disassembling (red, from Figure 3B) conditions; also indicated are the peak-to-peak distances in nanometers. decreased while the fibrillar structures became increasingly prevalent (Figure 2B). We carried out a statistical analysis of the average topographical height of the filamentous structures. Because the observed width is inflated by cantilever tip convolution, it is the height data that was correlated with the thickness of the filaments. The histogram of height data (Figure 4A) displayed normal distribution with a mean of 3.2 nm ( 0.8 nm SD, n ¼ 3505, 63 filaments), and a height variation amplitude of 1.0 nm (0.3 nm SD, n ¼ 607, 127 filaments). Based on the thickness of the filamentous structures, which is smaller than that of TTR amyloid fibrils which are typically 10–12 nm (Serpell et al., 1995; Serpell et al., 2000), we identify them as protofibrils and will refer to them as such. Analysis of protofibril contour lengths revealed a wide distribution between 30 and 300 nm (Figure 3C). In the contour-length histogram local maxima can be observed, which are separated by approximately 15 nm (arrowheads in Figure 3C). The discrete distribution indicates that the length of the protofibrils is an integer multiple of subunits with 15 nm dimensions. Protofibrils may therefore be considered as a linear assembly of 15-nm-long subunits. To further test for this possibility we analyzed the height topography of the protofibrils along the longitudinal axis. In the axial profile plot (Figure 3D, blue curve) periodically spaced peaks and valleys can be discerned. Statistical analysis of the peak-to-peak distance (Figure 4B, blue histogram) shows an average distance of 15 nm (0.2 nm SD, n ¼ 266, 45 protofibrils). Thus, the protofibrils indeed seem to be linear chains of subunits with axial dimensions of 15 nm. Given their large dimensions compared with the size of monomers, these aggregation subunits are likely to be oligomers themselves. Already within the first week, but extending throughout the whole observation period, protofibrils showed a tendency to associate into higher-order aggregates. An example of such an aggregate, recorded in the second week of incubation, is shown in Figure 2C. The figure displays an elongated structure with approximately 650 nm in length that appears to result from the association of several 100-nm-long protofibrils. Protofibrils associate mostly via their termini, forming axial aggregates with additional protofibrils annealing in a quasi-radial symmetry and extending away from central nodules (Figure 2C, circles). This type of architecture increased in size and complexity as a function of incubation time, but it was not accompanied by an increase in order. This is seen for example on an image obtained on the 10th week of incubation where an arrangement that is more similar to a protofibril bundle, rather than an amyloid fibril, can be seen (Figure 2D). Although the sample was imaged for up to one year after the start of incubation, no noteworthy changes where observed henceforth; isolated protofibrils became scarcer and only large bundles (1–4 mm2) were routinely present. 471 J. Mol. Recognit. 2011; 24: 467–476 Copyright ß 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jmr R. H. PIRES ET AL. Figure 4. Morphological analysis of protofibrils obtained on the pathways to assembly (acidic conditions, blue) and disassembly (upon incubation in PBS, red). (A) Height distribution of protofibrils. (B) Histogram of periodicities obtained by measuring peak-to-peak distances. (C) Normalized autocorrelation of statistically representative segments of protofibril cross-sections. (D) Power spectral density (PSD) of statistically representative segments of protofibril cross-sections obtained by calculating the discrete Fourier transform (DFT) from the nCAC shown in Figure 4C. To investigate whether the protofibril bundles display canonical amyloid-like structure, we carried out spectroscopic measurements to test for thioflavin-T (ThT) and Congo red binding (Figure 2E and 2F). Protofibril bundles were positive for ThT binding as observed by the increased fluorescence emission (Figure 2E). The Congo red red-shift assay was also positive (Figure 2F), as seen from the shoulder at 540 nm in the UV/vis spectrum (red and black spectra), which is missing when only Congo red is present (green spectrum) or when WT TTR was added (blue and gray spectra). However, not only is the extent of the shift much smaller than that obtained for ex-vivo fibrils (Benditt et al., 1970), but the shift rapidly decayed on a the time scale of a few minutes in the assay buffer, indicating instability of the bundle structure at near-physiological pH. To test for possible structural changes, we investigated the morphology of the protofibril bundles with AFM following exposure of the sample to more physiological conditions (PBS buffer) for 1 min as described in the Materials and Methods section. The results are shown in Figure 3, where a sample imaged on the 8th week of incubation (Figure 3A) clearly exhibits a dense association of protofibrils into a bundled structure. When the sample was subsequently incubated briefly in PBS, the protofibril bundles disassembled, protofibrils became more dispersed and showed a uniform average thickness (Figure 3B). Analysis of protofibril contour length following bundle disassembly again showed a distribution containing several local maxima, but in this case the peaks were separated by 12 nm (Figure 3C, arrowheads in red histogram). Statistical analysis of the height distribution of these protofibrils (Figure 4A, red histogram) indicates an average topographical height of 4.5 nm (0.8 nm SD, n ¼ 3535, 50 protofibrils). An enlarged view of the protofibrils resulting from bundle disassembly (Figure 3B, inset) shows that, similarly to protofibrils in the assembly conditions (pH 3.6), an axial topographical height periodicity is present. However, the histogram of axial periodicity of these protofibrils (Figure 4B, red histogram) shows a much narrower distribution with a mean of 14 nm (0.01 nm SD, n ¼ 133, 25 protofibrils). Although the difference in axial periodicity between the assembly and disassembly conditions is small, it is nevertheless significant according to statistical analysis (unpaired Student’s t-test, p < 0.0001). Thus, protofibrils obtained upon disassembly of bundles appear wider (by 1 nm) and with a shorter periodicity (by 1 nm) suggesting that radial expansion coupled with axial tightening of the protofibril structure has taken place. In order to further evaluate if indeed an axial compaction of protofibrils occurred upon bundle disassembly, we carried out a detailed analysis of the axial topographical periodicity along the protofibrils. After calculating a normalized circular autocorrelation (nCAC) function of the axial topographical waveforms (Figure 4C) followed by discrete Fourier transformation (DFT) we obtained the power spectral density function (Figure 4D) in which major periodicities were identified and the relative amplitudes compared. Three major periodicities could be identified: 28, 17, and 12 nm for assembling protofibrils (blue curve), and 26, 13, and 8 for the disassembling ones (red curve). These values are in close agreement with those obtained from the histogram of periodicities and provide evidence 472 wileyonlinelibrary.com/journal/jmr Copyright ß 2011 John Wiley & Sons, Ltd. J. Mol. Recognit. 2011; 24: 467–476 WILD-TYPE TTR PROTOFIBRIL STRUCTURE that upon disassembly of bundles the resulting protofibrils are compacted and display reduced periodicities. DISCUSSION In the present work we investigated the assembly–disassembly pathway of transthyretin amyloidogenesis with high-resolution non-contact atomic-force microscopic imaging under liquid conditions. The commitment of WT TTR towards the amyloidogenetic pathway was evoked by lowering the pH of the buffer to 3.6. Acidification has been shown in the past to induce the assembly of WT TTR into filamentous aggregates that exhibit morphological, structural, and chemical properties similar to those of tissue-formed amyloid fibrils (Bonifacio et al., 1996). In recent years it has become clear that the aggregation reaction in amyloidogenic systems is more complex than previously thought, involving the assembly of oligomeric and protofibril structures with very heterogeneous morphologies. The involvement of these species in amyloid fibril assembly is still debated (Kodali and Wetzel, 2007). Because these pre-fibrillar species appear to be the key agents in amyloid cytotoxicity, it is important to characterize their structural properties in full detail. Here we described the appearance of nodular protofibrillar species in the amyloidogenic aggregation of TTR (Figure 2B and 2C), with a topographical height that averaged 3.2 nm and ranged between 2.7 and 3.7 nm (1 nm height amplitude) (Figure 4A). The length repeat is consistent with the observed topographical height periodicity (Figure 4B) and suggests that protofibrils grow in stepwise fashion by the fusion of 15 nm oligomers with the free end of the protofibril. Such an oligomer fusion mechanism has been observed for other types of amyloid protofibrils (Modler et al., 2003; Modler et al., 2004; Carrotta et al., 2005; Hill et al., 2009). Similarly, we observe that the early protofibrillar stages are characterized by the presence of small oligomers (Figure 2A) that tend to disappear as protofibrils grow in number. In this aggregation mechanism an oligomer is one periodic unit. Interestingly, in an earlier work, the pre-fibrillar state in the aggregation of WT TTR was reported to be populated by 8-nm globular oligomers (Cardoso et al., 2002) the size of which is approximately one half of the periodicity observed here. The 8 nm oligomers were found to disappear as 4–5 nm wide protofibrils were formed, giving rise to higher order aggregates of amyloid-like fibrils through hierarchical assembly (Cardoso et al., 2002). We did not observe the formation of amyloid fibrils. Instead, protofibrils appeared to bundle via their termini forming clusters displaying quasi-radial symmetry rather than by lateral association (Figure 2C). Protofibril bundles have been suggested to form an intermediate state leading to the assembly of amyloid fibrils (Arimon et al., 2005). This does not appear to be the case with WT TTR under the conditions used in this study, where a transition from protofibril bundles to mature fibrils was never observed even after 1 year of study. The samples nevertheless were positive for ThT (Figure 2E) and Congo red (Figure 2F), which are classical amyloid markers. However, the spectral red-shift of the Congo red absorption maximum decayed on the time-scale of a few minutes, suggesting that disaggregation of the bundle structure under conditions of the assay (neutral pH) may have occurred. This observation is in accordance with several reports that show that amyloid protofibrils and fibrils formed in acidic medium can undergo depolymerization under neutral or alkaline conditions (Yamaguchi et al., 2001; Yamamoto et al., 2005). The possibility of instability of TTR amyloid protofibril bundles at neutral pH conditions prompted us to undertake a morphological study. As seen from the AFM images (Figure 3A and 3B), by incubation of protofibrils for 1 min in PBS resulted in the disassembly of bundles into individual protofibrils. In addition, small oligomers and monomers which populated the background when imaging was performed at acidic buffer are no longer present, suggesting that, since TTR is an acidic protein with pI 5 (Connors et al., 1998), increasing the pH might affect the protein surface charge to an extent that inhibits their adsorption to the surface. The protofibrils thus obtained reveal slightly altered structural features. The changes are characterized by radial expansion, with the average height of protofibrils increasing from 3.2 to 4.5 nm after PBS exposure (Figure 4A), which can be partly explained by differences in electrostatic interactions arising from the differences in pH and ionic strength in the two samples (Muller and Engel, 1997). However, we also observed that upon disassembly an axial compaction also took place, as seen from the decrease in axial periodicity from 15 to 14 nm (Figure 4B). Although this difference is relatively small, it is statistically significant ( p < 0.0001). In addition, while the contour-length histogram of assembling protofibrils displays a multimodal distribution with local maxima spaced at integer multiples of 15 nm (Figure 3C, blue histogram), upon disassembly of bundles each mode of the distribution becomes separated by 12 nm (Figure 3C, red histogram). Both results suggest that, upon disassembly a structural transition takes place that induces a small but significant axial compaction of the protofibrils. A similar observation can be made from the analysis of the power spectral density (PSD) of the statistical representation of protofibrils (Figure 4D). The identified periodicities, 12, 17, and 28 nm (on assembly) and 8, 13, and 26 nm (on disassembly), are comparable to the values obtained from the statistical measurement of peak-to-peak distances (Figure 4B). Since the two types of analyses are fundamentally different is not possible to directly compare the magnitude of the power in a PSD curve with the magnitude in a periodicity histogram (counts/ frequency). However, the Fourier transform analysis of waveforms is an extremely sensitive approach to study periodic structures, allowing the identification of periods that could otherwise be unnoticeable in a histogram derived from peak-to-peak distances. While the histograms show a sensible shortening of periodicities, these changes are far more noticeable in the PSD analysis where periods identified on protofibril disassembly are generally shorter than those in the assembly route (Figure 4D). On the assembly and disassembly PSD curves, only a central period at 12–13 nm appears to be a common feature, albeit with different power. Upon disassembly, the 17 nm periodicity present in protofibrils during assembly is no longer apparent, and this change is accompanied by a noticeable increase in the power of the 12–13 nm period. This shortening in periodicity by 4.5 nm is within the dimensions of a single TTR monomer (Blake et al., 1978). Thus, as suggested by the periodicity histogram, transient incubation of protofibrils in PBS leads to a tightening of the protofibril structure, a process that might be coupled to the observed radial expansion. Additional periodicities were also identified by the PSD analysis that are weakly represented in the periodicity histogram, notably the 28 nm as well as the 8 and 26 nm periods found in assembling and disassembling protofibrils, respectively. The shorter periods are likely to be associated with very small height variations as indicated by their smaller power in the PSD curves, and thus may have been 473 J. Mol. Recognit. 2011; 24: 467–476 Copyright ß 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jmr R. H. PIRES ET AL. occasionally overlooked during peak-to-peak distance measurements. On the other extreme are the large periods of 26 and 28 nm which are relatively large when compared to the measured peak-to-peak distances, and may reflect a more global morphological feature of the protofibril structure on binding to the surface. Altogether, the morphological changes observed likely stem from molecular rearrangements at the level of individual monomers, underlying the structural dynamism that is present within the TTR protofibril that could lead to the disassembly of the protofibril bundles. Since it has been proposed that protofibrils could be the direct precursors of the protofilament, it is important to compare our morphological data obtained here with the existing structural models for the TTR amyloid protofilament. Analysis of electron micrographs of ex vivo TTR amyloid fibril cross sections has revealed that the protofilament is 4–5 nm in diameter (Serpell et al., 1995). For the in vitro assembled TTR amyloid-like fibrils, protofibrils measuring 4 nm were also found to be the fundamental polymeric unit (Cardoso et al., 2002). In this latter work, mass-per-length measurements in STEM data suggest that the unitary filamentous structure is a linear array of monomers. This is in agreement with a previous model for the TTR protofilament based on X-ray diffraction data obtained on ex vivo fibrils (Inouye et al., 1998). In the report by Cardoso and others, no periodic substructure was resolved for protofibrils (Cardoso et al., 2002). However, molecular dynamics simulations for a TTR protofilament modeled as a series of monomers that form two extended b-sheets have proposed the existence of a helical structure with a 48 b-strand repeat (Correia et al., 2006), or a 23.04 nm periodic unit considering 0.48 nm as the inter-strand distance. There is, however, another model for the TTR protofilament also based on X-ray diffraction of ex vivo amyloid fibrils, which is described as double-helical structure containing a 11.55 nm repeat and measuring 5 nm in height (Blake and Serpell, 1996; Sunde et al., 1997). While the height of the protofibrils is in good agreement with the diameter of the TTR protofilament, the periodicity of 12–13 nm detected on the PSD analysis is very close to the 11.55 nm repeat of the double-helical model, with a difference of 1 nm that is within the error of the measurements of our AFM images in the XY-plane (1024 1024 pixels for 1 1 mm images). Assuming an overall helical conformation for the protofibrils described here, as it has been proposed for protofibrils from lithostathine (Gregoire et al., 2001) the 15–17 nm periodicity found in protofibrils in assembly conditions, may result from a more relaxed conformation than that of the protofilaments which are likely to have their structure more constrained by the remaining fibril structure. In addition, transient incubation of the same protofibrils in near-physiological conditions of pH and ionic strength, lead to a structural transition where the height and periodicities match quite remarkably the dimensions proposed for the double-helical model of the protofilament. Despite the morphological similarities between the TTR protofibrils and the double-helical model of the protofilament, the protofibrils did not assemble into higher order aggregates that would resemble mature amyloid fibrils as seen from ex vivo preparations. Thus, protofibrils, despite some morphological similarity to current protofilament models, may have a very distinct structural organization that effectively precludes their hierarchical assembly into amyloid fibrils and may therefore be considered ‘off-pathway’ products. However, it may also happen that the protofibrils obtained here simply require an additional factor that promotes their self-assembly into amyloid fibrils. For example, ex vivo TTR amyloid fibrils are often found to contain fragments of the protein that appear to influence fibrillar morphology (Bergstrom et al., 2005). In addition, it is known that the in vitro and in vivo assembly can be stimulated by a variety of cellular components, including proteoglycans, glucosaminoglycans, lipids, or collagen (Relini et al., 2006; Relini et al., 2008; Naiki and Nagai, 2009). Inclusion of these elements in future experiments will yield further insights into the mechanisms of TTR amyloid fibrillogenesis. Since the acidification of TTR induces the formation of cytotoxic species before the pre-fibrillar state, it is very likely that the protofibrils described here have a cytotoxic activity. The fact that they manifest instability at physiological conditions is therefore likely to be of relevance for the etiology of TTR-related amyloidoses. CONCLUSIONS We have described the morphological features of transthyretin protofibrillar amyloid intermediates by using atomic force microscopy under liquid conditions. The protofibrils, obtained by mild acidification of the sample, display an axial periodicity related possibly to the oligomeric fusion mechanism of their assembly. The protofibrils aggregate into irregular bundles on a time-scale of several weeks, but disperse into axially compacted and radially expanded structures upon neutralizing the pH. The periodicity observed here in protofibrils agrees with the predictions of the double-helical model of TTR assembly. Despite displaying the hallmarks of amyloid features, protofibrils failed to aggregate into mature fibrils even during extended time periods. Thus, the protofibrils seen here likely represent a kinetically trapped structural variant, for which a precise structural description awaits further high-resolution investigations. The axial compaction and radial expansion indicate that the protofibrils are nevertheless structurally dynamic, a fact that may be relevant for their potentially cytotoxic activity. Acknowledgements The authors are thankful to András Kaposi for insightful comments and suggestions. This work was supported by grants from the Hungarian Science Foundation (OTKA K73256), the Hungarian National Office of Research and Technology (NANOAMI KFKT-1– 2006–0021, OMFB-380/2006), and the Hungarian Medical Research Council (ETT-229/09) to MSZK; and Project GRICES&FCT – Proj. 4.1.1-Hungary, Portugal and Project n. 037525 EURAMY (FP6-LIFESCIHEALTH-6) from EU to AMD. RHP acknowledges the award of a short-term travel grant by the Calouste Gulbenkian Foundation of Portugal. REFERENCES Almeida MR, Damas AM, Lans MC, Brouwer A, Saraiva MJ. 1997. Thyroxine binding to transthyretin Met 119. Comparative studies of different heterozygotic carriers and structural analysis. Endocrine 6: 309–315. Arimon M, Diez-Perez I, Kogan MJ, Durany N, Giralt E, Sanz F, FernandezBusquets X. 2005. Fine structure study of Abeta 1-42 fibrillogenesis with atomic force microscopy. Faseb J. 19: 1344–1346. 474 wileyonlinelibrary.com/journal/jmr Copyright ß 2011 John Wiley & Sons, Ltd. J. Mol. Recognit. 2011; 24: 467–476 WILD-TYPE TTR PROTOFIBRIL STRUCTURE Benditt EP, Eriksen N, Berglund C. 1970. Congo red dichroism with dispersed amyloid fibrils, an extrinsic cotton effect. Proc. Natl Acad. Sci. USA 66: 1044–1051. Bergstrom J, Gustavsson A, Hellman U, Sletten K, Murphy CL, Weiss DT, Solomon A, Olofsson BO, Westermark P. 2005. Amyloid deposits in transthyretin-derived amyloidosis: cleaved transthyretin is associated with distinct amyloid morphology. J. Pathol. 206: 224–232. Blake C, Serpell L. 1996. Synchrotron X-ray studies suggest that the core of the transthyretin amyloid fibril is a continuous beta-sheet helix. Structure 4: 989–998. Blake CC, Geisow MJ, Oatley SJ, Rerat B, Rerat C. 1978. Structure of prealbumin: secondary, tertiary and quaternary interactions determined by Fourier refinement at 1.8 A. J. Mol. Biol. 121: 339–356. Blake CC, Serpell LC, Sunde M, Sandgren O, Lundgren E. 1996. A molecular model of the amyloid fibril. Ciba Found Symp. 199(6-15): discussion 15-21, 16–40. Bonifacio MJ, Sakaki Y, Saraiva MJ. 1996. ‘In vitro’ amyloid fibril formation from transthyretin: the influence of ions and the amyloidogenicity of TTR variants. Biochim. Biophys. Acta 1316: 35–42. Cardoso I, Goldsbury CS, Muller SA, Olivieri V, Wirtz S, Damas AM, Aebi U, Saraiva MJ. 2002. Transthyretin fibrillogenesis entails the assembly of monomers: a molecular model for in vitro assembled transthyretin amyloid-like fibrils. J. Mol. Biol. 317: 683–695. Carnally SM, Dev HS, Stewart AP, Barrera NP, Van Bemmelen MX, Schild L, Henderson RM, Edwardson JM. 2008. Direct visualization of the trimeric structure of the ASIC1a channel, using AFM imaging. Biochem. Biophys. Res. Commun. 372: 752–755. Carrotta R, Manno M, Bulone D, Martorana V, San BiagioPL., 2005. Protofibril formation of amyloid beta-protein at low pH via a non-cooperative elongation mechanism. J. Biol. Chem. 280: 30001– 30008. Caughey B, Lansbury PT. 2003. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu. Rev. Neurosci. 26: 267–298. Chiti F, Dobson CM. 2006. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75: 333–366. Colon W, Kelly JW. 1992. Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro. Biochemistry 31: 8654–8660. Connors LH, Ericsson T, Skare J, Jones LA, Lewis WD, Skinner M. 1998. A simple screening test for variant transthyretins associated with familial transthyretin amyloidosis using isoelectric focusing. Biochim. Biophys. Acta 1407: 185–192. Correia BE, Loureiro-Ferreira N, Rodrigues JR, Brito RM. 2006. A structural model of an amyloid protofilament of transthyretin. Protein Sci. 15: 28–32. Fleming CE, Nunes AF, Sousa MM. 2009. Transthyretin: more than meets the eye. Prog. Neurobiol. 89: 266–276. Foss TR, Wiseman RL, Kelly JW. 2005. The pathway by which the tetrameric protein transthyretin dissociates. Biochemistry 44: 15525–15533. Gosal WS, Morten IJ, Hewitt EW, Smith DA, Thomson NH, Radford SE. 2005. Competing pathways determine fibril morphology in the selfassembly of beta2-microglobulin into amyloid. J. Mol. Biol. 351: 850–864. Gregoire C, Marco S, Thimonier J, Duplan L, Laurine E, Chauvin JP, Michel B, Peyrot V, Verdier JM. 2001. Three-dimensional structure of the lithostathine protofibril, a protein involved in Alzheimer’s disease. Embo J. 20: 3313–3321. Habicht G, Haupt C, Friedrich RP, Hortschansky P, Sachse C, Meinhardt J, Wieligmann K, Gellermann GP, Brodhun M, Gotz J, Halbhuber KJ, Rocken C, Horn U, Fandrich M. 2007. Directed selection of a conformational antibody domain that prevents mature amyloid fibril formation by stabilizing Abeta protofibrils. Proc. Natl Acad. Sci. USA 104: 19232–19237. Hill SE, Robinson J, Matthews G, Muschol M. 2009. Amyloid protofibrils of lysozyme nucleate and grow via oligomer fusion. Biophys. J. 96: 3781–3790. Hou X, Parkington HC, Coleman HA, Mechler A, Martin LL, Aguilar MI, Small DH. 2007. Transthyretin oligomers induce calcium influx via voltage-gated calcium channels. J. Neurochem. 100: 446–457. Inouye H, Domingues FS, Damas AM, Saraiva MJ, Lundgren E, Sandgren O, Kirschner DA. 1998. Analysis of x-ray diffraction patterns from amyloid of biopsied vitreous humor and kidney of transthyretin (TTR) Met30 familial amyloidotic polyneuropathy (FAP) patients: axially arrayed TTR monomers constitute the protofilament. Amyloid 5: 163–174. Kad NM, Thomson NH, Smith DP, Smith DA, Radford SE. 2001. Beta (2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro. J. Mol. Biol. 313: 559–571. Karsai Á, Grama L, Murvai Ü, Soós K, Penke B, Kellermayer MSZ. 2007. Potassium-dependent oriented growth of amyloid ß 25-35 fibrils on mica. Nanotechnology 18: 345102. Karsai Á, Mártonfalvi Z, Nagy A, Grama L, Penke B, Kellermayer MSZ. 2006. Mechanical manipulation of Alzheimer’s amyloid ß 1-42 fibrils. J. Struct. Biol. 155: 316–326. Karsai A, Murvai U, Soos K, Penke B, Kellermayer MS. 2008. Oriented epitaxial growth of amyloid fibrils of the N27C mutant beta 25-35 peptide. Eur. Biophys. J. 37: 1133–1137. Karsai Á, Nagy A, Kengyel A, Mártonfalvi Z, Grama L, Penke B, Kellermayer MSZ. 2005. Effect of lysine-28 side chain acetylation on the nanomechanical behavior of Alzheimer amyloid ß25-3 fibrils. J. Chem. Info. Mod. 45: 1641–1646. Kellermayer MS, Grama L, Karsai A, Nagy A, Kahn A, Datki ZL, Penke B. 2005. Reversible mechanical unzipping of amyloid beta-fibrils. J. Biol. Chem. 280: 8464–8470. Kellermayer MS, Karsai A, Benke M, Soos K, Penke B. 2008. Stepwise dynamics of epitaxially growing single amyloid fibrils. Proc. Natl Acad. Sci. USA 105: 141–144. Khurana R, Ionescu-Zanetti C, Pope M, Li J, Nielson L, Ramirez-Alvarado M, Regan L, Fink AL, Carter SA. 2003. A general model for amyloid fibril assembly based on morphological studies using atomic force microscopy. Biophys. J. 85: 1135–1144. Kodali R, Wetzel R. 2007. Polymorphism in the intermediates and products of amyloid assembly. Curr. Opin. Struct. Biol. 17: 48–57. Lai Z, Colon W, Kelly JW. 1996. The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can selfassemble into amyloid. Biochemistry 35: 6470–6482. Modler AJ, Fabian H, Sokolowski F, Lutsch G, Gast K, Damaschun G. 2004. Polymerization of proteins into amyloid protofibrils shares common critical oligomeric states but differs in the mechanisms of their formation. Amyloid 11: 215–231. Modler AJ, Gast K, Lutsch G, Damaschun G. 2003. Assembly of amyloid protofibrils via critical oligomers–a novel pathway of amyloid formation. J. Mol. Biol. 325: 135–148. Muller DJ, Engel A. 1997. The height of biomolecules measured with the atomic force microscope depends on electrostatic interactions. Biophys. J. 73: 1633–1644. Naiki H, Nagai Y. 2009. Molecular pathogenesis of protein misfolding diseases: pathological molecular environments versus quality control systems against misfolded proteins. J. Biochem. 146: 751–756. Nixon RA. 2007. Autophagy, amyloidogenesis and Alzheimer disease. J. Cell Sci. 120: 4081–4091. Relini A, Canale C, De Stefano S, Rolandi R, Giorgetti S, Stoppini M, Rossi A, Fogolari F, Corazza A, Esposito G, Gliozzi A, Bellotti V. 2006. Collagen plays an active role in the aggregation of beta2-microglobulin under physiopathological conditions of dialysis-related amyloidosis. J. Biol. Chem. 281: 16521–16529. Relini A, De Stefano S, Torrassa S, Cavalleri O, Rolandi R, Gliozzi A, Giorgetti S, Raimondi S, Marchese L, Verga L, Rossi A, Stoppini M, Bellotti V. 2008. Heparin strongly enhances the formation of beta2microglobulin amyloid fibrils in the presence of type I collagen. J. Biol. Chem. 283: 4912–4920. Serpell LC, Sunde M, Benson MD, Tennent GA, Pepys MB, Fraser PE. 2000. The protofilament substructure of amyloid fibrils. J. Mol. Biol. 300: 1033–1039. Serpell LC, Sunde M, Fraser PE, Luther PK, Morris EP, Sangren O, Lundgren E, Blake CC. 1995. Examination of the structure of the transthyretin amyloid fibril by image reconstruction from electron micrographs. J. Mol. Biol. 254: 113–118. Sousa MM, Cardoso I, Fernandes R, Guimaraes A, Saraiva MJ. 2001. Deposition of transthyretin in early stages of familial amyloidotic 475 J. Mol. Recognit. 2011; 24: 467–476 Copyright ß 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jmr R. H. PIRES ET AL. polyneuropathy: evidence for toxicity of nonfibrillar aggregates. Am. J. Pathol. 159: 1993–2000. Sousa MM, Fernandes R, Palha JA, Taboada A, Vieira P, Saraiva MJ. 2002. Evidence for early cytotoxic aggregates in transgenic mice for human transthyretin Leu55Pro. Am. J. Pathol. 161: 1935–1948 . Sousa MM, Saraiva MJ. 2003. Neurodegeneration in familial amyloid polyneuropathy: from pathology to molecular signaling. Prog. Neurobiol. 71: 385–400. Sunde M, Serpell LC, Bartlam M, Fraser PE, Pepys MB, Blake CC. 1997. Common core structure of amyloid fibrils by synchrotron X-ray diffraction. J. Mol. Biol. 273: 729–739. Westermark P, Sletten K, Johansson B, Cornwell GG 3rd. 1990. Fibril in senile systemic amyloidosis is derived from normal transthyretin. Proc. Natl Acad. Sci. USA 87: 2843–2845. Yamaguchi I, Hasegawa K, Takahashi N, Gejyo F, Naiki H. 2001. Apolipoprotein E inhibits the depolymerization of beta 2-microglobulinrelated amyloid fibrils at a neutral pH. Biochemistry 40: 8499–8507. Yamamoto S, Hasegawa K, Yamaguchi I, Goto Y, Gejyo F, Naiki H. 2005. Kinetic analysis of the polymerization and depolymerization of beta(2)-microglobulin-related amyloid fibrils in vitro. Biochim. Biophys. Acta 1753: 34–43. 476 wileyonlinelibrary.com/journal/jmr Copyright ß 2011 John Wiley & Sons, Ltd. J. Mol. Recognit. 2011; 24: 467–476